The T cell reactivity against the major merozoite protein of Plasmodium falciparum

Immunology Letters, 25 (1990), 143-148 Elsevier I M L E T 01444

The T cell reactivity against the major merozoite protein of

Plasmodium falciparum A n d r e a Crisanti 1, 2, Klaus Frfih 1, H a n s M i c h a e l Mfiller I a n d H e r m a n n B u j a r d ~ IZMBH, Zentrum f~r Molekulare Biologie Heidelberg, Universit6t Heidelberg, Heidelberg, ER.G., and 2Istituto di Parassitologia, Universit~ de Roma "La Sapienza", Rome, Italy

1. Summary We have undertaken a systematic search for T cell epitopes within the sequence of the major merozoite surface antigen (GP190) of Plasmodium falciparum. Recombinant polypeptides expressed in E. coli were used to evaluate the reactivity of peripheral blood monuclear cells (PBMC) from both inhabitants of a rural community of West Africa exposed to P. falciparum transmission and from German patients with diagnosis of acute malaria. Although the proliferative response of the PBMC was in most cases very low, several T cell clones could be established. Deletion analysis of each gpl90-derived polypeptide allowed the identification of six different T cell epitopes. Epitopes could be mapped within the dimorphic region of gpl90, which also contains the sequences most frequently recognized by sera from adult individuals living in endemic areas. 2. Introduction

The major surface proteins of the merozoite of Plasmodium falciparum are processed products of a 190-kilodalton precursor glycoprotein, gpl90 [1]. Comparison of the gpl90 sequences of different P falciparum isolates reveals three degrees of variability: isolate-specific stretches of amino acids, dimorphic parts which are either MAD20 or K1 like, and clusters of highly conserved sequences [2-5]. Key words." Plasmodium falciparum gpl90; Dimorphic sequence; T cell epitopes

Correspondence to: Andrea Crisanti, Z M B H , Zentrum for Molekulare Biologie Heidelberg, Universit/it Heidelberg, Im Neuenheimer Feld 282, 6900 Heidelberg, F.R.G. Fax: + 49-6221-566809.

When used for the immunization of monkeys, gpl90 modifies the course of infection by the parasite [ 6 - 8 ] . Homologous proteins in other animal models were also shown to be involved in protective immunity [9-11]. These protective properties candidate gpl90 as a component of a malaria vaccine. The analysis of the humoral immune response against the gpl90 protein has shown that: (1) both qualitative and quantitative differences exist among groups of individuals with different susceptibility to P.falciparum infection. (2) The regions of the molecule may differ in the ability to induce protective immunity. (3) Both MAD20 and K1 dimorphic regions are recognized by nearly all the sera from adult individuals when the parasite carrying the corresponding gpl90 allele is transmitted in a given area [12-13]. An association between intensity of transmission and antibody level against the dimorphic region was observed in both adults and children living in a malaria endemic area [13]. In children the humoral response against the dimorphic region was found to be shortlived, the level of specific antibodies decreased dramatically after the end of the transmission season, [13]. Furthermore, naturally acquired antibodies to polymorphic region of the gpl90 were shown to be predictive for resistance to P. falciparum infection whereas the presence of antibodies to the amino proximal invariant region inversely correlated with protection [14]. Specific T helper cells should play a crucial role in eliciting the humoral immunity that has been observed in humans against the different region of the gpl90. The gpl90 regions should be candidated for vaccine development on the basis of the human humoral response and of the presence within the sequences of T cell epitopes that would induce a cellular helper immune response. We analyzed the T cell reactivity against recombinant poly-

0165-2478 / 90 / $ 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

143

peptides corresponding to the regions of the gpl90 sequences that are most frequently recognized by sera from individuals exposed to malaria transmission. The T cell epitopes have been identified by testing a panel of human T cell clones against the polypeptides, each produced as a set of deletion fragments progressively truncated either at the amino-terminus or at the carboxyl terminus.

cleavage of plasmid DNA, the coding sequences were digested with exonuclease III, and then treated with S1 and Klenow enzyme. Proper linkers were [igated to the truncated ends, fragments of the desired length were isolated from polyacrylamide gels and recloned into the vector pDS789. The progressively shortened fusion proteins differed from each other by 1 0 - 2 0 amino acids.

3. Materials and Methods

3.4. Lymphocyte proliferation assay

3.1. Donors

PBMC were isolated from heparinized whole blood by Ficoll-Hypaque centrifugation. PBMC were adjusted to a concentration of 1 x 106 cells/ml in RPMI supplemented with AB + serum and added to 96-well flat-bottomed plates in 100 ffl aliquots. gpl90 recombinant polypeptides (i mg/ml in 6M urea) were diluted to 5/xg/ml and added in triplicate in 100 #1 volumes. After 5 days the cultures were pulsed with 1 #Ci of [3H]thymidine, and incorporation of labeled nucleotide was determined after additional 16 h.

Blood ( 2 0 - 3 0 ml) was obtained from 15 adults living in rural community in Mali, West Africa, at three time points: June 88, Nov. 88, May 89. Transmission season lasted from June to the beginning of December. Samples were processed in Heidelberg within 24 h from the time of blood withdrawal. In addition blood was obtained from 12 German patients that had been infected by P falciparum during a visit to malaria endemic areas.

3.2. Recombinant GP190 polipeptides. The expression of recombinant gp190 fusion polypeptides to CAT and DNA in Escheriehia coli has been described [12]. In short, genomic DNA fragments were inserted into the pDS789, 6xHis, D H F R vector by replacing the D H F R coding sequences. In this way a tail of six histidines resulted fused to the amino-terminus of the GP190 polypeptides. The presence of the histidine tail allows to purify the recombinant proteins in a single step by nickel chelate chromatography [15]. Total bacterial protein was dissolved in 6 M guanidinium hydrochloride, pH 8.0, and directly applied to the nickel column. Bound protein was eluted in the same buffer by lowering the pH stepwise to pH 4. In this study the following gpl90 polypeptides were used: M6 AA 384-595, M7 AA 596-898, both derived from the amino-proximal dimorphic region (DR I) of MAD20, F2 AA106-321, F2 corresponds to the amino proximal constant region (CR I) of K1.

3.5. Cloning and maintenance of gpl90-specific T cells PBMC were adjusted to a concentration of 1 x 10 6 cells/ml in RPMI supplemented with AB + serum and incubated with the purified GP 190 recombinant polypeptides (M6-His, M7-His, F2 CAT, 1 #g/ml) for 6 days at 37 °C in 5% CO2/air. The cells were washed twice and transferred to medium containing 100 U/ml rIL-2 (Hoffmann-La Roche) and, after another 7 days, cloned by limiting dilution. For cloning, T cells were seeded at 0.5 cells/well in the presence of 5 #g/ml gpl90 polypeptides, 104 autologous irradiated PBMC and IL-2 100 U/ml. All the clones were subcloned, for each subclone the probability of being generated from a single precursor was 98o70 as determined by Poissons analysis. Antigen stimulation of the clones was measured by harvesting a coculture of 2 x 10 4 cloned T cells with 2 x l0 s autologous irradiated (3000 rad) PBMC as source of antigen presenting cells after exposure to [3H]thymidine for 12-18 h on day 3.

3.3. Generation of deletion fragments 3.6. Media Progressively truncated polypeptides from M6, M7 and F2, were generated as described, [16]. After 144

The culture medium was RPMI 1640 (Gibco

GP190 M A D 20 R

CR II

CRI

DR I

CR Ill

DR II

'

'"

I

CR IV

I I AA 1702

AAI M2

I

M3

M4

M6

M7

M6

M9

MIO

M1 1

F8

F9

FIO

I

GP190 KI

•

I :llll]lll I

AAI

I

AA1631

F2

F4

F5

F7

Fig. 1. Schematic representation of the K1 and MAD20 precursor proteins showing the relative position of the gpl90 polypeptides used here and in the studies described at ref. 12, 13, 14. Blocks of constant and dimorphic regions are progressively numerated from the amino terminus of the molecule.

Laboratories, Paisley, U.K.) supplemented with Lglutamine (2 mM) 1°70 non essential amino acids from 100 × stock solution (Gibco), 1 mM sodium pyruvate, 50 U/ml penicillin, 50/zg/ml streptomicin, 5×10 -5 M 2ME and 10°70 AB + human serum. To support the antigen independent growth of T cell clones, the medium was supplemented with 100 U/ml rIL-2 (Hoffmann-La Roche, Nutley, N J).

4. Results

4.1. Expression of gpl90 polypeptides and generation of deletions For the analysis of the T cell response against the gpl90 in individuals exposed to P. falciparum infection we have used the fragments F2, M6 and M7 (Fig. 1). These fragments have been chosen on the

TABLE 1 Identification of T cell epitopes in gpl90 CR I using progressively deleted F2 fragments. T cell clones

cpm × 103 Cat

F2

2.1

2.8

2.22

2.17

3.7

AC68 AC69 AC71 AC75

0.2 0.1 0.1 0.3

45.0 55.6 68.0 98.9

35.4 39.5 36.2 96.6

29.6 53.4 48.5 85.7

30.4 62.0 39.6 98.4

0.2 0.7 0.3 0.5

0.2 0.3 0.3 0.4

AC AC AC HD

63 66 74 11.3

0.1 0.1 0.1 0.7

30.3 36.8 19.6 25.4

26.6 28.6 17.8 19.6

28.0 25.6 10.9 13.1

9.0 8.5 5.0 6.2

35.0 34.4 14.9 23.6

H D 11.9 H D 7.8

0.9 0.7

32.3 15.9

30.6 14.0

ND ND

5.8 4.9

37.8 16.3

3.2

4.20

4.19

4.5

5.22

0.2 0.4 0.2 0.5

0.3 0.2 0.3 0.3

ND ND ND ND

0.2 0.1 0.2 0.4

0.3 0.2 0.2 0.3

29.0 32.3 18.7 24.9

16.0 23.3 17.6 21.0

38.0 21.6 12.0 20.9

0.2 0.4 0.2 0.8

0.2 0.4 0.2 1.1

0.2 0.2 0.2 0.g

35.5 15.2

33.0 14.4

39.6 16.8

34.2 15.7

0.8 1.2

0.9 1.1

F2 progressively shortened at the a m i n o terminus ( F 2 - F 5 . 2 2 ) stimulated the T cell clones until the epitopes were partially or totally deleted. Three T cell epitopes have been mapped: (a) within the amino termini of F2.22 and F2.17 (AA 1 7 0 - 183), (b) within F4.20 and F4.19 (AA 219 - 232); (c) within F4.19 and F4.5 (AA 237 - 252). The mean incorporation (round value) of [3H]thymidine into triplicate cultures is given.

145

TABLE 2 Identification of T cell epitopes in gpl90 DR I using deleted fragments originating from M6 (A) and M7 (B). (A) T cell clones

cpm x 103 M6 AA 384 595

0301/6 0301/27 HD 15/23

M6-30 541

M6-60 516

M6-80 508

M6-20 487

9.35

7.83

7.58

5.06

7.35

7.66

M6-34 467

M6-31 452

M6-45 424

M6-40 419

6.90

7.42

3.38

6.70

5.41

7.77

8.23

7.27

0.17

3.54

3.34

5.96

6.~4

6.11

0.44

0 18

0.45

0.25

ND

ND

ND

(B)

T cell clones

cpm × 103 M7 AA 596- 898

H D 15/3.1

23.5

M7-45 826

M7-60 796

M7-64 766

M7-91 748

M7-92 745

M7-99 743

M7-107 728

10.55

11.55

11.90

8.90

2.05

1.80

2.20

Both gpl90 M6 and M7 were progressively shortened at their carboxyl termini. The position in gpl90 MAD 20 of the amino acid at the corboxyl terminus is given under each fragment. Three T cell epitopes have been identified. Two epitopes are located within M6 at the carboxyl termini of AA 516 508 and of AA424 - 419 respectively. The third epitope is placed in M7 within the carboxyl termini of AA 748 745.

basis b o t h of the h u m o r a l response against the molecule a n d o f the gpl90 p o l y m o r p h i s m expressed by the parasites t r a n s m i t t e d in Safo: (a) most sera from G e r m a n patients react against F2 a n d M6; (b) nearly all adult i n h a b i t a n t s o f Safo have high a n t i b o d y levels against M6 a n d M7 [12]; (c) the gpl90 sequences expressed by the parasites t r a n s m i t t e d in Safo has been shown to be o n l y o f the M A D 2 0 d i m o r p h i c type [13]. The fragments F2, M6 a n d M7 were expressed in E. coli fused to a six-histidine tail at their a m i n o termini. The presence of the histidines allowed efficient p u r i f i c a t i o n (95 °70)in a single step by nickel chelate chromatography. In order to m a p the T cell reactivity, the coding sequences of the fragments were digested with exonuclease III to generate a set o f progressively t r u n c a t e d fragments. A n u m b e r of deletions from M6 a n d M7 were generated a n d expressed in E. coli with the histidine tail. The a m i n o acid p o s i t i o n along the g p l 9 0 sequences for each deletion used is shown in Tables 1 a n d 2. The deletions from the F2 f r a g m e n t have already been described [16].

146

4.2. Proliferative response to antigens Responses to gpl90 (F2, M6, M7) a n d to tetanus toxoid (TT) are s u m m a r i z e d in Fig, 2 as m e a n stimuSl i

30

- !

20

-

i i

lO

I

July 88

[]

F2,

• M6,

1

I

Nov88

May 89

[] M71 o TT

Fig. 2. Proliferative response of PBMC in Safo Mall against gpl90 derived polypeptides and tetanus toxoid. The proliferative response is shown as the mean of the stimulation index (SI) of all the individuals tested at each time point. Bars represent standard deviations.

lation indices. P B M C from the majority of the individuals analyzed in Safo dit not show, at three time points during the year, a significant proliferative response (SI > 5) to the gpl90 recombinant polypeptides. Only at the end of the dry season in May 89, few individuals from Safo showed a response to M6 and F2 with a SI higher than 5, and T cell clones could be established. The proliferative response to a control antigen like TT was clearly demonstrable through all the year although it was depressed during the time of malaria transmission (July and Nov.) when compared with the response observed at the end of the dry season in the same individuals. Similarly, P B M C from G e r m a n patients that had been infected with P falciparum during a visit to malaria endemic areas did not show any proliferative response against the gpl90 polypeptides, (not shown). PBMC from three patients ( H D 7, H D l l , H D 15) were assayed four months after recovery from malaria. At this time a proliferative response and specific

F2 K1 A A 1 0 6 - 3 2 1 :

70

185

v T-~A
2!9

232

LDN'KDNV@K~IEDY

c~ helix

c~ helix

237

K

!

252

E-'~I~,,EL

epitopes T cell clones were obtained from the immune donor 0301 against the polypeptide M6 that maps to DR I of MAD20 gpl90. The reactivity against M6 and the derived deletions of two clones, 0301/6 and 0301/27, is shown in Table 2A. The results indicate the presence o f a T cell epitope at the carboxyl termini of M6-45 and M6-40 around position 424-419 of gpl90. The DR I of MAD20 gpl90 does not contain only the T cell epitope recognized by 0301 clones. As shown in Table 2A and B, two clones derived from the G e r m a n patient H D 15 react with DR I. The clone H D 15/23 recognizes an epitope in M6 at the carboxyl termini of residues 516-508 whereas the clone H D 15/3.1 reacts with an epitope in M7 placed around residues 7 4 8 - 7 4 5 as indicated by the reactivity against the deletion fragments. The sequences of the epitopes so far identified within the gpl90 both in the gpl90 regions CR I and DR I are shown in Figure 3. All the sequences of the T cell epitope contain an o~-helical structure that in most of the cases is amphypathic [17]. Furthermore, four out of six epitopes contain a motive described to be peculiar to sequences that function as T cell epitopes [18]. 5. Discussion

M5-M7 M A D 20 A A 380-1059 423 'v v p L s L T D I H N S L AVA D N ez h e i ×

L E K+F'Y'E ~ K ~

NV N

518

N F D K D- *V V. - D K+ . I

!

*

c~ h e l i x

+--

_..__v : Y E V T~_E T V G o~ h e l i x

4.3. T cell clones and identification of T cell

EESKK

o. h e l i x

ct h e l i x

T cell clones against F2 and M6 were obtained.

H TTTV

7a8 T: ~L Pa

:

Fig. 3. Sequences and structural features of T cell epitopes identified within the sequences of the gpl90 in the CR I region (F2 KI A A 106-321) and in the DR I region (M6-M7 M A D 20 AA3801050). Underlined sequences correspond to regions that have an high probability of being ~ helix. Alternated charged ( + ; - ) and hydrophobic (*) amino acid and amphipaticity (!) in a helix are indicated. In F2, synthetic peptides contain the T cell epitopes in the sequences shown. In M6 and M7 the carboxyl terminal sequences of the smallest deletion that stimulates the T cell clones to proliferate together with the position of the closest deletion that abrogates the stimulating activity are shown.

We have analyzed the T cell reactivity against recombinant polypeptides corresponding to the region of the gpl90 that are the most frequently recognized by sera from individuals exposed to P. falciparum infection. Our results show that PBMC from inhabitants of an endemic area as well as acutely infected patients from a Western country do not proliferate against the region of the gpl90 we have tested. Depressed or absent proliferative response for other blood stage antigens has already been reported and it has been attributed to immune suppression during malaria [19-20]. In our case it would be difficult to interpret the lack of proliferative response of the P M N C as the result of immune suppression. By analysing the humoral response of the same group of individuals from Safo at the same time points, we have observed, during the peak of the transmission season, a marked increase of antibody 147

level (IgG) a g a i n s t M6 a n d M7 [13] thus suggesting the presence, in these subjects, o f an in vivo T helper cell response. T cell clones reacting a g a i n s t the g p l 9 0 p o l y p e p t i d e s c o u l d be o b t a i n e d f r o m Safo i n h a b i tants d u r i n g the d r y season a n d from G e r m a n patients few m o n t h s after the acute e p i s o d e o f m a l a r i a thus i n d i c a t i n g t h a t the absence o f the proliferative response is n o t due to lack o f T cell e p i t o p e s in g p l 9 0 sequences. F u r t h e r m o r e , using a set o f d e l e t i o n s progressively t r u n c a t e d at the a m i n o - t e r m i n i o r at the c a r b o x y l - t e r m i n i o f the g p l 9 0 fragments, several T cell e p i t o p e s c o u l d be i d e n t i f i e d in the c o n s e r v e d a n d in the d i m o r p h i c region o f the gpl90. As a l t e r n a tive e x p l a n a t i o n to i m m u n e suppression, the a b s e n c e o f p r o l i f e r a t i o n o f the P B M C m a y be due to a m a r k e d r e d u c t i o n o f g p l 9 0 specific T cells in the circ u l a t i n g p o o l o f T l y m p h o c y t e s in p a r a s i t i s e d individuals. T h e i d e n t i f i c a t i o n o f T cell e p i t o p e s within the sequence o f M 6 a n d M7 has, to o u r o p i n i o n , also implications on m a l a r i a vaccine design. H u m o r a l response a g a i n s t the d i m o r p h i c regions o f g p l 9 0 m a y a c c o u n t for the r e d u c e d susceptibility to clinical m a l a r i a o f a d u l t i n h a b i t a n t s f r o m e n d e m i c areas [12-13]. T h e presence o f T cell e p i t o p e s in M 6 a n d M7 w o u l d p e r m i t the d e v e l o p m e n t o f a h u m o r a l imm u n e response a g a i n s t the d i m o r p h i c regions o f R falciparum g p l 9 0 by using these f r a g m e n t s for imm u n i z a t i o n . T h e h u m o r a l response against the polym o r p h i c regions o f the g p l 9 0 (M2 a n d M4 in Fig. 1) has been shown to be predictive for parasite i m m u n i ty [14], however the v a r i a b i l i t y a n d the limited sequences length o f the p o l y m o r p h i c regions m a y det e r m i n e the need for a d d i t i o n a l T cell epitopes. Such e p i t o p e s c o u l d be p r o v i d e d by the M6 a n d M7 sequences from D R I. A l s o the a m i n o p r o x i m a l region o f g p l 9 0 c o n t a i n s several T cell e p i t o p e s within its sequences. However the C R I region is recognized at high frequency o n l y by sera f r o m n o n - i m m u n e ind i v i d u a l s like G e r m a n p a t i e n t s a n d A f r i c a n infants [12]. F u r t h e r m o r e , the h u m o r a l reactivity a g a i n s t C R I was f o u n d to correlate inversely to parasite imm u n i t y in a d u l t i n d i v i d u a l s living in a m a l a r i a end e m i c area [14]. T h e r e m a y be the p o s s i b i l i t y to induce an h u m o r a l response t h a t facilitate P. falcipa-

148

rum infection by using gp190 C R I T cell e p i t o p e s if e n o u g h sequences are shared with B cell epitopes. References [1] Holder, A. A. and Freeman, R. R. (1984) J. Exp. Med. 160, 624. [2] Mackay, M., Goman, M., Bone, J., Hyde, J. E., Scaife, J., Certa, U., Stunneberg, H. and Bujard, H. (1985) EMBO J. 4, 3823. [3] Peterson, M. G., Coppel, R. L., McIntire, R, Langford, C. J., Woodrow, G., Brown, G. V., Anders, R. F. and Kemp, D. J. (1988) Mol. Biochem. Parasitol. 27, 291. [4] Tanabe, K., Mackay, M., Goman, M. and Scaife, J. (1987) J. Mol. Biol. 195, 273. [5] Weber, J., Leininger, M. W. and Lyon, J. A. (1986) Nucleic Acids Res. 14, 3311. [6] Hall, R., Hyde, E. J., Goman, M., Simmons, D. L., Hope, 1. A., Mackey, M., Scaife, J., Merkli, B., Richle, R. and Stocker, J. (1984) Nature 311, 379. [7] Perrin, L. H., Loche, M., Dedet, J. P., Roussilhon, C. and Fandeur, T. (1988) Clin. Exp. Immunol. 56, 67. [8] Siddiqui, W., Tam, Q. L., Kramer, K. J., Hui, G. S. N., Case, S. E., Yamaga, K. M., Chang, S. P., Chart, E. B. T. and Kan, S. C. (1987) Proc. Natl. Acad. Sci. USA 84, 3014. [9] Boyle, D. B., Newbold, C. I., Smith, C. C. and Brown, K. N. (1982) Infect. Immun. 8, 94. [10] Epstein, N., Miller, L. H., Kaushel, D. C., Udeynya, I. J., Rener, J., Howard, R. J., Asosfsky, R., Aikawa, M. and Hes, R. L. (1981) J. Immunol. 127, 212. [11] Holder, A. A. and Freeman, R. R. (1981) Nature 294, 361. [12] MOiler,H. M., Fr0h, K., yon Brunn, A., Esposito, F., I.ombardi, S., Crisanti, A. and Bujard, H. (1989) Infect. lmmun. 57, 3765. [13] Friih, K., Doumbo, O., Mciller, H. M., Koita, O., McBride, J., Crisanti, A., Bujard, H. (1990) manuscript submitted. [14] Frtih, K., MOiler,H. M., Crisanti, A., yon Brunn, A., Huffman, S., Oster, C. N., Chulay, J., Mugabi, M., Bujard, H. and Lyon, J. (1990), manuscript submitted. [15] Hochuli, E., Banwarth, W., Dabeli, H., Gentz, R. and Stuber, D. (1988) Biotechnology 1321. [16] Crisanti, A., Muller, H.-M., Hilbich, C., Sinigaglia, F., Matile, H., MacKay, J., Scaife, J., Geyreuther, K., Bujard, H. (1988) Science 240, 1324. [17] De Lisi, C. and Berzofsky, A. (1985) Proc. Natl. Acad. Sci. USA 85, 7048. [18] Rothbard, J. B. (1986), Ann. Inst. Pasteur 137E, 518. [19] Ho, M., Webster, H. K., Looareesuwan, S., Supanaranond, W., Phillips, R. E., Chanthavanich, R and Warrel, D. A. (1986) J. Infect. Dis. 153, 763. [20] Troye-Blomberg, M., Perlmann, H., Patarruyo, M. E. and Perlmann, P. (1983) Clin. Exp. Immunol. 53, 345. (Accepted for publication 15 May 1990)