Intern. Rev. Immunol. 6 , 1990, pp. 61-73 Reprints available directly from the publisher Photocopying permitted by license only 01990 Hanvood Academic Publishers GmbH Printed in the United States of America
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Peptide-MHC Interaction: A Rational Approach to Vaccine Design PAOLA ROMAGNOLI, BELA TAKACS, JOCHEN KILGUS, J. RICHARD L. PINK, and FRANCESCO SINIGAGLIA Central Research Units, E Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland KEYWORDS: vaccination, synthetic peptide, MHC, T cell epitope
The goal of vaccination is to induce a protective immune response without inducing the disease itself or other undesirable effects. In the past this was achieved by giving inactivated or attenuated pathogens, with notable success in some cases, but problems of safety, efficacy or production in others. A currently pursued alternative is the construction of synthetic peptide vaccines that contain pathogen-derived determinants able to induce an immune response both at the B and the T cell level. Because T cells recognize peptide fragments which are derived from the processing of soluble proteins [l], the technical problems of epitope conformation can be largely avoided in the design of synthetic T cell sites. However the constraints presented by the extensive polymorphism of MHC antigens on one hand [ 2 ] , and the obligate interaction between the antigenic peptide and the MHC molecule on the other [3], create a problem for the immune system. When an antigenic determinant does not interact favorably with the MHC molecules possessed by a given individual, T cell clones specific for this particular combination of determinant and MHC molecules cannot be activated and therefore not all individuals will respond to any one antigenic determinant. Thus in order to develop a synthetic peptide vaccine applicable to an entire population, individuals with each MHC haplotype would have to be studied to ascertain which peptides they predominantly recognize as T cell determinants. In this review, through the work we have done on malaria sporozoite vaccine development, we should like to propose new strategies for identifying pathogen-derived sequences whose characteristics offer possible solutions to the problem of MHC restriction of the immune response for peptide vaccines.
DEFINING EPITOPES OF THE CS PROTEIN WHICH BIND TO HLA-DR MOLECULES In recent years, considerable progress has been made in understanding the interaction between peptides and MHC molecules. Several groups have shown that immunologically active peptides can specifically bind to purified, detergent-solubilized class I1 proteins. The first direct evidence that antigen and MHC can interact with one another, independently of 61
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62
F? ROMAGNOLI er al.
the T cell receptor, was obtained by Emil Unanue’s group at Washington University. Using equilibrium dialysis, they demonstrated that an antigenic peptide derived from chicken lysozyme could bind to the class I1 MHC molecule that served as a restriction element in the immune response to that antigen [4]. Buus etal. confirmed and extended this observation by measuring the binding of several antigenic peptides to a variety of mouse MHC class I1 alleles [ 5 ] . Following these early studies, we recently developed a biochemical binding assay, based on the method described by Buus el al. [6], to analyze the binding of peptides derived from the malaria f? falciparum parasite circumsporozoite protein (CS) to HLA-DR molecules. Affinity purified preparations of DR1 molecules (Fig. 1) were used in these experiments. The influenza virus matrix peptide Mat 17-31 [7] binds to the DR1 molecule (Fig. 2). Binding of labelled peptide was saturable and could be inhibited by an excess of cold matrix peptide. We then examined the interactionof DRl with CS peptides by competitionwith the labelled Mat 17-31 peptide (Table I). Peptide CS.T3, whose sequence DIEKKIAKMEKASSVFNVVNS corresponds to the conserved sequence of the CS protein residues 378-398 except that the two cysteines at positions 384 and 389 were substituted with alanine residues [8], competes very efficiently with the binding of the matrix peptide to DR1 molecules. The other peptides tested, comprising together about 50% of the CS protein sequence, did not significantly inhibit the binding. In a previous study we showed that peptide CS.T3 could bind to two other different DR antigens by testing it in a functional peptide competition assay. In this assay, we asked whether the CS.T3 could inhibit the proliferative response of a T cell clone to its specific antigen. We found that CS.T3 and, to a lesser extent, CS 325-341 were the only peptides out of the seven tested able to compete with the binding of the stimulator peptide to DRwll(5). Only CS.T3 competed for binding to DRw14(w6) molecules (Fig. 3) [9]. In a second step, we examined the ability of the CS.T3 peptide to induce primary T-cell responses in human peripheral blood mononuclear cells (PBMC) and to induce T cell clones able to respond to the native CS protein in the presence of appropriate antigen-presenting cells (APCs) [lo, 111. We therefore challenged PBMC of 20 malaria non-immune donors with the peptide in vitro. From the stimulatedcells of 8 donors, close to 300 peptide-specific T lymphocyte clones (TLC) were derived. Analysis of the restriction specificity of the clones revealed that the peptide could be recognized in combination with at least 8 different DR molecules including DR1, 2, 4, 5, 6, 7, 8 and 9 (Fig. 4) [lo, 121. These results suggested that it may therefore not be necessary to search for a cocktail of different, T cell epitopes to stimulate an anti-sporozoite response in most vaccinated individuals, since CS.T3 alone would be recognized in association with one or more DR alleles present in 80-90% of typical African or European populations.
STRUCTURAL ANALYSIS OF THE PERMISSIVE ASSOCIATION BETWEEN CS.T3 AND DR MOLECULES The CS.T3 peptide is recognized in association with at least eight different DR antigens (possibly more, as we lack information about the other DR alleles). The structural basis for this broad binding capacity to DR molecules is unclear. We initially proposed the possibility that the binding of the peptide might be dependent on the DRa chain, which shows limited allelic polymorphism. However, analyses of the fine specificity of the interaction of CS.T3 with different DR alleles make it clear that the chain also play an important role in the binding of the CS.T3 peptide. For example, using a series of peptides truncated at either the Nor C-terminus of the
63
PEPTIDE-MHC INTERACTION
92 69 46 W
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2
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I
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-s
30 W
14
__
a
b
C
FIGURE 1 SDS-PAGE analysis of immuno-affinity purified HLA-DR1 molecules. For the immunoaffinity purification of DR antigens (lane c). detergent extracts (lane b ) obtained from about 1010 DRI-homozygous EBV-B cells were applied to a column containing Sepharose 4B-coupled specific mouse monoclonal anti-HLA-DR Ab. Lane a contains coomassie blue stained M.W. standards. The two noncovalently associated a (Mr 34000) and p (Mr 29000) chains are indicated.
200000
E a 0
'I 00000
0
0
10 Elution
20
30
volume (ml)
FIGURE 2 Gel liltration analysis of peptide-DR1 complexes. The DR molecules were isolated in solution using detergent solubilization from DR-homozygous EBV-transformed B cell lines (Fig. 1). The DR1-binding influenza virus Matrix peptide Mat 17-31 was modified by the addition of a tyrosine residue to the amino terminus to allow 1251 labelling. The peptide was labelled using Iodo-Beads, purified and used within 1 week. Binding assays were done following the basic protocol of Buus er al. with some modifications (B. T., manuscript in preparation). 'The incubation mixture contained 100-300 pmoles of purified DRI and 1-3 pmoles of ~2SI-radiolabelledpeptide. After incubation at room temperature for 48 hours, the DRI-peptide complexes were separated from free peptide by gel filtration on a Sephadex G50 column and two ml fractions were collected and assayed for radioactivity in a gamma Mat 17-31; (A-A) DR1/12~I-labelled Mat 17-31+ cold Mat 17-31; (H-H) 125Ispectrometer. ( X - X ) DR1/~251-lahelled labelled Mat 17-31
64
I! ROMAGNOLI et af. TABLE I The Binding of CS Peptides to DRl Peptide
Sequence
DR1 Binding
CS(23-43) CS( 103-122) CS(297-309) CS(3 10-3 18) CS(325-341) CS(378-398)
YQCYGSSSNTRVJJJELNYDNA EKLRKPKHIUUKQPGJXNPD GHNMPNDPNRNVD ENANANNAV EPSDKHEEQYL,KKIKNS DIEKKIAKMEKASSVFNVVNS NANF'NWNANF'
-
+
-
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CS peptides were tested for the ability to inhibit the binding of **Wabeledinfluenza matrix peptide Mat 17-31 to detergent-dubilid DRl molecules.
HH.12 DRwll(5) Percent of response to 1 $4 antgenic peptide competitor (IOOrM)
20
0
40
60
100
80
120
CS 23-43 CS 103-122 CS 297-309 CS310-318 CS 325-341
cs 378-398 NANP3
I
1 AC.129 DRw14(w6)
competitor (100rM)
i
Percent of response to 1 )IM antigenicpeptide
,
2p
,
4p
,
6p
,
8p
,
1 7
,
170
CS 23-43 CS 103-122
CS 297-309
CS 310-318 CS 325-341
cs 378-398 NANP3
FIGURE 3 In vitro competition by CS peptides for interaction with DRwll(5) and DRw14(w6). Inhibition of antigen presentation was determined as previously described [91.
65
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PEPTIDE-MHC INTERACTION
DR1 DR2 DR4 DR5 DRw6 DR7 DR8 DR9 FIGURE 4 Definition of CST3 determinants recognized by T cell clones restricted by different DR alleles. The CS.T3 peptide, whose sequence is shown, and peptides which were truncated either from the amino or the carboxy terminus of CST3 were tested for their ability to stimulate T cell clones restricted by the DR alleles shown. The shortest sequences identified by this truncation analysis which could stimulate different DR-restricted T cell clones are shown.
CS.T3 sequence we have determined the binding profile for the two DR molecules, DRwll(5) and DRw14(w6) (Table 11). The critical residues required for interaction with DRwll(5) are contained within residues 380-391, and for interaction with DRw14(w6) within residues 381392 [13]. In order to identify the contribution of each aminoacid residue to the binding of CS.T3 to the two different DR molecules, analogs of CST3 were synthesized. At least 3 substitutions were made at each position (Table ID),most of them selected in order to change the size, charge or polarity of this residue. Each peptide analogue was tested for its capacity to bind to DRwll(5) and DRw14(w6) as well as to activate CS.T3-specific T cell clones. Although the two different DR alleles examined showed a nearly identical fine-specificity in the truncation analysis, when we analyzed the relative binding capacity of the 51 analogues used in this study, it was clear that distinct sites in the CS.T3 sequence are interacting in different ways with the two DR molecules analyzed (Fig. 5). For example, while substitution 389A -+ D drastically reduced binding in the DRwll(5) molecule, the same substitution resulted in a nearly twofold increase in binding to DRw 14(w6). Positions 383 and 388 appear to be the most critical for the binding to DRwll(5), whereas 392 is critical for binding to DRw14(w6). All the nonconservative changes at these positions abrogated binding to the respective DR antigen. Only when the isoleucine at position 383 was replaced by the very similar leucine was this change tolerated, albeit with an eightfold decrease in potency. The substitution of an aspartic acid for methionine 386 and a phenylalanine for glutamic acid 387 also destroyed the capacity to compete in the DRwll(5) functional assay. Again this effect could not be observed for DRw14(w6) binding, where, in addition to the substitutions 389 A += E, the replacement 391 S --+ F was no longer tolerated. The peptides containing point mutations were also assayed for their ability to activate TLC specific for the CST3 peptide (Table IV and J.K. submitted). As previously seen for other
P. ROMAGNOLI et al.
66
TABLE 11 Effect of CS.T3 Truncations on Binding to DRwll(5) and DRwIqw6) Binding to
CS Peptide 378-398
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379-398 380-398 381-398 382-398 383-398 378-396 378-395 378-394 378-393 378-392 378-391 378-390
Amino Acid Sequence
D I E K K I E K K E K K K K K D D D D D D D
I I I I I I I
E E E E E E E
K K K K K K K
K K K K K K K
I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E
K K K K K K K K K K K K K
A A A A A A A A A A A A A
S S S S S S S S S S S S S
S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V S V F N V S V F N S V F S V S
DRwll(5)
DRw14w6 15
It
0.7 2 >200 >200 >200
7 2 32 28 35 160
1200
12
15 67 >200 >200 5
8 15
17 100 >200 >200
tConcentration (pM) of truncated peptide required for 50% inhibition of antigen presentation.
peptides recognized by either class I or class 11-restricted T cells, the majority of substitutions affected T cell recognition significantly more than the ability of the peptide to bind the MHC protein [ 5 ] .Of the 51 analogues examined, more than 30%prevented recognitionof the peptides by TLC restricted by one of the three DR molecules. In contrast only 1045% of the substitutions abrogated binding to the DRs examined. The structuralconformationof the peptide bound to class 11moleculesis still the subject of an ongoing debate. Several authors have provided evidence for or against an a-helical structure of peptide bound to the MHC molecules (14-20). NMR analysis of the CS.T3 peptide in water shows that this peptide can indeed adopt an a-helical structure in solution (Labhardt A., unpublished results). However, when we compared the relative binding capacity of the peptide analogs with their ability to stimulateT cell clones specificfor the parental peptide, we observed stretches of four consecutive residues (384-387) where the peptide analogs could not activate the appropriateT cell clone (Table IV).Sincethe vast majority of the peptide analogs substituted within this stretch (31 out of 33) are still capable of binding the two DR molecules, the most likely explanationis that these residues make contact with the antigen receptor of the T cells. In a regular a-helix there are 3.6 residues per turn, so the fact that 4 consecutive residues are involved in contactingthe T cell receptor providesevidence against an a-helical conformationof the CS.T3 peptide in its bound state for the DR alleles examined. Thus, at least in its central region, the peptide seems to have an extended conformation.Clearly experimentalproof of this will require the crystallographic determination of the structure of the class 11-peptide complex. Of the 51 analogs tested, the peptide substituted at position 387 E + G showed enhanced binding to the DR alleles tested (Fig. 5). Because the CS.T3peptide, as discussed below, is able to induce helper T cells which could be boosted by immunization with the whole malaria parasite, these results may have implications for a malaria vaccine. It might be possible, as illustrated here, to i d e n w analogs that bind more strongly to MHC antigens than does the natural sequence. Such a result would be interesting, as it would suggest that the naturallyoccurring sequences might be improved as vaccine candidates by judicious selection of crossreactive, more immunogenic variant peptides.
PEPTIDE-MHC INTERACTION
67
TABLE IU Sequences of Peptide Analogs Tested ~
Analogs
Parental
(abbreviations)
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CS(380-396) 380E-+K 380E-F 380E-+G 381K-+G 38 1K-+D 381K-+V 382K-+D 382K-+G 382K-+Y 383I-+G 383IP.D 383IP.K 383I-+L 384A-+V 384A-+I 384A-+Y 384A-+K 385K-+D 385K-+F 385K-+G 386M-+G 386M-+K 386M-+D 387E-+F 387E-+G 387E-+K 388K-+G 388K-+D 388K-+F 389A-+E 389A-+D 389A-+Y 389A-+V 390S-+F 390S-+K 390S-+E 391S-F 391 S-+K 39 1S-+E 392V-+G 392V-+K 392V-+D 393F+G 393F+D 393F+K 394N-+K 394N-+E 394N-+F 395V-+G 395V-+K 395V-+D
380
385
390
395
€? ROMAGNOLI et al.
68 380
P a r e n t CS s e q u e n c e aa. S U b S t l t U t l o n
E
385
K
K
I
A
K
390
M
E
K
A
S
395
S
V
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0.01
380
aa' SUbSt'tUt'on
N
V
V
KFG GDV DGY GDKL VIYK DFG GKD FGK GDF EDYV FKE FKE GKD GDK KEF
T Parental CS sequence
F
I
A
K
395
390
385
K
M
E
K
A
S
1
S
V
F
N
V
V
KFG GDV DGY GDKL VIYK DFG GKD FGK GDF EDYV FKE FKE GKD GDK KEF
0.0 1
FIGURE 5 Binding of the 51 CS(380-396) analogs to DRwll(5) and DRw14(w6) molecules. The series of analogs shown in Table III was tested for binding to the two DR alleles by the functionalcompetition assay previously described [9].Each bar represents the arithmetic mean of three independent experiments.
THE PEPTIDE CAN ACT AS A HELPER DETERMINANT To examine whetherthe CS.T3 peptide could induce helper T cell function in vivo,we coupled it to the B cell determinant(NANP),, the repetitive NANP sequence in the central domain of the CS protein which is the major epitoperecognized by anti-sporozoiteantibodies [21]. Mice from severalstrains which were normallyunable to respond to the NANP repeatsproduced antibodies to the repeats, as well as to sporozoites, when injected with the ("P),-CS.T3 conjugate [lo]. This shows that theCS.T3 sequenceis also recogiuzed in associationwith many differentmouse Ia molecules, and that it can indeed function, at least in mice, as a carrier determinant for responses to other parts of the CS protein. Since the synthetic T cell determinant CS.T3 was capable of priming T cell help for antibody production to the B cell determinant (NANP),, and since antibody reacted with the native CS protein, this system provides evidencethat syntheticT and B cell recognition sites can be combined to yield a functional malaria immunogen. Such a
PE€TIDE-MHC INTERACTION
69
TABLE IV Evidence Against an a-Helical Conformation DRwl l(5) 380
385
390
395
MHC
TCR JK18
-
-
-
-
-
-
DRw 14(w6) MHC
TCR BR14
-
-
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parental 380E-K 380E-+F 380E+G 383I+G 3831-D 3831-K 3831-L 384A+V 384A-I 384A-Y 384A+K 38SK-D 385K-F 385K-G 386M-G 386M-K 386M+D 387E-F 387E+G 387E-K 388K-+G 388K-D 388K-F 392V-43 392V-+K 392V+D 393-G 393FiD 393hK 394N-K 394N-E 394N-F 395V-G 395V-K 395V+D
+ + ? + + + + + +
+
+ +
+
+ + + + + +
+ + + + +
+ ++ ++ + ?
+ + + +
+ + + + + + + + +
Peptide analogs were compared w~tbthe parental sequence (38&3%) for Tcell receptor recognition by clones JK18 and BR14 and binding to DRwll(5) and DRw14(w6). + + better binder (relative binding capacity >lo); + equal binder (relative binding capacity 10-0.1); ? poorer binder (relative binding capacity 0.01-0.1);- nonbinder (relative binding capacity
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Peptide-MHC Interaction: A Rational Approach to Vaccine Design PAOLA ROMAGNOLI, BELA TAKACS, JOCHEN KILGUS, J. RICHARD L. PINK, and FRANCESCO SINIGAGLIA Central Research Units, E Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland KEYWORDS: vaccination, synthetic peptide, MHC, T cell epitope
The goal of vaccination is to induce a protective immune response without inducing the disease itself or other undesirable effects. In the past this was achieved by giving inactivated or attenuated pathogens, with notable success in some cases, but problems of safety, efficacy or production in others. A currently pursued alternative is the construction of synthetic peptide vaccines that contain pathogen-derived determinants able to induce an immune response both at the B and the T cell level. Because T cells recognize peptide fragments which are derived from the processing of soluble proteins [l], the technical problems of epitope conformation can be largely avoided in the design of synthetic T cell sites. However the constraints presented by the extensive polymorphism of MHC antigens on one hand [ 2 ] , and the obligate interaction between the antigenic peptide and the MHC molecule on the other [3], create a problem for the immune system. When an antigenic determinant does not interact favorably with the MHC molecules possessed by a given individual, T cell clones specific for this particular combination of determinant and MHC molecules cannot be activated and therefore not all individuals will respond to any one antigenic determinant. Thus in order to develop a synthetic peptide vaccine applicable to an entire population, individuals with each MHC haplotype would have to be studied to ascertain which peptides they predominantly recognize as T cell determinants. In this review, through the work we have done on malaria sporozoite vaccine development, we should like to propose new strategies for identifying pathogen-derived sequences whose characteristics offer possible solutions to the problem of MHC restriction of the immune response for peptide vaccines.
DEFINING EPITOPES OF THE CS PROTEIN WHICH BIND TO HLA-DR MOLECULES In recent years, considerable progress has been made in understanding the interaction between peptides and MHC molecules. Several groups have shown that immunologically active peptides can specifically bind to purified, detergent-solubilized class I1 proteins. The first direct evidence that antigen and MHC can interact with one another, independently of 61
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62
F? ROMAGNOLI er al.
the T cell receptor, was obtained by Emil Unanue’s group at Washington University. Using equilibrium dialysis, they demonstrated that an antigenic peptide derived from chicken lysozyme could bind to the class I1 MHC molecule that served as a restriction element in the immune response to that antigen [4]. Buus etal. confirmed and extended this observation by measuring the binding of several antigenic peptides to a variety of mouse MHC class I1 alleles [ 5 ] . Following these early studies, we recently developed a biochemical binding assay, based on the method described by Buus el al. [6], to analyze the binding of peptides derived from the malaria f? falciparum parasite circumsporozoite protein (CS) to HLA-DR molecules. Affinity purified preparations of DR1 molecules (Fig. 1) were used in these experiments. The influenza virus matrix peptide Mat 17-31 [7] binds to the DR1 molecule (Fig. 2). Binding of labelled peptide was saturable and could be inhibited by an excess of cold matrix peptide. We then examined the interactionof DRl with CS peptides by competitionwith the labelled Mat 17-31 peptide (Table I). Peptide CS.T3, whose sequence DIEKKIAKMEKASSVFNVVNS corresponds to the conserved sequence of the CS protein residues 378-398 except that the two cysteines at positions 384 and 389 were substituted with alanine residues [8], competes very efficiently with the binding of the matrix peptide to DR1 molecules. The other peptides tested, comprising together about 50% of the CS protein sequence, did not significantly inhibit the binding. In a previous study we showed that peptide CS.T3 could bind to two other different DR antigens by testing it in a functional peptide competition assay. In this assay, we asked whether the CS.T3 could inhibit the proliferative response of a T cell clone to its specific antigen. We found that CS.T3 and, to a lesser extent, CS 325-341 were the only peptides out of the seven tested able to compete with the binding of the stimulator peptide to DRwll(5). Only CS.T3 competed for binding to DRw14(w6) molecules (Fig. 3) [9]. In a second step, we examined the ability of the CS.T3 peptide to induce primary T-cell responses in human peripheral blood mononuclear cells (PBMC) and to induce T cell clones able to respond to the native CS protein in the presence of appropriate antigen-presenting cells (APCs) [lo, 111. We therefore challenged PBMC of 20 malaria non-immune donors with the peptide in vitro. From the stimulatedcells of 8 donors, close to 300 peptide-specific T lymphocyte clones (TLC) were derived. Analysis of the restriction specificity of the clones revealed that the peptide could be recognized in combination with at least 8 different DR molecules including DR1, 2, 4, 5, 6, 7, 8 and 9 (Fig. 4) [lo, 121. These results suggested that it may therefore not be necessary to search for a cocktail of different, T cell epitopes to stimulate an anti-sporozoite response in most vaccinated individuals, since CS.T3 alone would be recognized in association with one or more DR alleles present in 80-90% of typical African or European populations.
STRUCTURAL ANALYSIS OF THE PERMISSIVE ASSOCIATION BETWEEN CS.T3 AND DR MOLECULES The CS.T3 peptide is recognized in association with at least eight different DR antigens (possibly more, as we lack information about the other DR alleles). The structural basis for this broad binding capacity to DR molecules is unclear. We initially proposed the possibility that the binding of the peptide might be dependent on the DRa chain, which shows limited allelic polymorphism. However, analyses of the fine specificity of the interaction of CS.T3 with different DR alleles make it clear that the chain also play an important role in the binding of the CS.T3 peptide. For example, using a series of peptides truncated at either the Nor C-terminus of the
63
PEPTIDE-MHC INTERACTION
92 69 46 W
I
2
- a
x
L
I
20 Int Rev Immunol Downloaded from informahealthcare.com by Mcgill University on 11/24/14 For personal use only.
-s
30 W
14
__
a
b
C
FIGURE 1 SDS-PAGE analysis of immuno-affinity purified HLA-DR1 molecules. For the immunoaffinity purification of DR antigens (lane c). detergent extracts (lane b ) obtained from about 1010 DRI-homozygous EBV-B cells were applied to a column containing Sepharose 4B-coupled specific mouse monoclonal anti-HLA-DR Ab. Lane a contains coomassie blue stained M.W. standards. The two noncovalently associated a (Mr 34000) and p (Mr 29000) chains are indicated.
200000
E a 0
'I 00000
0
0
10 Elution
20
30
volume (ml)
FIGURE 2 Gel liltration analysis of peptide-DR1 complexes. The DR molecules were isolated in solution using detergent solubilization from DR-homozygous EBV-transformed B cell lines (Fig. 1). The DR1-binding influenza virus Matrix peptide Mat 17-31 was modified by the addition of a tyrosine residue to the amino terminus to allow 1251 labelling. The peptide was labelled using Iodo-Beads, purified and used within 1 week. Binding assays were done following the basic protocol of Buus er al. with some modifications (B. T., manuscript in preparation). 'The incubation mixture contained 100-300 pmoles of purified DRI and 1-3 pmoles of ~2SI-radiolabelledpeptide. After incubation at room temperature for 48 hours, the DRI-peptide complexes were separated from free peptide by gel filtration on a Sephadex G50 column and two ml fractions were collected and assayed for radioactivity in a gamma Mat 17-31; (A-A) DR1/12~I-labelled Mat 17-31+ cold Mat 17-31; (H-H) 125Ispectrometer. ( X - X ) DR1/~251-lahelled labelled Mat 17-31
64
I! ROMAGNOLI et af. TABLE I The Binding of CS Peptides to DRl Peptide
Sequence
DR1 Binding
CS(23-43) CS( 103-122) CS(297-309) CS(3 10-3 18) CS(325-341) CS(378-398)
YQCYGSSSNTRVJJJELNYDNA EKLRKPKHIUUKQPGJXNPD GHNMPNDPNRNVD ENANANNAV EPSDKHEEQYL,KKIKNS DIEKKIAKMEKASSVFNVVNS NANF'NWNANF'
-
+
-
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CS peptides were tested for the ability to inhibit the binding of **Wabeledinfluenza matrix peptide Mat 17-31 to detergent-dubilid DRl molecules.
HH.12 DRwll(5) Percent of response to 1 $4 antgenic peptide competitor (IOOrM)
20
0
40
60
100
80
120
CS 23-43 CS 103-122 CS 297-309 CS310-318 CS 325-341
cs 378-398 NANP3
I
1 AC.129 DRw14(w6)
competitor (100rM)
i
Percent of response to 1 )IM antigenicpeptide
,
2p
,
4p
,
6p
,
8p
,
1 7
,
170
CS 23-43 CS 103-122
CS 297-309
CS 310-318 CS 325-341
cs 378-398 NANP3
FIGURE 3 In vitro competition by CS peptides for interaction with DRwll(5) and DRw14(w6). Inhibition of antigen presentation was determined as previously described [91.
65
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PEPTIDE-MHC INTERACTION
DR1 DR2 DR4 DR5 DRw6 DR7 DR8 DR9 FIGURE 4 Definition of CST3 determinants recognized by T cell clones restricted by different DR alleles. The CS.T3 peptide, whose sequence is shown, and peptides which were truncated either from the amino or the carboxy terminus of CST3 were tested for their ability to stimulate T cell clones restricted by the DR alleles shown. The shortest sequences identified by this truncation analysis which could stimulate different DR-restricted T cell clones are shown.
CS.T3 sequence we have determined the binding profile for the two DR molecules, DRwll(5) and DRw14(w6) (Table 11). The critical residues required for interaction with DRwll(5) are contained within residues 380-391, and for interaction with DRw14(w6) within residues 381392 [13]. In order to identify the contribution of each aminoacid residue to the binding of CS.T3 to the two different DR molecules, analogs of CST3 were synthesized. At least 3 substitutions were made at each position (Table ID),most of them selected in order to change the size, charge or polarity of this residue. Each peptide analogue was tested for its capacity to bind to DRwll(5) and DRw14(w6) as well as to activate CS.T3-specific T cell clones. Although the two different DR alleles examined showed a nearly identical fine-specificity in the truncation analysis, when we analyzed the relative binding capacity of the 51 analogues used in this study, it was clear that distinct sites in the CS.T3 sequence are interacting in different ways with the two DR molecules analyzed (Fig. 5). For example, while substitution 389A -+ D drastically reduced binding in the DRwll(5) molecule, the same substitution resulted in a nearly twofold increase in binding to DRw 14(w6). Positions 383 and 388 appear to be the most critical for the binding to DRwll(5), whereas 392 is critical for binding to DRw14(w6). All the nonconservative changes at these positions abrogated binding to the respective DR antigen. Only when the isoleucine at position 383 was replaced by the very similar leucine was this change tolerated, albeit with an eightfold decrease in potency. The substitution of an aspartic acid for methionine 386 and a phenylalanine for glutamic acid 387 also destroyed the capacity to compete in the DRwll(5) functional assay. Again this effect could not be observed for DRw14(w6) binding, where, in addition to the substitutions 389 A += E, the replacement 391 S --+ F was no longer tolerated. The peptides containing point mutations were also assayed for their ability to activate TLC specific for the CST3 peptide (Table IV and J.K. submitted). As previously seen for other
P. ROMAGNOLI et al.
66
TABLE 11 Effect of CS.T3 Truncations on Binding to DRwll(5) and DRwIqw6) Binding to
CS Peptide 378-398
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379-398 380-398 381-398 382-398 383-398 378-396 378-395 378-394 378-393 378-392 378-391 378-390
Amino Acid Sequence
D I E K K I E K K E K K K K K D D D D D D D
I I I I I I I
E E E E E E E
K K K K K K K
K K K K K K K
I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E I A K M E
K K K K K K K K K K K K K
A A A A A A A A A A A A A
S S S S S S S S S S S S S
S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V N S S V F N V V S V F N V S V F N S V F S V S
DRwll(5)
DRw14w6 15
It
0.7 2 >200 >200 >200
7 2 32 28 35 160
1200
12
15 67 >200 >200 5
8 15
17 100 >200 >200
tConcentration (pM) of truncated peptide required for 50% inhibition of antigen presentation.
peptides recognized by either class I or class 11-restricted T cells, the majority of substitutions affected T cell recognition significantly more than the ability of the peptide to bind the MHC protein [ 5 ] .Of the 51 analogues examined, more than 30%prevented recognitionof the peptides by TLC restricted by one of the three DR molecules. In contrast only 1045% of the substitutions abrogated binding to the DRs examined. The structuralconformationof the peptide bound to class 11moleculesis still the subject of an ongoing debate. Several authors have provided evidence for or against an a-helical structure of peptide bound to the MHC molecules (14-20). NMR analysis of the CS.T3 peptide in water shows that this peptide can indeed adopt an a-helical structure in solution (Labhardt A., unpublished results). However, when we compared the relative binding capacity of the peptide analogs with their ability to stimulateT cell clones specificfor the parental peptide, we observed stretches of four consecutive residues (384-387) where the peptide analogs could not activate the appropriateT cell clone (Table IV).Sincethe vast majority of the peptide analogs substituted within this stretch (31 out of 33) are still capable of binding the two DR molecules, the most likely explanationis that these residues make contact with the antigen receptor of the T cells. In a regular a-helix there are 3.6 residues per turn, so the fact that 4 consecutive residues are involved in contactingthe T cell receptor providesevidence against an a-helical conformationof the CS.T3 peptide in its bound state for the DR alleles examined. Thus, at least in its central region, the peptide seems to have an extended conformation.Clearly experimentalproof of this will require the crystallographic determination of the structure of the class 11-peptide complex. Of the 51 analogs tested, the peptide substituted at position 387 E + G showed enhanced binding to the DR alleles tested (Fig. 5). Because the CS.T3peptide, as discussed below, is able to induce helper T cells which could be boosted by immunization with the whole malaria parasite, these results may have implications for a malaria vaccine. It might be possible, as illustrated here, to i d e n w analogs that bind more strongly to MHC antigens than does the natural sequence. Such a result would be interesting, as it would suggest that the naturallyoccurring sequences might be improved as vaccine candidates by judicious selection of crossreactive, more immunogenic variant peptides.
PEPTIDE-MHC INTERACTION
67
TABLE IU Sequences of Peptide Analogs Tested ~
Analogs
Parental
(abbreviations)
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CS(380-396) 380E-+K 380E-F 380E-+G 381K-+G 38 1K-+D 381K-+V 382K-+D 382K-+G 382K-+Y 383I-+G 383IP.D 383IP.K 383I-+L 384A-+V 384A-+I 384A-+Y 384A-+K 385K-+D 385K-+F 385K-+G 386M-+G 386M-+K 386M-+D 387E-+F 387E-+G 387E-+K 388K-+G 388K-+D 388K-+F 389A-+E 389A-+D 389A-+Y 389A-+V 390S-+F 390S-+K 390S-+E 391S-F 391 S-+K 39 1S-+E 392V-+G 392V-+K 392V-+D 393F+G 393F+D 393F+K 394N-+K 394N-+E 394N-+F 395V-+G 395V-+K 395V-+D
380
385
390
395
€? ROMAGNOLI et al.
68 380
P a r e n t CS s e q u e n c e aa. S U b S t l t U t l o n
E
385
K
K
I
A
K
390
M
E
K
A
S
395
S
V
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0.01
380
aa' SUbSt'tUt'on
N
V
V
KFG GDV DGY GDKL VIYK DFG GKD FGK GDF EDYV FKE FKE GKD GDK KEF
T Parental CS sequence
F
I
A
K
395
390
385
K
M
E
K
A
S
1
S
V
F
N
V
V
KFG GDV DGY GDKL VIYK DFG GKD FGK GDF EDYV FKE FKE GKD GDK KEF
0.0 1
FIGURE 5 Binding of the 51 CS(380-396) analogs to DRwll(5) and DRw14(w6) molecules. The series of analogs shown in Table III was tested for binding to the two DR alleles by the functionalcompetition assay previously described [9].Each bar represents the arithmetic mean of three independent experiments.
THE PEPTIDE CAN ACT AS A HELPER DETERMINANT To examine whetherthe CS.T3 peptide could induce helper T cell function in vivo,we coupled it to the B cell determinant(NANP),, the repetitive NANP sequence in the central domain of the CS protein which is the major epitoperecognized by anti-sporozoiteantibodies [21]. Mice from severalstrains which were normallyunable to respond to the NANP repeatsproduced antibodies to the repeats, as well as to sporozoites, when injected with the ("P),-CS.T3 conjugate [lo]. This shows that theCS.T3 sequenceis also recogiuzed in associationwith many differentmouse Ia molecules, and that it can indeed function, at least in mice, as a carrier determinant for responses to other parts of the CS protein. Since the synthetic T cell determinant CS.T3 was capable of priming T cell help for antibody production to the B cell determinant (NANP),, and since antibody reacted with the native CS protein, this system provides evidencethat syntheticT and B cell recognition sites can be combined to yield a functional malaria immunogen. Such a
PE€TIDE-MHC INTERACTION
69
TABLE IV Evidence Against an a-Helical Conformation DRwl l(5) 380
385
390
395
MHC
TCR JK18
-
-
-
-
-
-
DRw 14(w6) MHC
TCR BR14
-
-
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parental 380E-K 380E-+F 380E+G 383I+G 3831-D 3831-K 3831-L 384A+V 384A-I 384A-Y 384A+K 38SK-D 385K-F 385K-G 386M-G 386M-K 386M+D 387E-F 387E+G 387E-K 388K-+G 388K-D 388K-F 392V-43 392V-+K 392V+D 393-G 393FiD 393hK 394N-K 394N-E 394N-F 395V-G 395V-K 395V+D
+ + ? + + + + + +
+
+ +
+
+ + + + + +
+ + + + +
+ ++ ++ + ?
+ + + +
+ + + + + + + + +
Peptide analogs were compared w~tbthe parental sequence (38&3%) for Tcell receptor recognition by clones JK18 and BR14 and binding to DRwll(5) and DRw14(w6). + + better binder (relative binding capacity >lo); + equal binder (relative binding capacity 10-0.1); ? poorer binder (relative binding capacity 0.01-0.1);- nonbinder (relative binding capacity
