benign and malignant proliferative lesions of cutaneous epithelium (zur Hausen ... Furthermore, as the different papilloma- virus types demonstrate a high ...
Journal of General Virology (1990), 71, 423-431.
423
Pr&ted & Great Britain
Human T cell responses to human papillomavirus type 16 L1 and E6 synthetic peptides: identification of T cell determinants, HLA-DR restriction and virus type specificity George Strang, l*t Julian K. Hickling, 1 G. Angus J. Mclndoe, 1 Kevin Howland, ~ David Wilkinson, 2 Hitoshi Ikeda 2 and Jonathan B. Rothbard 1 1Laboratory of Molecular Immunology and ZLaboratory of Human lmmunogenetics, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, U.K.
Four T cell determinants in the major capsid protein of human papillomavirus (HPV) type 16 L1 and one in the E6 protein associated with cellular transformation were defined using synthetic peptides to stimulate peripheral blood mononuclear cells from asymptomatic individuals. H L A - D R restriction was defined using murine L cells transfected with H L A - D R genes to present antigen. Responses to two of the five determinants by T cell lines and clones were shown to be specific for HPV-16 based on the lack of cross-
recognition of the corresponding sequences of other known papillomavirus sequences (types 1a, 5, 6b, 8, 11, 18 and 33). The T cells raised against two of the other peptides cross-reacted with corresponding peptides from other strains to varying extents, depending on their structural homology. The implications of these results regarding the prevalence of HPV-16 infection in the population and the possible diagnostic role of these responses in papillomavirus infection is discussed.
Introduction
cinomas of the cervix, evidence from transfection experiments shows HPV infection to be necessary but not in itself sufficient for transformation (Storey et al., 1988). In addition, HPV DNA has been identified in a significant percentage of asymptomatic individuals (Pfister, 1987), which is a characteristic of a latent viral infection. Experimental and clinical evidence suggests that cellular immune responses are important in the control of other latent viral infections such as those with herpesviruses and hepatitis B virus (Wildy & Gell, 1985; Rickinson et al., 1985; Sissons et al., 1986; Gelb, 1985; Zuckerman & Howard, 1979). The observation that immunosuppressed individuals have an increased incidence of HPV-induced lesions, including condyloma and CIN (Kirchner, 1986; Porreco et al., 1975) suggests that this may also be the case for HPV. However, knowledge of the human immune response to papillomaviruses is rudimentary. Circulating antibodies that recognise HPV-16 fusion proteins have been detected in sera from women with HPV-16 DNA positive biopsies (Jenison et al., 1988). Recently, lymphoproliferative responses to purified HPV virions have been demonstrated (Cubie & Norval, 1988), but no attempt was made to analyse the antigen specificity of the T cell response. An understanding of the immune response to HPV-16 is required if
Papillomaviruses are small D N A viruses associated with benign and malignant proliferative lesions of cutaneous epithelium (zur Hausen & Schneider, 1987; Pfister, 1987). Over 50 different types of papillomavirus have been isolated from human tissue and can be distinguished by differences in DNA sequence. They can be segregated into logical groups by sequence homology and the specific lesion with which they are associated. D N A of human papillomavirus (HPV) types 6, 11, 16, 18 and 33 have been found in genital lesions, whereas types 1, 2 and 4 are predominantly found in flat warts on the hands and feet (Pfister, 1987; Jablonsky et al., 1985). The strains found in anogenital lesions can be further distinguished by their association with invasive carcinoma. HPV types 6 and 11 are most commonly found in cervical condyloma and low grade cervical intraepithelial neoplasia (CIN). In contrast, HPV-16, -18 and -33 DNA is associated with more severe CIN and invasive carcinoma of the cervix (Pfister, 1987). The development of CIN and progression to invasive carcinoma is a multistep process. Although HPV D N A has been identified in the majority of invasive cart Present address: New England Biolabs, 32 Tozer Road, Beverly, Massachusetts, U.S.A. 0000-9148 © 1990 SGM
424
G. S t r a n g a n d others
effective strategies are to be developed to p r e v e n t H P V 16 infection, or to m o d u l a t e the course of existing infections. R e c e n t studies have identified H P V - 1 6 D N A in a p p r o x i m a t e l y 7 0 ~ of w o m e n with n o r m a l cervical cytology (Young et al., 1989). If H P V - 1 6 infection is as widespread in the p o p u l a t i o n as these results suggest, a n d the cellular i m m u n e response is i m p o r t a n t in the control of infection, t h e n HPV-specific T cell responses m i g h t be a p p a r e n t in the peripheral blood of a s y m p t o m a t i c individuals. F u r t h e r m o r e , as the different p a p i l l o m a virus types d e m o n s t r a t e a high degree of sequence homology, it is possible t h a t T cells specific for a p a r t i c u l a r strain m a y well be able to recognize the c o r r e s p o n d i n g sequences from\ other strains. Therefore the a i m of this study was to e x a m m e w h e t h e r H P V specific T lymphocytes could be detected in asymptom a t i c individuals, a n d if so to identify the epitopes recognized by these cells, a n d to d e t e r m i n e w h e t h e r T cells cross-reactive b e t w e e n different H P V types were present. T h e absence of a permissive culture system for H P V a n d the c o r r e s p o n d i n g difficulties in o b t a i n i n g infected cells or i n d i v i d u a l viral proteins p r e v e n t s the use of s t a n d a r d t e c h n i q u e s for p r o b i n g i m m u n e responses. T h e ability to stimulate both helper a n d cytotoxic T cells with peptides c o r r e s p o n d i n g to f r a g m e n t s of viral proteins, as d e m o n s t r a t e d for influenza virus ( T o w n s e n d et al., 1986), has provided a n alternative strategy for i n v e s t i g a t i n g the cellular i m m u n e response. I n this study, peptides c o r r e s p o n d i n g either to sequences of the m a j o r capsid p r o t e i n L1 or the t r a n s f o r m a t i o n - a s s o c i a t e d p r o t e i n E6 of H P V - 1 6 (Seedoff et al., 1985) were chosen, based on structural similarities with previously defined T cell d e t e r m i n a n t s ( R o t h b a r d & Taylor, 1988), a n d used as a n t i g e n s i n lymphoproliferative assays. P e r i p h e r a l blood m o n o n u clear cells ( P B M C ) from a p a n e l of healthy donors with n o a p p a r e n t H P V - a s s o c i a t e d disease were tested for their ability to respond to these peptides. P B M C were found to proliferate in response to i n d i v i d u a l peptides, suggesting that H P V - 1 6 infection m a y be w i d e s p r e a d in the population. S u b s e q u e n t l y i n t e r l e u k i n 2 (IL-2)-dependent, peptide-specific, T l y m p h o c y t e lines a n d clones were established a n d used to define the H L A restriction of the responses a n d to e x a m i n e a n y possible crossreactive recognition of c o r r e s p o n d i n g sequences from other H P V types.
Methods Blood donors. Venous blood samples (60 ml) were taken from healthy donors with no obvious papillomavirus-associated disease. The blood was dispensed into 40 ml Iscove's modified Dulbecco's medium
(Gibco) containing either preservative-free heparin (600 units) or 0.66~ trisodium citrate anticoagulants. PBMC were separated by isopycnic centrifugation on lymphocyte separation medium (Flow Laboratories). Purified PBMC were then used as required, or were cryopreserved in liquid nitrogen in 90~ foetal calf serum and 10~ dimethyl sulphoxide. T lymphocyte culture. T lymphocyte lines were established by incubating either freshly purified or cryopreserved PBMC in Iscove's modified Dulbecco's medium supplemented as below, at a cell concentration of 2 x 106 ml in 24-well Falcon plates (Becton-Dickinson) with synthetic peptide antigen at a concentration of 10 to 50 gM and 10~ autologous serum. Fresh antigen and irradiated autologous PBMC (5 x 105 per well) were added 7 days and 14 days later. Recombinant IL-2 (20 units/ml) (Boehringer)was added, beginning on day 14 and thereafter fresh medium containing 20 units/ml IL-2 and 10~ autologous serum was added every 3 to 4 days, with antigen and irradiated feeder cells being supplied weekly. Peptide-specific T lymphocyte clones were isolated from these cell lines by the method of Lamb et al. (1982). Briefly, T lymphocytes were seeded at a density of 0.3 cells per well in Terasaki plates with irradiated PBMC as feeder cells (2000 fads) (1 x 104 per well) in Iscove's medium containing 10~ human serum, 20 units/ml IL-2 and 10 gm peptide. When outgrowth of clones was observed the T cell clones were expanded in 0.2 ml wellsand then 2 ml wells. L cells transfected with human HLA-DR genes. Before use in an assay as antigen-presenting cells the transfected fibroblasts (Young et al., 1987; Ikeda et al., 1988; Lock et al., 1988) were treated with 25 gg/ml mitomycin C (Sigma) for 30 min in serum-free Iscove's modified Dulbecco's medium, then washed three times in 10~ calf serum. Lymphoprotiferation assays. T lymphocyte lines or clones (1 x 104 to 2 x 104 per well) were assayed against serial dilutions of peptide in Iscove's modified Dulbecco's medium, supplemented with 2 mM-Lglutamine, 100IU/ml penicillin, 100gg/ml streptomycin and 10~ human serum in 96-well flat-bottom plates at 37 °C, in a humidified atmosphere with 5~ CO2. Control wells, which had only medium and cells with no peptide, were also prepared. Irradiated autologous PBMC, Epstein-Barr virus-transformed B cells or mitomycin Ctreated human HLA-DR-transfected mouse fibroblasts (5 x 104 to 10 x 104 per well) were used as antigen-presenting cells. The plates were pulsed with [Me-3H]thymidine (1 gCi/well; [3H]TdR; Amersham) after 2 days and harvested onto glass fibre filters on the third day. Proliferation as correlated with [3H]TdR incorporation was measured by liquid scintillation spectroscopy using the LKB Betaplate system (Pharmacia). Synthetic peptides. Peptides were synthesized by using solid-phase techniques (Barany & Merrifield, 1979) on an Applied Biosystems Peptide Synthesizer with commercially available Pare resins, t-Bocprotected amino acids and commercially available reagents (Applied Biosystems). The peptides were cleaved from the resin and the side chain-protecting groups were simultaneously removed with anhydrous hydrofluoric acid, with anisole as a free radical trap. The side chainprotecting groups were extracted with ether. Subsequently, the peptides were dissolved in 15% acetic acid, filtered from the resin and lyophilized. The crude peptides were analysed by HPLC on a C-8 reverse-phase column (Aquapore RP-300, Brownlee Laboratories) and by amino acid analysis. Peptides that were not of greater than 90~ purity, as judged by analytical HPLC analysis, were purified by using a preparative RP-300 column and a water-trifluoroacetic acid-acetonitrile gradient. Principal peaks were collected, lyophilized and analysed by amino acid analysis. The particular regions of the protein were selectedon the basis of the presence of a pattern of either four or five amino acids (charged or
Human T cell responses to HPV-16 L1 and E6 peptides glycine followed by two or three hydrophobic residues and then a polar amino acid) previously shown to be present in many helper and cytotoxic T cell determinants (Rothbard & Taylor, 1988).
425
stimulating antigen in order to avoid selecting particular subpopulations of lymphocytes. The pattern o f recognition seen with the lines confirmed the general pattern of recognition observed with assays using P B M C s (Fig. 1). Both lines responded to peptide 279 to 294, consistent with either D R 7 , D Q w 2 or D R w 5 3 restricted recognition. The line from H D 2 proliferated in response to 91 to 106, whereas the line from H D 1 recognized 219 to 244 and 40 to 63.
Results Proliferative responses of P B M C to HPV-16 L1 peptides Eight peptides corresponding to sequences o f the H P V 16 L1 open reading frame and four corresponding to sequences of the E6 open reading frame (Seedorf et al., 1985) were synthesized, based on the presence o f patterns of four or five amino acids previously shown to be present in a high percentage of helper and cytotoxic T cell determinants ( R o t h b a r d & Taylor, 1988) (Table 1). Type 16 was selected because of its association with invasive cervical carcinoma. L1 was chosen because it is believed to encode the major structural protein of the H P V virion and E6 was selected as it is associated with the transformation o f cells. E a c h peptide was assayed for its ability to stimulate P B M C s both from H L A - t y p e d healthy donors with no s y m p t o m s of H P V infection and from donors with various stages o f C I N . Four of the eight L1 peptides (40 to 63, 91 to 106, 219 to 244 and 279 to 294) were found to elicit T cell proliferation from several healthy donors. However, the pattern o f responses to the peptides varied between donors; no one peptide was recognized by all donors and no single d o n o r responded to all four peptides (results not shown). In order to allow a more detailed examination of the specificity and possible H L A restriction of the peptide recognition, IL-2-dependent T cell lines were established from healthy donors ( H D ) H D 1 and H D 2 using a cocktail o f either the eight L1 peptides or the E6 peptide as an antigen. Cocktails of all the peptides were used as a
Presentation of peptide by L cells transfected with HLADR genes Mouse fibroblast cell lines transfected with H L A - D R genes were used to d e f n e further the H L A restriction of the proliferative responses to the peptides. L cells transfected with either H L A - D R 1 D w l , D R 4 D w 4 , or D R 7 D w 7 were used as antigen-presenting cells for peptide-specific cell lines (Fig. 2). Only D R 4 D w 4 - t r a n s f e c t e d L cells were able to present 40 to 63 to a line from H D 1 specific for this peptide. Similar assays using T cell clones from H D 1 , which recognized 219 to 244, demonstrated that recognition o f this peptide was also restricted through D R 4 D w 4 . A line from H D 2 previously shown to be specific for 279 to 294 and 91 to 106, proliferated to 279 to 294 only in the presence o f H L A - D R 7 - t r a n s f e c t e d L cells. N o n e of the transfectants was able to present 91 to 106. Proliferation to this peptide was only observed when either P B M C or B lymphoblastoid cell lines from D R 3 donors were used as antigen-presenting cells (data not shown). L cells transfected with D R 3 were not available. T cell lines from HD2, which were specific for E6 42 to 57, only proliferated when that peptide was presented by D R 7 D w 7 transfected L cells.
Table 1. Amino acid sequences of L1 peptides Peptide
Sequence
L1 29 to 44
L
W L
P
Lla0to63
P
P
V
P
L191to106
V
S
G
L
L1209to224
G
D
C
P
L1219to244 L1279to294
T E
V Q
LI 295 to 310
E
L1382to397
K
S [E
A
T
V
Y I*L
V
S [K
V
V
Q
Y [R
V
F
P
L [E
L
I Q M F
D V
G [D R [H
N
V
P
D [D
N
T
N
F
P
P
v
P
v
S ]T
D IE
R ]1
H
L
Y
V
A
P
D
P
]
N ]Y
V
I
Q
M L
V F
H IT N IR
G A
L
Y
1
K ]G
K [E
Y
L
R ]H
R IT
N
I
D
G
F IG G T
A V
M ID [F G
T
T]L
S
G
S
T
A
G
E
E
Y
D
* Boxed residues represent the four or five amino acid sequence motif used to predict T cell determinants.
Y
Y
H
A
Q
A
N
K
S
426
G. Strang and others
t0 000
HD1
8000 6000 "-7. 4000 E
.g
2000 o
.~ 20 000 HD2 -= 15 000 10 000 5000 0
40 to 63 29 to 44
209 to 224 279 to 294 382 to 397 91 to 106 219 to 224 295 to 310 Control Stimulating peptide
Fig. 1. Proliferative response of T cell lines from HD1 and HD2 against the panel of eight HPV-16 L1 peptides. Bars represent proliferation obtained to serial threefold dilutions of peptide starting at 30 ~tM,with the lowest peptide concentration on the left of each group. Proliferation obtained in the absence of peptide is also shown (control). Each bar represents the mean of three wells. The HLA types of donors used in this experiment and elsewhere are as follows. HD1 : A24, B39 and -57, C6 and -7, DR4 and -7, DRw53, DQw2 and -w3; H D 2 : A 2 and -3, B18, C5, DR3(wl7) and -7, DRw52 and -w53, DQw2.
(a) DR7Dw7
LI
~
1
(b)
40 to 63 I
I
I
I
I
L1 I
219 to 244
I
I
I
I
I
DR4DW4 DR1Dwl L Cell
~
I
0
2000
(c)
L1
I
4000
~ I 6000
279 to 294
10 000 (d)
E6
I
I
20 000
30 000
42 to 57
DR4DW4 ~ DRIDwl ~ L Cell I
0
I
2O00
t
I
4000
I
I
6000
l
0
I
2000
I
I
4000
I
6000
['HlThymidine uptake (c.p.m.) Fig. 2. Assignment of HLA-DR restriction of peptide recognition using transfected murine L cells. Proliferative responses of peptidespecific T cell lines obtained using a panel of mitomycin C-treated L cells transfected with HLA-DR genes as antigen-presenting cells. Results from representative experiments are shown. Peptide concentrations used were : L1 40 to 63, 50 gM; L1 91 to 106, 30 ~tM; L1 219 to 244, 10 ~tM,L 1 279 to 294, 30 O.M;E6 42 to 57, 100 ~M. T cell clones from HD 1 were used in the assays against peptides L 1 40 to 63 and L1 219 to 244. T cell lines from HD2 were used in the other assays against peptides L1 279 to 294 and E6 42 to 57.
Human T cell responses to HPV-16 L1 and E6 peptides
427
Refinement of T cell epitopes
peptides being only weakly recognized
B e f o r e d e t e r m i n a t i o n o f t h e s t r a i n s p e c i f i c i t y o f t h e T cell responses, truncated peptides were examined for their stimulatory capacity. Initially experiments were performed using PBMC, but the results obtained were not easily interpretable, possibly due to the presence of m u l t i p l e T cell p o p u l a t i o n s e x h i b i t i n g v a r i a t i o n s i n f i n e specificity for different regions on the peptide. Therefore experiments were carried out using peptide-specific T cell l i n e s o r c l o n e s . T h e r e s u l t s a r e s h o w n i n T a b l e 2. T h e p e p t i d e c o r r e s p o n d i n g to r e s i d u e s 4 0 t o 63
receptor. L i n e s g e n e r a t e d f r o m H D 2 i n r e s p o n s e t o 91 t o 106 recognized peptides truncated at either the amino or carboxyl termini. Interestingly, the proline aspartic acid-proline at the carboxyl termini appeared to be important in the response of both the lines and clones. T h e p a t t e r n o f r e c o g n i t i o n o f p e p t i d e 219 t o 2 4 4 w a s complex. The only peptide shorter than the full-length s e q u e n c e c a p a b l e o f s t i m u l a t i n g T cell l i n e s f r o m H D 1 w a s 231 t o 244. P h e n y l a l a n i n e a t p o s i t i o n 231 a p p e a r e d t o b e i m p o r t a n t b e c a u s e 232 t o 244 w a s s i g n i f i c a n t l y less p o t e n t a n d 233 t o 2 4 4 h a d n o r e a c t i v i t y . C o n s e q u e n t l y , s e q u e n c e s h o m o l o g o u s t o 231 t o 2 4 4 w e r e a s s a y e d f o r c r o s s - r e a c t i o n . T h e p h e n o m e n o n o f loss o f r e c o g n i t i o n b y deleting residues of a long peptide, followed by recovery o f t h e a c t i v i t y , a n d t h e n l o s i n g it a g a i n h a s a l s o b e e n d e s c r i b e d f o r t h e r e c o g n i t i o n o f a T cell d e t e r m i n a n t
s t i m u l a t e d T cell l i n e s f r o m H D 1 b e t t e r t h a n a n y o f t h e s h o r t e r a n a l o g u e s . H o w e v e r , 48 t o 63, 50 t o 63 a n d 52 t o 63 w e r e all r e c o g n i z e d to a l e s s e r d e g r e e , p o s s i b l y b e c a u s e t h e r e w e r e t w o p r e d i c t e d s i t e s f o r T cell r e c o g n i t i o n i n t h e f u l l - l e n g t h p e p t i d e , o n e o f w h i c h is a b s e n t i n p e p t i d e 48 t o 63. P e p t i d e s 54 to 63, 50 t o 61 a n d 51 t o 61 i n d u c e d very weak proliferation, possibly due to the shortened
b y t h e T cell
T a b l e 2. Proliferative response obtained from T cell lines and clones stimulated with peptides from HP V-16 L1 L1 40 48 5O 52 54 5O 51
40 to 63 Series
L1 91 91 91 91 92 93
91 to 106 Series
P
L1 219 to 244 Series 219 T V I 224 226 228 229 230 231 232 233 L1 279 to 294 Series 279 282 284 279 280 281 279 280 281 282
P
V P V S K V V S T D E Y V S T D E Y T D E Y E Y
V A R T V A R T V A R T V A R T VA RT DE YV ART E YVART
V S
G L Q Y
N I N I N I N 1 NI N1 NI
H A H A H A H A HA
63 63 63 63 63 61 61
D P
106 105 104 103 103 103
-F+++ ++-4++ ++ ++ +
244 244 244 244 244 244 244 244 244
+++
294 294 294 293 293 293 292 292 292 292
+++t +-f+++ +++ ++ + + + -
R V F
R I
H L P
Q Q Q Q Q
Y Y Y Y Y
R R R R R
V V V V V
F F F F F
R R R R R
I I I I I
H H H H H
L P D L P L L L
A A A A A A A A A
M M M M M M M M M
D D D D D D D D D
F F F F F F F F F
T T T T T T T T T
T T T T T T T T T
L L L L L L L L L
Q Q Q Q Q Q Q Q Q
A A A A A A A A A
N N N N N N N N N
E Q M F V R F V R R E Q M F V R Q M F V R M F V R E Q M F V R Q M F V R M F V R F V R
H H H H H H H H H H
L L L L L L L L L L
F F F F F F F F F F
N N N N N N N N N N
R R R R R R R R R R
A A A A A A A A A A
G G G G G G G G G G
T T T T T T T T T T
V G V G V G V V V
V S G L V S G L V S G L S G L G L Q D G D M V H T G D M V H T M V H T H T T
G G G G G G
F F F F F F F
G G G G G G G G
Lines* +++ ++ ++ ++ + + +
Y Y Y Y Y Y Y Y YY Y Y YY
K K K K K K K K K
S S S S S S S S S
+ -
* Proliferative responses were scored as the minimal peptide concentration required to induce maximal proliferation: + + + + , 10-6 to 10-5 M; -F -+-A-, 10-5 to 10-4 M; +- + , 10-4 to 10-3 M; +, 10-3 to 10-2 M; , no proliferation observed at any concentration tested. t Assays performed using T cell clone from HD2.
428
G. Strang and others
from pp89 protein of murine cytomegalovirus (Reddehase et al., 1989), and could be explained by the inability of the inactive sequences to adopt a critical conformation required for binding to M H C proteins. Recognition of 279 to 294 was less ambiguous. Residue 294 was not necessary for stimulation because 279 to 293 was equally potent. A T cell clone from HD2 was fully stimulated by 280 to 293, allowing this sequence to be defined as the minimal peptide for maximal stimulation. Further deletions at either end resulted in reduced stimulation. Assays using a nested set of E6 42 to 57 peptides to stimulate a panel of T cell clones from HD2 revealed differences in the minimum length of peptide required to stimulate maximum proliferation (Fig. 3). Clones 12 and 15 responded strongly to peptides 42 to 57 and 42 to 55. However, whereas clone 15 also responded strongly to 43 to 55, clone 12 hardly responded at all. Maximum levels of proliferation were low from clones 13 and 16 when compared with those of clones 12 and 15. In the case of clone 13 peptide 46 to 57 stimulated as strong a response as peptide 42 to 57. Removal of two amino acids from the carboxyl terminus of peptide 42 to 57 completely prevented the response of clone 13, but was tolerated by
clone 16. Therefore each of the clones appeared to have different requirements in the minimum peptide recognized by their T cell receptor. To determine whether the proliferative responses observed could be due to cross-reaction of T cells elicited by a different strain of HPV, the T cells were assayed for their ability to recognize the corresponding regions in L1 of the seven further strains of papillomavirus whose sequence has been determined, namely types la, 5, 6b, 8, 11, 18 and 33 (Danos et al., 1982; Zachow et al., 1987; Schwarz et al., 1983 ; Fuchs et al., 1986; Dartmann et al., 1986; Cole and Danos, 1987; Cole & Streek, 1986). The corresponding sequences shown in Table 3 were easily identified due to the high homology among the L1 proteins, with the exception of H P V - l a L1, which contains no sequence with close homology to HPV-16 L1 91 to 106. When tested in proliferation assays the T cell lines reactive with 231 to 244 and 279 to 294 were strainspecific. No proliferation was seen when any of the homologous sequences were used other than the HPV-16 sequence. In contrast, significant cross-reaction was seen when T cells specific for 50 to 63 and 91 to 106 were assayed. Lines raised against 91 to 106 responded to the
30 000
100 000
i
i
,
,lll~
I
,
T
i
i,l,,
I
i
i
,
i,iii
I
i
i
i
i
,i
r
,11,,11
,
i
Tfl,,,
I
]
i
i
,
}
[
ill,i
i
,
r,,,i,
Clone 13
Clone 12 80 000 20 000 60 000 40 000 10 000 E eq
20 000 •
0 10 7 ~3
100 000
~fT
i
i
~T,~lr
I
T
THI
10 5
~
I
I,IIll
I
1
,
,
10-~
,
,
illl,,
I
10
i
0 10 -~
30 000
1,1,1,,
Clone
e-
~Z
T
1°0 ~
J
~ i~11][
I111~
10 ~
,
p
i
?]llil[
10 -~
. . . . . . .
10 -4
10 -3
, ,i,q
i,
Clone 16
80 000 20 000 60 000 40 000 10 000 20 000 0 10 7
0
10 -6
10 5
10-4
10 3
i
i
10-'
i
iiiill
i
10 ~
1[[1111]
i
10 -~
J
iiiiii1
i
10 ~
i
i t IHI
10
Proliferative response (M) Fig. 3. Proliferative response of four T cell clones from HD2 (clones 12, 13, 15 and 16), which were expanded against peptide E6 42 to 57 and then assayed against a nested set of shorter peptides from the same region. The symbols used for the peptides are as follows; 42 to 57 (O), 44 to 57 (F1), 46 to 57 (A), 42 to 55 (It), 43 to 55 (O), 44 to 55 (A), 48 to 57 ( + ) , and no peptide ( × ) .
429
Human T cell responses to HPV-16 L1 and E6 peptides
Table 3. Proliferative response obtained from T cell lines stimulated with peptide analogues Jrom other H P V types HPV type
Position
16
50 to 63
la
20 to 33
5/8
25 to 38
Sequence T -
D
-
I
-
23 to 36 23 to 36
-
18
85 to 98
-
33
24 to 37
-
-
91 t o 1 0 6 6 6 to 81
6/11
62 to 77
V
S
126 to 141 6 6 t o 81
-
16
231 to 244
F
G
209 to 222
-
-
5/8
204 to 217
6b
210 to 223
-
-
11
202 to 215
-
18
266 to 279
Y
33
205 to 218
-
279 to 294
E
Q
257 to 272
-
-
5/8
252 to 267
6b
249 to 264
11
250 to 265
18
314 to 329
33
253 to 268
* Proliferative
-
H
A
+ +
F
-
-
-
+ +
Q
.
.
-
D
-
T
P
-
-
-
S
G
A
A
-
-
+ +
-
-
-
S
F
-
-
-
-
S
-
Y
-
+ +
F
L
P
Y
R
V
-
H
-
-
Y
.
M
.
. D
.
.
.
T
T
A
A
N
N
K
-
-
N
A
D
A
D
-
S
-
-
K
.
H
M C
-
H K
V
V
V
Q
-
V
R
-
D
P
.
.
.
.
-
Q
A
N
K
S
+
-
Q
D
-
-
-
-
Q
R
--
-
T
-
-
T -
D
.
T
.
.
-
-
-
C
-
.
.
L
F
N
R
A
G
T
V
G
T
F
-
T
-
G
-
S
-
-
Y
A
F
V
-
G
K
T
L
-
A -
as described from
.
.
.
.
F -
-
in the footnotes
F F F
to Table
.
.
.
. .
-
.
-
-
-
-
-
.
. -
+ + +t -
E
. W
+ + + + + +
V
-
+++ -
F
A
R
L
I L
Y
A
using a T cell clone
-
R K
-
N
F
-
. -
.
.
F
Q
Y
. F
L
.
N
-
N
-
scored
Y
L
-
-
responses
performed
Y
-
T
-
T
K
C
16
R
-
-
la
I
T -
.
la
A
A
-
18
t Assays
A
-
33
V
-
11
16
Y -
6b
5/8
E
-
Response*
M -
L
-
2.
HD2.
corresponding peptides from two other oncogenic strains, HPV-18 and -33, as strongly as to the original HPV-16 peptide. They did not respond to peptides from HPV-5, -6, -8 or -11. However, T cells which recognized 50 to 63 cross-reacted not only with the corresponding peptide from HPV-33 but also to peptides from H P V - l a and -6b. These results indicate that the responses seen in peripheral blood lymphocytes to HPV-16 peptides could not be attributable to proliferation of T cells crossreactive with and primed by the other HPV strains. Obviously we cannot exclude the possibility that these responses may have been stimulated in vivo by a strain that has not yet been sequenced. The amino acid differences responsible for either the presence or absence of cross-reaction are currently being examined.
Discussion A variety of phenomenological results have indicated that the human immune response and in particular the cellular immune response might be important in modulating the growth of warts, both those in the skin and in
the genital tract. Patients with cell-mediated immune deficiencies (AIDS, Hodgkin's disease, malignant lymphoma and chronic lymphocytic leukaemia) are more susceptible to warts (Morison, t975). Individuals taking immunosuppressant drugs, particularly renal allograft recipients, are prone to widespread resistant warts and also CIN (Porreco et al., 1975). Next to herpesvirus infections, HPV infections are the most frequent viral complications affecting immunosuppressed individuals (Kirchner, 1986). As provocative as these associations are, deficient immune responses have not been demonstrated in "women with cervical carcinoma, primarily because little work has been done on the cellular immune response to papillomavirus and the concern that patients might be poor responders. However, because of the strong association of HPV-16 with cervical cancer, an understanding of the immune response to the virus is essential, particularly if vaccines to prevent HPV-16 infection, or to modulate existing infection are to be successfully developed. Recently, HPV-16 D N A has been demonstrated in a high percentage of asymptomatic individuals (Young et al., 1989). If the cellular immune response has a role in controlling infection, HPV-16-specific T cell responses
430
G. Strang and others
might be detectable in such subjects. Therefore, we have screened asymptomatic individuals for T cell responses to papillomavirus peptides. In this report PBMC from healthy asymptomatic subjects were shown to respond to peptides corresponding to sequences from both the major structural protein L1, and E6 one of the proteins involved in cellular transformation of HPV-16. The peptides tested in this study were selected on the basis that they contained a motif of four or five amino acids found to be present in a high percentage of previously identified helper and cytotoxic T cell determinants (Rothbard & Taylor, 1988). The determinants were initially identified by screening the panel of peptides from the L1 and E6 open reading frames for the ability to stimulate PBMC. The fact that four out of the eight L1 peptides tested proved to be stimulatory indicates that use of the predictive algorithm and the subsequent screening of peptides for the ability to stimulate T cell proliferation provides a legitimate approach for defining T cell determinants. This strategy is particularly valuable in cases such as papillomavirus, in particular HPV-16, where the lack of a suitable culture system makes it impossible to screen for T cell responses against whole virus or virus-infected cells, as has been possible with other systems. In addition, use of synthetic peptides enabled the H L A - D R restriction of four of the responses to be mapped to a particular H L A - D R allele with the aid of L cells transfected with H L A - D R genes. The fact that T cells recognizing two of the HPV-16 sequences (51 to 53 and 91 to 106) also recognized sequences from other HPV types raises the possibility that infection with a relatively benign HPV type may induce protective immunity against other HPV types which are associated with malignant disease. Responses to the other two peptides from L1 were specific for HPV-16, in that corresponding sequences from seven other HPV types were not recognized. Although the possibility that the responding cells had not been primed in vivo by an as yet unsequenced or unidentified HPV cannot be ruled out, these results suggest that the asymptomatic individuals had previous exposure to HPV-16, suggesting that HPV-16 infection may be widespread in the population. This conclusion is consistent with recent studies using the polymerase chain reaction to detect the presence of HPV D N A in cervical smears. Of the women with no history of cytological abnormality 7 0 ~ were found to be positive for the presence of HPV-16 D N A (Young et al., 1989). At this stage no definitive serological assays are available to confirm or deny previous exposure to HPV and cervical biopsies are not available from the asymptomatic individuals used in this study. However, a study o f T cell responses in healthy women who have been demonstrated as having HPV-16 D N A is now being initiated.
An alternative explanation for these lymphoproliferative responses to the papillomavirus peptides would be that the observed responses were due to the generation of a primary response in vitro. PBMCs from seronegative individuals have recently been shown to proliferate in vitro to human immunodeficiency virus gpl20 (Siliciano et al., 1988). In addition, PBMCs from non-immune individuals and also from cord blood have been reported to proliferate in response to sexual stage antigens and peptides from the major circumsporozoite antigen of Plasmodium falciparum (Good et al., 1987; Guttinger et al., 1988), although whether this was due to crossreactivity at the T cell level was not investigated. Experiments are currently being performed to determine whether the proliferative responses seen to the HPV peptides were indeed due to restimulation of memory T cells, or due to in vitro priming of T cells. In summary, PBMC from healthy asymptomatic individuals have been shown to proliferate in response to synthetic peptides corresponding to sequences from HPV-16 L1 and E6. This has enabled T cell determinants from these proteins to be identified and furthermore suggests that HPV-16 infection may be more widespread in the population than previously imagined. Experiments are being undertaken using as antigens cloned HPV proteins, HPV-infected cell lines and series of peptides covering the complete open reading frame of the smaller proteins. Although the use of synthetic peptides has enabled the determination of several T cell epitopes and the long-term culture of T cell lines, ultimately the identification of biologically relevant T cell epitopes requires the use of HPV proteins in conjunction with synthetic peptides. The definition of the T cell determinants and their HLA restriction will enable us to go on to compare the proliferative T cell responses between normal individuals, women with CIN and those with invasive carcinoma of the cervix to determine whether any correlations between disease state and level of specific cellular immunity can be detected. In addition, monitoring the cellular immune response to papillomavirus peptides might also prove useful as a diagnostic tool in studying cervical neoplasia. We would like to thank Mr Steven Marsh (Tissue Antigen Laboratory, ICRF) for HLA typing and Mrs Wendy Senior for preparation of the manuscript.
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(Received 28 June 1989; Accepted 12 October 1989)
