Supplementary Material: Strategy for detection of CD8 responses and analysis of the degree
of cross-genotype reactivity
Strategy for detection of CD8 responses
Overlapping peptides were combined in 9 pools for each genotype (9-10 peptides/pool).
PBMC (3 x 106) were resuspended in 1 ml of complete medium (RPMI 1640 containing 10%
fetal calf serum (FCS), 1% streptomycin/ penicillin, and 1.5% Hepes buffer 1mol/l) on a
24well plate and stimulated with peptide pools (each peptide 10 μg/ml) and anti-CD28 (0.5
μg/ml; BD Biosciences). On day 3 and 7, 1 ml of complete medium and recombinant IL-2 (20
U/ml; Hoffmann-La Roche) were added. On day 10, the cells were tested for interferon
gamma secretion after stimulation with the same peptide pool by intracellular cytokine
staining. For this purpose an aliquot of 100µl of each cell suspension was incubated in the
presence or absence of the peptide pool (10 µg/ml) on a 96well plate at 37°C. After 60min 5µl
Brefeldin A (BD Biosciences) were added. After 5 hours cells were washed twice in PBS
containing 1% FCS and stained with 1µl anti-CD4-PE, 1µl anti-CD8-APC and 4µl Viaprobe
(all BD Biosciences) for 30min at 4°C. Cells were washed once in cold PBS containing 1%
FCS and incubated in Fixation/Permeabilization solution (BD Biosciences) for 20min at 4°C.
Cells were then washed three times in BD Perm/Wash buffer (BD Biosciences) before adding
0.25µl anti-IFNg-FITC. Cells were then analyzed on the FACS Calibur (BD Biosciences).
Pools were considered positive when the frequency of CD8+IFNg+ cells in the presence of
peptides was at least three times higher as the frequency of CD8+IFNg+ cells in the absence
of peptides. The individual reactive peptide in positive pools was identified the next day by
re-stimulation with single peptides followed by IFNg staining. An example for identification
of a reactive peptide (from subject C78) is provided as supplementary material.
Analysis of the degree of cross-genotype reactivity
All individual responses were confirmed in a second series of cultures utilizing cryopreserved
PBMC and fine-mapped if possible based on predicted motifs within the reactive peptide
matching the subject’s HLA-type. To determine the degree of cross-genotype reactivity
PBCM were cultivated in the presence of the GT1b and GT3a sequence of every reactive
peptide. After 10 days both cultures were re-simulated with the GT1b and the GT3a peptide
before intracellular IFNg-staining as described. Lack of cross-reactivity between genotypes
was assumed when no specific T cells were expanded in the presence of one peptide in at
least two attempts or the frequency of CD8+IFNg+ T cells upon re-stimulation in the
presence of the highest concentration (10µg/ml) of the non-reactive peptide was more than
10-fold below the frequency of the reactive peptide. Partial cross-genotype reactivity was
assumed when specific T cells were expanded in the presence of both peptides and when the
frequency of CD8+IFNg+ T cells in the presence of the highest concentration (10µg/ml) of
one peptide was less than 10-fold reduced but there was a more then 10-fold difference in the
peptide concentration needed to stimulate 50% of the maximum response (SD50). Full crossgenotype reactivity was assumed when specific T cells were expanded in the presence of both
peptides and when the frequency of CD8+IFNg+ T cells in the presence of the highest
concentration (10µg/ml) of one peptide was less than 10-fold reduced and there was a less
then 10-fold difference in the SD50.
Table S1: complete list of subjects and CD8 epitopes
Table S1: complete list of subjects and CD8 epitopes
ID age gender genotype serotype
VL a
ALT
21 41
male
1a
1
1,278,890
23
24
30
34
43
46
57
68
GT1
45
43
46
38
47
50
29
28
male
male
female
male
female
male
male
female
1b
1a
1b
1a
1a
1a
1a
1a
1
1
NT
1
4
1
1
1
73
43
male
1b
NT
74
45
male
1b
1
78
29
male
1b
1
HLA
A1,3 B7,8
CD8 epitopes detected with overlapping peptides
GT1b cons sequence strength GT3a cons sequence strength
58,557 27 A2, 29 B15, 44
206,462 337
A1,3 B8,27
1,777,000 23
A1,3 B8,35
132,783
A2 B40,51
2,923 69
A1,3 B7,8
1,674,380 187 A2,25 B7,39
1,580,680
A3,30 B7,13
1,064 448 A2,32 B15,44
1,785
18 A24,30 B13,55
178,565
RLGAVQNEV
........A
8.3
RLGPVQNEI
...A....A
23.42
VTPGERPSGMFDSSVL
................
-
VAPGERPSGMFDSVVL
.T...........S..
1.29
A1, 24 B8
83,420 105
A2,32 B35,41
additional CD8 epitopes detected with optimal peptides
prototype sequence strength GT3a cons sequence strength
ATDALMTGY
3.98
ATDALMTGF
........F
.........
CVNGVCWTV
.I.......
KLSGLGLNAV
..VA..V...
8.95
ATDALMTGY
........F
HPNIEEVAL
.S.......
KLSGLGLNAV
..........
GT3
83
84
33
49
female
female
1b
1b
1
1
85
33
male
1a
1
86
89
11
48
25
35
male
male
male
1b
1a
3a
1
NT
3
498,921 122
male
male
3a
3a
3
2
26
28
34
38
male
male
3a
3a
NT
3
<615 135
94,572
32
36
39
44
59
60
63
56
51
33
43
40
53
42
male
male
female
female
male
female
male
3a
3a
3a
3a
3a
3a
3a
3
3
3
3
3
NT
1
3,507,000 261
1,284,000 44
1,103 69
1,536,000 80
600,973 329
84,660
27,530 30
32
36
33
male
female
female
3a
3a
3a
3;1
3
3
A2,25 B7,44
A1,2 B40,51
A2 B7,15
1,150,150 118 A29,32 B40,55
65,058 22 A3,30 B15,39
<615 45 A3,26 B39,40
32
52
439,544 48
779,254 132
2,387,000
231,100
20,407
male
3a
3
A2,24 B18,37
71
76
40
40
male
female
3a
3a
3
3
77
27
female
3a
1
222,312
A2 B44
79
88
28
25
male
male
3a
3a
NT
3
340,600
92,879
A2 B8,13
A2,32 B51,55
90
9
10
43
44
37
male
male
male
3a
neg
neg
3
3;1
3
188,924
-
7
21
A2 B15, 40
A23,33 B7,18
A30,31 B8,35
12
44
female
neg
NT
-
68
A2,3 B13,15
13
41
female
neg
NT
- 236
14
42
female
neg
NT
-
19
A2,30 B13,44
29
47
male
neg
1
-
33
A2,11 B27,44
37
40
42
38
49
48
male
male
male
neg
neg
neg
NT
1
1
-
19 A33,68 B40,44
30
A2 B27,44
14
A2 B14,51
58
50
male
neg
NT
-
61
62
39
43
female
male
neg
neg
NT
NT
-
31
66
57
male
neg
3
-
33
35
male
neg
1
-
A2,3 B7,15
A2,31 B39,40
A1,3 B7,13
A3,26 B7,38
27
5.2
0.5
GAKHPALQMYTNVDQDLV
SS.H.VI...........
LQFIPVETL
.........
DLQPAETTVRLRAYL
...............
GKSTKVPAAYVAQGYNVL
..........A.......
IPFYGKAI
V.......
FLATCVNGVCWTVYHGA
..G.T.G..M.......
5.35
FLGTTVGGVMWTVYHGA
.................
-
EFWESVFTGLTHIDAHFL
D.................
2.88
DFWESVFTGLTHIDAHFL
..................
4.09
-
GKSTKVPAAYVAQGYNVL
AATLGFGSFMSRAYGI
0.45
8.42
RLGAVQNEV
VTLTHPITK
HLHAPTGSGK
GFGAYMSKAH
KLSALGLNAVAYYRGLDV
GVDPNIRTGV
SWDQMWKCLIRLKPTLH
RLGAVQNEV
31.92
11.51
12.68
12.38
1.12
51.85
RLGPVQNEI
ICLTHPVTK
YLHAPTGSGK
GFGSFMSRAY
KLRGMGLNAVAYYRGLDV
GIDPNIRTGN
SWDETWKCLVRLKPTLH
RLGPVQNEI
52.22
20.12
5.2
5.66
38.95
TLGFGAYMSK
VTLTHPITK
GRGRRGIYRF
3.73
5.43
-
TLGFGSFMSR
ICLTHPVTK
GRGRLGTYRY
14.99
HAVGIFRAAV
13.38
HVAGIFRAAV
7.56
GVDPNIRTGV
IPFYGKAI
2.05
11.9
GIDPNIRTGN
IPFYGKAI
10.83
1.27
2.88
GKAIPIALLKGGRHLIF
ICLTHPVTK
-
A2,33 B7,14
A1,2 B40,51
IPFYGKAI
11.95
IPFYGKAI
18.52
A1,3 B8,35
LIRLKPTL
39.51
LVRLKPTL
43.89
2.64
TVGGVMWTV
-
HSNIEEVAL
.........
KLRGMGLNAV
..S.L.....
3.8
2.56
-
KLSGLGLNAV
......I...
12.91
KLRGMGLNAV
..S.L.....
-
KLSGLGLNAV
..VA..V...
1.41
KLRGMGLNAV
..VAL.V...
-
CVNGVCWTV
T.G..M...
5.62
TVGGVMWTV
.........
-
ATDALMTGY
........F
0.31
ATDALMTGF
.........
-
CVNGVCWTV
T.G..M...
KLSGLGLNAV
..R.M.....
11.01
TVGGVMWTV
.........
KLRGMGLNAV
..........
-
CVNGVCWTV
T.G..M...
4.05
TVGGVMWTV
.........
CVNGVCWTV
T.G..M...
CVNGVCWTV
T.G..M...
8.11
TVGGVMWTV
.........
TVGGVMWTV
.........
-
CVNGVCWTV
T.G..M...
5.18
TVGGVMWTV
-
HPNIEEVAL
0.41
HSNIEEVAL
-
KLSGLGLNAV
0.44
KLRGMGLNAV
-
CVNGVCWTV
28.87
TVGGVMWTV
-
CVNGVCWTV
0.79
TVGGVMWTV
-
CVNGVCWTV
14.06
TVGGVMWTV
-
CVNGVCWTV
8.24
TVGGVMWTV
-
ATDALMTGY
HSKKKCDEL
HPNIEEVAL
29.26
62.5
31.47
ATDALMTGF
HSKKKCDEI
HSNIEEVAL
59.38
11.95
KLSGLGLNAV
1,69
KLRGMGLNAV
-
1.75
-
-
GKSTKVPAAYAAQGYKVL
AATLGFGSFMSRAYGI
CVNGVCWTV
-
9.98
GKAIPIEVIKGGRHLIF
VTLTHPITK
A2 B35,39
ATDALMTGF
.........
-
3.23
viral load was determined with the Versant HCV-RNA 3.0 assay (bDNA) with a limit of detection of 615IU/ml
5.47
-
-
GKSTKVPAAYAAQGYKVL
...........V...N..
IPFYGKAI
V.......
9.64
0.57
-
A2 B18
A3,24 B15,55
A2,11 B15,35
A2,24 B40,51
A3,24 B7,35
A3,33 B35,38
A2 B7,40
35
1,320,000
2,009,000 240
VDFVPVESM
LQ.I...TL
ELTPAETSVRLRAYL
D.Q....T.......
1.03
A1,11 B57
A1,3 B7,44
A3,24 B15,35
A2,24 B39,40
A2,68 B38,51
293,700
GPKGPITQMYTNVDQDLV
SS...VI...........
A3,24 B35
A2 B39,52
70
82
a
46
31
18
19
64
65
67
RNA
neg
46,920
61,550
TVGGVMWTV
CIG..M...
KLRGMGLNAV
..ALG.V...
0.68
6.83
-
-
11
|
RGL LGCIITS
... ..T.V..
161
|
RGV AKAVDFV
... ...LQ.I
311
|
ETA GARLVVL
... .V..T..
461
|
RRG RTGRGRR
... ......L
611
|
LTH PITKYIM
... .V.....
1
|
GT1b cons APITAYSQQT
GT3a cons ......A...
151
|
GT1b cons VGIFRAAVCT
GT3a cons A.........
301
|
GT1b cons LGIGTVLDQA
GT3a cons ..........
451
|
GT1b cons VPQDAVSRSQ
GT3a cons A.........
601
|
GT1b cons RLGAVQNEVT
GT3a cons ...P....IC
621
|
ACMSADLEVV
.........T
471
|
GIYRFVTPGE
.T..Y.A...
321
|
ATATPPGSVT
........I.
171
|
PVESMETTMR
...TLS.QA.
21
|
LTGRDKNQVE
.......V.T
631
|
T
.
481
|
RPSGMFDSSV
........V.
331
|
VPHPNIEEVA
...S......
181
|
SPVFTDNSSP
..S.S...T.
31
|
GEVQVVSTAT
.....L....
491
|
LCECYDAG CA
........ .S
341
|
LSNTGEIP FY
.GSE.... ..
191
|
PAVPQTFQ VA
.....SY. .G
41
|
QSFLATCV NG
.T..G.T. G.
501
|
WYELTPAETS
..D.Q....T
351
|
GKAIPIETIK
......ALL.
201
|
HLHAPTGSGK
Y.........
51
|
VCWTVYHGAG
.M........
511
|
VRLRAYLNTP
.......S..
361
|
GGRHLIFCHS
..........
211
|
STKVPAAYAA
........V.
61
|
SKTLAGPKGP
.R....A.H.
521
|
GLP VCQDHLE
... ......D
371
|
KKK CDELAAK
... ...I.S.
221
|
QGY KVLVLNP
... N......
71
|
ITQ MYTNVDQ
AL. .......
531
|
FWESVFTGLT
..........
381
|
LSGLGLNAVA
.R.M......
231
|
SVAATLGFGA
.........S
81
|
DLVGWQAPPG
.....P....
NS3 GT1b consensus sequence aligned to NS3 GT3a consensus sequence
541
|
HIDAHFLSQT
..........
391
|
YYRGLDVSVI
..........
241
|
YMSKAHGVDP
F..R.Y.I..
91
|
ARSLTPCTCG
.K..E..A..
551
|
KQAG DNFPYL
..Q. L..S..
401
|
PTSG DVVVVA
..T. ....C.
251
|
NIRT GVRTIT
.... .N..V.
101
|
SSDL YLVTRH
.A.. .....D
561
|
VAYQATVCAR
T.........
411
|
TDALMTGFTG
..........
261
|
TGAPITYSTY
...KL.....
111
|
ADVIPVRRRG
.....A....
571
|
AQAPPPSWDQ
.........E
421
|
DFDSVIDCNT
.........V
271
|
GKFLADGGCS
..........
121
|
DSRGSLLSPR
..TA......
581
|
MWK CLIRLKP
T.. ..V....
431
|
CVT QTVDFSL
A.E .Y.....
281
|
GGA YDIIICD
... ..V....
131
|
PVS YLKGSSG
.LA C......
591
|
TLHGPTPLLY
..........
441
|
DPTFTIETTT
....S...R.
291
|
ECHSTDSTSI
...AQ.A...
141
|
GPLLCPSGHA
..VM.....V
C73
negative
GT1 pool 9
positive
0,21
9,30
Peptide Pool
IFNg
GT1-83
GT1-80
GT1-77
2,76
0,39
0,23
GT1-84
GT1-81
GT1-78
0,37
0,34
0,21
Single Peptides
Strategy for detection of CD8 responses
GT1-85
GT1-82
GT1-79
0,19
14,54
0,23
CD8