Download Antibody-Selected Mimics of Hepatitis C Virus Hypervariable Region

Antibody-Selected Mimics of Hepatitis C Virus
Hypervariable Region 1 Activate Both Primary and
Memory Th Lymphocytes
Loredana Frasca,1 Cristiano Scottà,1 Paola Del Porto,1 Alfredo Nicosia,2 Caterina Pasquazzi,3 Ilaria Versace,3
Anna Maria Masci,4 Luigi Racioppi,4 and Enza Piccolella1
An ideal strategy that leads to a vaccine aimed at controlling viral escape may be that of
preventing the replication of escape mutants by eliciting a T- and B-cell repertoire directed
against many viral variants. The hypervariable region 1 (HVR1) of the putative envelope 2
protein that presents B and T epitopes shown to induce protective immunity against hepatitis C virus (HCV), might be suitable for this purpose if its immunogenicity can be improved by generating mimics that induce broad, highly cross-reactive, anti-HVR1 responses.
Recently we described a successful approach to select HVR1 mimics (mimotopes) incorporating the variability found in a great number of viral variants. In this report we explore
whether these mimotopes, designed to mimic B-cell epitopes, also mimic helper T-cell
epitopes. The first interesting observation is that mimotopes selected for their reactivity to
HVR1-specific antibodies of infected patients also do express HVR1 T-cell epitopes, suggesting that similar constraints govern HVR1-specific humoral and cellular immune responses. Moreover, some HVR1 mimotopes stimulate a multispecific CD4ⴙ T-cell repertoire
that effectively cross-reacts with HVR1 native sequences. This may significantly limit effects
as a T-cell receptor (TCR) antagonist frequently exerted by natural HVR1-variants on
HVR1-specific T-cell responses. In conclusion, these data lend strong support to using
HVR1 mimotopes in vaccines designed to prevent replication of escape mutants.
(HEPATOLOGY 2003;38:653-663.)
H
epatitis C virus (HCV) displays a high rate of
mutation during replication, mainly accumulated in a few restricted regions, referred to as
hypervariable. Of these, the 27 amino acid long N-termi-
Abbreviations: HCV, hepatitis C virus; HVR1, hypervariable region 1; ELISA,
enzyme-linked immunosorbent assay; EBV, Epstein-Barr virus; PCR, polymerase
chain reaction; MAP, multiple antigenic peptide; APCs, antigen-presenting cells;
PBMC, peripheral blood mononuclear cell; HLA, human leukocyte antigen; TCR,
T-cell receptor; BV, TCR ␤ variable gene segment.
From the 1Department of Cellular and Developmental Biology and the 3Department of Tropical and Infectious Diseases, “La Sapienza” University, Rome, Italy;
the 2Istituto di Ricerche Biologia Molecolare P. Angeletti, Pomezia, Rome, Italy;
and the 4Department of Cellular and Molecular Biology and Pathology, “Federico
II” University, Naples, Italy.
Received January 18, 2003; accepted June 20, 2003.
Supported by grants from the University of Rome “La Sapienza,” Ateneo Project,
the Ministry for the University and Research (MIUR-COFIN), and the National
Council of Research (Consiglio Nazionale delle Ricerche, Progetto Finalizzato Biotecnologie).
Address reprint requests to: Enza Piccolella, Ph.D., Dipartimento di Biologia
Cellulare e dello Sviluppo, Università “La Sapienza,” Via dei Sardi 70, 00185
Roma, Italy. E-mail: [email protected]; fax: (39) 6-49917594.
Copyright © 2003 by the American Association for the Study of Liver Diseases.
0270-9139/03/3803-0016$30.00/0
doi:10.1053/jhep.2003.50387
nal segment of the putative envelope protein E2, hypervariable region 1 (HVR1), displays the highest degree
of sequence variability.1 Recent data have shown that
acute resolving hepatitis is associated with an evolutionary stasis of HVR1 quasispecies, whereas progressive hepatitis correlates with sequence variability.2
Anti-HVR1 antibodies have been shown to impair viral attachment in vitro and infectivity in vivo,3-6 and a
significantly higher frequency of anti-HVR1 CD4⫹
and CD8⫹ T-cell responses has been detected in patients who recovered from HCV infection.7,8 In this
scenario, it is reasonable to deduce that variations
within HVR1-specific B and T epitopes may concur to
elicit immune evasion.8-11
To prevent the replication of escape mutants, we recently selected HVR1 synthetic variants (mimotopes)
from a vast repertoire of HVR1 surrogates displayed on
M13 bacteriophage by using antibodies of infected patients.12 Such reagents were found to induce highly crossreacting anti-HVR1 Ab in animal models.13-15 Given the
fundamental role of helper T cells in antiviral immunity
and vaccine efficacy, here we explored whether these an653
654
FRASCA ET AL.
HEPATOLOGY, September 2003
ants. This phenomenon significantly decreases the occurrence of antagonistic effects frequently exerted by natural
HVR1 variants.9
Table 1. Clinical and Virologic Characteristics of the Patient
Population
Individual
Patient
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Healthy
controls
20
Age
Sex
Genotype*
Liver
Histology†
ALT (IU/L)
69
63
59
65
63
62
60
57
39
62
60
39
58
47
59
39
39
39
47
28
M
F
F
F
F
F
F
F
M
F
M
M
F
M
M
M
M
M
M
M
1b
1b
1b
ND
1b
2a
2a/2c
1b
1a
3a
ND
1a
1b
1b
1b
3a
1b
2a/2c
4c/4d
ND
CAH-C
C
C
C
C
C
C
CAH-C
CAH
CAH
CAH
CAH
C
CAH
CAH
CAH
CAH
CAH
CAH-C
CAH-C
⬍30
80
118
150
125
100
⬍30
100
66
70
90
ND
136
⬍30
88
90
120
66
110
95
34 ⫾ 8
10F/10M
-
-
-
Patients and Methods
Patients. We enrolled 20 patients with chronic HCV
infection and 20 healthy individuals (Table 1). The diagnosis of HCV infection was based on standard clinical
parameters and serologic assays. Liver biopsies were performed and the histologic status was defined according to
conventional classification. Twenty control healthy subjects showed no sign of past or present HCV infection
(enzyme-linked immunosorbent assay [ELISA] tests negative for HCV reactivity).
Human Leukocyte Antigen-DR Typing. DNA extracted from patients’ Epstein-Barr virus transformed B
(EBV-B) cell lines was typed by sequence-specific amplification using polymerase chain reaction (PCR)-SSP system (Dynal SSP DR low resolution kit; Dynal Biotech
Ltd., Oslo, Norway).
Mimotopes and Natural HVR1 Peptides. Thirtytwo HVR1 natural sequences and 12 mimotopes12 (Table
2) were synthesized as multiple Ag peptide (MAP).7 Accession numbers and sequences of HVR1 peptides (residues 384-410 of HCV) are reported in Table 3.
We also synthesized linear 13-mer peptides corresponding to the C-terminal region of MAPs 455, 320,
877, 296 (residues 15-27 of the HVR1 region), and analogues of 876 (879), substituting residues 21 (R3 S), 22
(Q3 P), and 24 (P3 A), and of 455 (875) substituting
residues 21 (S3 R), 22 (P3 Q), and 24 (A3 P).
B Cell Lines. Both EBV-B cells generated from patients and healthy donors9 and homozygous EBV-B
SWEIG007 (DRB1*1101, DRB3*0202), SA (DR1,
Abbreviations: ALT, alanine aminotransferase; M, male; CAH, chronic active
hepatitis; F, female; C, cirrhosis; ND, not determined.
*Genotype was determined according to Simmonds et al.27
†Histologic status of liver specimens.
tibody-selected mimotopes could be stimulatory for
CD4⫹ T cells in humans.
We found that not only were HVR1 mimotopes recognized by helper T cells of infected individuals, but they
also induced the ex vivo priming of helper T-cell responses
in healthy donors. In addition, some of the mimotopeinduced helper T cells showed a degree of cross-reactivity
never observed after stimulation with natural HVR1 vari-
Table 2. Amino Acid Sequence of Mimotopes
Name
1
440
441
443
444
445
988
990
876
877
455
316
320
Q
Q
T
Q
Q
T
T
T
T
Q
T
Q
5
T
T
T
T
T
T
T
T
T
T
T
T
T
H
H
T
T
T
T
R
H
H
T
T
V
T
T
T
V
T
T
T
T
T
T
T
V
T
V
T
T
V
T
T
T
T
T
T
10
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
S
V
S
S
Q
Q
Q
S
S
Q
Q
Q
Q
V
V
A
A
A
A
A
A
A
V
V
S
G
A
S
S
S
G
S
S
G
G
S
NOTE. Boxed residues correspond to conserved amino acids.
15
H
H
R
H
H
H
H
R
H
H
H
H
T
A
Q
A
T
T
Q
Q
Q
Q
Q
A
V
T
V
V
T
T
A
T
T
A
T
T
R
S
H
S
S
S
H
S
S
H
S
A
G
G
S
S
S
S
S
R
R
S
G
G
20
L
L
L
L
L
L
L
L
L
L
L
L
T
T
T
T
T
T
T
V
V
T
T
T
S
S
G
G
G
G
S
S
S
G
G
G
L
L
L
L
L
L
L
L
L
L
L
L
F
F
F
F
F
F
F
F
F
F
F
F
25
S
S
S
S
S
S
S
R
S
S
S
S
P
P
P
P
P
P
P
Q
P
P
P
L
G
G
G
G
G
G
G
G
G
G
G
G
A
P
P
S
A
S
A
P
A
A
A
P
S
S
Q
K
S
Q
S
Q
Q
K
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
N
K
K
N
K
N
K
N
N
N
N
K
HEPATOLOGY, Vol. 38, No. 3, 2003
FRASCA ET AL.
655
Table 3. HVR1 Natural Sequences and Accession Numbers
Name
Amino Acid Sequence
Accession Number
Sequence Location
265
266
268
269
270
271
272
274
275
277
278
279
280
281
282
283
285
289
290
292
294
295
296
297
298
299
300
301
302
303
304
305
STHVTGALQGRAAYGITSFLSHGPSQK
HTRVTGGVQGHVTSTLTSLFRPGASQK
ETHVTGGSAGRTTAGLVGLLTPGAKQN
ATYTTGGSAAKTAHRLASFFTVGPKQD
DTHVVGGATERTAYSLTGLFTAGPKQN
GTTCQGGVYARGAGGIASLFSVGANQK
RTLSFGGLPGHTTHGFASLSAPGAKQN
NTHAMGGVVARSAYRITSFLSPGAAQN
STRITGGSMARDVYRFTGFFARGPSQN
NTYVTGGAAARGASGITSLFSRGPSQK
NTYASGGAVGHQTASFVRLLAPGPQQN
ETHTTGGEAARTTLGIASLFTSGANQK
ETHTTGGSAARATFGIANFFTPGAKQN
ETYTSGGSAAHTTSGFVSFFSPGAKQN
GTTRVGGAAARTTSSFASLLTHGPSQN
NTHTVGAAASRSTAGLTSLFSIGRSQK
NTHVSGGRVGHTTRSLTSFFTPGPQQK
ETRVTGGAAGHTAFGFASFLAPGAKQK
NTYVTGGSAGRAVAGFAGLLQPGAKQN
ETHVTGGSAASTTSTLTKLFMPGASQN
GTTTVGSAVSSTTYRFAGMFSQGAQQN
NTHTVGGTEGFATQRLTSLFALGPSQK
NTHVTGGVVARNAYRITTFLNPGPAQN
HTYTTGGTASRHTQAFAGLFDIGPQQK
KTHVTGMVAGKNAHTLSSIFTSGPSQN
GTHVTGGKVAYTTQGFTSFFSRGPSQK
ETYTSGGNAGHTMTGIVRFFAPGPKQN
STYSMGGAAAHNARGLTSLFSSGASQR
ETHVTGGSAGRSVLGIASFLTRGPKQN
ETYIIGAATGRTTAGLTSLFSSGSQQN
ETHVTGGNAGRTTAGLVGLLTPGAKQN
ETHVTGGSAGHTAAGIASFFAPGPKQN
PIR:PC1193
Genbank:D00574
Genbank:M62381
Genbank:U24616
PIR:C48776
Genbank:U24607
PIR:D48766
Genbank:D43650
PIR:PQ0835
Genbank:D10934
Genbank:D31972
Genbank:U14231
Genbank:U24602
Genbank:L19380
Genbank:M74888
Genbank:L12354
PIR:A48776
Genbank:D14853
Genbank:S24080
Genbank:S62395
Genbank:D88472
Genbank:D10687
Genbank:D43651
Genbank:D14305
Genbank:X60590
Genbank:D30613
Genbank:X53131
Genbank:U24619
Genbank:M62382
Genbank:D88474
(H77-1)
(H79)
aa 16-42
bp 1240-1320
bp 1426-1506
bp 22-102
aa 13-39
bp 22-102
aa 13-39
bp 1-81
aa 6-32
bp 1491-1571
bp 1409-1489
bp 103-183
bp 22-102
bp 46-126
bp 1147-1227
bp 1468-1548
aa 13-39
bp 1491-1571
bp 46-120
bp 43-123
bp 1485-1565
bp 1180-1260
bp 39-119
bp 1427-1507
bp 46-126
bp 1491-1571
bp 802-882
bp 22-102
bp 1426-1506
bp 1488-1568
bp 1-81
bp 1-81
DQ1), and BOLETH (DRA*0101, DRB1*0401,
DRB4*0103, DRB7*0101, DRB8*0101) were used as
antigen-presenting cells (APCs).
Induction of HVR1 Natural Variant- and Mimotope-Specific T-Cell Lines. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured
with autologous dendritic cells matured with cross-linking of CD40 plus interferon ␥ (500 U/mL) as described.16
Briefly, 1 ⫻ 105 PBMCs were cultured with 1 ⫻ 104
irradiated mature dendritic cells prepulsed overnight with
10 ␮g/mL of the selected MAP, in RPMI 1640 10%
human serum medium, without interleukin 2. After 7
days, cells were restimulated with irradiated autologous
PBMCs pulsed with MAPs and 5 U/mL of human recombinant interleukin 2 (Roche Molecular Biochemicals,
Mannheim, Germany). T-cell lines from HCV chronically infected patients were generated by stimulating PBMCs for 7 days with 10 ␮g/mL of MAP. Cells were
maintained in culture with irradiated autologous PBMCs, peptide, and 10 U/mL of human recombinant interleukin 2. T-cell phenotype was determined by flow
cytometry as described.9 Peptide specificity and human
leukocyte antigen (HLA) restriction of T-cell lines were
tested in proliferation assays after 2 rounds of stimulation
by using irradiated autologous EBV-B as APC. Crossreactivity, antagonistic assays, and RNA extraction were
performed on T-cell lines after 4 rounds of stimulation.
Proliferation Assays. PBMCs (1 ⫻ 105), isolated
from freshly heparinized blood of HCV chronically infected patients and healthy controls, and T cells (2 ⫻ 104)
incubated with 4 ⫻ 104 mitomycin-C–treated autologous PBMCs or B-cell lines, were stimulated with different concentrations of MAPs (usually between 1-30 ␮g/
mL) for 5 and 2 days, respectively. Cells were labeled with
1 ␮Ci of [3H]-thymidine and harvested 18 hours later.
SDs of the mean counts per minute of triplicate cultures
of PBMCs and T-cell lines were consistently below 30%
and 10%, respectively. Stimulation index was calculated
as the ratio of [3H]-thymidine incorporation in the presence of antigen in relation to the control. A stimulation
index score greater than 3 was considered to indicate positive proliferative responses.
T-Cell Receptor Antagonism Assay. T-cell receptor
(TCR) antagonism was tested as described in a previous
report in which the antagonistic activity of HVR1 sequences was shown.9 Briefly, autologous EBV-B was pre-
656
FRASCA ET AL.
pulsed overnight at 37°C with different doses of agonist
peptide (usually between 10-30 ␮g/mL). After washing,
APCs were treated with mitomycin-C, plated out in flatbottomed microtitre plates, and variant peptides were
added directly into the wells at various concentrations.
After a further 5 hours of incubation, T cells were added
into the wells and proliferation was measured as described.
Analysis of Antibody Specificities by ELISA. Human HCV⫹ sera (100 ␮L), diluted 1:50, was incubated
overnight at 4°C in ELISA multiwell plates in the presence of each MAP (Immunoplate Maxisorp; Nunc, Roskilde, Denmark).12 Plates washed with phosphate-buffered
saline/Tween were incubated for 4 hours at 4°C in 100
␮L/well of goat anti-human immunoglobulin G (Fc-specific) alkaline phosphatase-conjugated Abs (Sigma Chemical Co., St. Louis, MO). Alkaline phosphatase was
revealed by incubation with 100 ␮L/well of 1 mg/mL
solution of p-nitrophenyl phosphate in substrate buffer
(10% diethanolamine buffer, .5 mmol/L MgCl2, adjusted
to pH 9.8 with HCl). Results were recorded as differences
between optical density (OD)405 nm and OD620 nm by an
automated ELISA reader (Labsystem Multiskan Bichromatic, Helsinki, Finland) and reported as absorbance/
cutoff ratio. The cutoff value for each peptide was
calculated as the mean plus 3 times the SDs of 20 healthy
controls. Values of 1 or less were considered negative.
Analysis of TCR Repertoire. RNA was extracted by
using the guanidium hydrochloride– containing Trizol
Reagent (Life Technologies, GIBCO-BRL, Gaithersburg, MD). First-strand complementary DNA synthesis
was performed by using oligo (dT) as a primer for reverse
transcription of 1 ␮g total RNA (SUPERSCRIPT II RT;
Life Technologies). PCR amplification was performed according to Yassai et al.17 Briefly, complementary DNA
was amplified for 30 cycles under nonsaturating PCR
conditions with a panel of 25 TCR ␤ variable gene segment (BV) family-specific primers, and a B-constant
primer in duplex. Simplex reactions were used for BV 6.1
and 6.2 amplifications. The common constant primer
was labeled at the 5⬘ end with 5⬘-6 carboxyfluorescein. To
normalize the results, the templates from different samples were titrated at different dilution points of starting
material by amplifying the TCR B-chain constant complementary DNA. TCR spectratyping was performed as
described by Yassai et al.17 Briefly, an equivalent volume
of PCR-labeled product was mixed with formamide dyeloading buffer and in the presence of TAMRA-labeled
size markers (Applied Biosystems, Foster City, CA),
heated at 94°C for 2 minutes, and applied to a pre-run 5%
acrylamide-urea sequencing gel. Gels were run on a 377
ABI Automatic DNA Sequencer (Applied Biosystems,
HEPATOLOGY, September 2003
Foster City, CA, USA) for 110 minutes at 40 W. After
resolution on the gel, the labeled PCR products were analyzed by Gene Scan software (Applied Biosystems). TCR
spectratyping of a healthy PBMC repertoire typically results in a banding pattern composed of between 7 and 8
bands at 3 nucleotide base intervals, reflecting the correct
in-frame nature of functionally rearranged BV-chain
TCR gene products. The limited number of PCR cycles
used leads to the generation of PCR products with a distribution representative of the starting material (i.e., a
Gaussian distribution).
Results
Analysis of T- and B-Cell Reactivity of HCV
Chronically Infected Patients to HVR1 Mimotopes.
To assess the presence of T-cell epitopes in HVR1-mimotopes, PBMCs of 20 HCV chronically infected patients
were cultured with 10 ␮g/mL of each of the 12 mimotopes. Twelve HVR1 natural sequences chosen from
those more frequently recognized by a group of HCVinfected patients as previously described7 also were included in the experiments. PBMCs of 20 HCV-negative
individuals were used as controls. The results of Table 4
show that 12 of 20 patients reacted to at least one mimotope (65%), whereas 6 patients recognized at least one
natural variant (30%). None of the serum-negative controls responded to any peptide used (data not shown).
Furthermore, we aligned the sequences of each mimotope
with those of the natural variants recognized by the same
T cells and evaluated the percentage of homology by an
ALIGN analysis (Genestream Network Server IGH,
Montpellier, France) (http://vega.igh.cnrs.fr). Table 5
shows that although the sequence homology between mimotope and natural variant is included in 50% to 63% of
similarity, each mimotope mimicked at least 4 natural
variants. An epitope mapping analysis also was performed
on PBMCs of 3 patients (patients 1, 3, and 4) responsive
to mimotopes 877, 320, and 455 by using the C-terminal
sequence encompassing residues 15 to 27. Comparative
proliferation assays using both the whole and truncated
sequences clearly showed that the mimotope carboxy-terminal sequence expresses T-cell epitopes (data not
shown), as previously described for HVR1 natural sequences.7,18
The presence of mimotope- and natural variant–specific antibodies in sera from 5 patients was tested and
reported in Fig. 1, where T-cell reactivity to the same
peptides also is shown. Interestingly, both patient sera and
T cells recognized mimotopes more frequently than natural variants. Moreover, 8 of 12 HVR1 mimotopes interacted with both T cells and antibodies isolated from the
HEPATOLOGY, Vol. 38, No. 3, 2003
FRASCA ET AL.
657
Table 4. Lymphoproliferative Responses to Mimotopes and to HVR1 Natural Variants
Mimotopes†
HVR1 Natural Variant†
Patient
HCVⴙ
No
Pep*
316
320
440
441
443
444
445
455
876
877
988
990
266
272
275
290
292
294
295
296
298
299
303
304
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
288‡
230
314
190
150
305
92
1919
1327
501
758
744
530
684
310
120
577
120
239
1490
2.4
0.8
2.2
1.8
1.8
0.9
1.1
0.8
1.1
0.9
0.7
1.0
2.1
1.8
2.1
1.1
1.1
3.0
1.5
0.9
3.0
1.0
5.6
3.0
16.0
0.8
1.3
0.6
1.1
0.7
1.0
0.8
2.2
1.0
1.8
0.8
1.2
1.8
1.3
0.6
1.6
0.7
3.0
1.1
2.0
0.6
1.1
0.7
1.3
0.8
1.1
1.8
1.8
0.9
1.9
0.9
0.9
1.4
1.6
0.7
3.0
0.9
3.3
0.8
1.3
0.6
1.4
0.5
1.2
1.1
0.8
2.1
5.0
3.4
2.4
1.2
1.4
1.3
1.0
0.4
1.4
1.1
2.1
1.5
1.8
0.8
1.7
0.9
0.9
1.3
1.3
1.7
1.0
0.7
1.0
0.8
1.1
1.6
1.0
0.5
1.0
0.8
3.5
1.3
2.2
0.9
1.5
1.3
1.1
1.0
0.6
3.0
1.7
1.0
1.1
1.0
1.6
1.5
1.1
0.8
1.1
0.8
3.0
1.1
1.0
1.0
1.5
0.8
1.4
1.0
0.7
1.4
0.9
1.0
1.8
3.1
0.8
1.1
1.2
1.0
1.5
1.2
3.0
1.0
1.0
0.8
1.6
1.2
1.2
1.3
0.6
1.5
1.0
2.0
1.7
2.1
0.7
1.4
1.1
1.1
3.5
0.9
3.2
1.3
1.1
1.1
1.3
1.2
1.1
3.0
0.6
1.0
1.0
1.2
3.1
1.5
4.2
3.2
0.9
1.3
3.3
3.0
13.0
3.0
1.8
1.0
1.9
1.5
0.8
1.2
1.1
2.1
1.4
1.8
2.1
1.4
0.7
1.2
0.8
1.4
1.6
0.8
1.4
0.9
1.4
0.7
1.2
0.8
0.9
1.3
1.3
1.9
0.8
1.0
1.9
0.9
0.9
1.4
1.1
0.7
1.6
0.9
1.1
1.1
1.3
0.8
1.1
0.9
0.9
1.4
1.1
3.5
0.9
1.1
1.7
0.7
1.0
1.8
1.3
0.6
1.4
1.6
1.4
1.2
0.7
1.1
1.1
1.1
1.5
1.1
0.7
0.8
1.1
0.8
2.1
1.2
1.2
1.2
1.3
1.4
1.0
1.1
1.8
0.9
1.1
0.8
1.3
0.8
1.2
0.7
1.3
1.0
2.2
0.6
1.5
1.5
1.7
1.1
1.2
0.4
0.9
0.8
1.3
0.8
0.5
0.6
1.6
1.0
0.8
1.0
0.6
1.2
1.0
0.7
1.6
0.7
0.7
2.1
2.1
0.6
3.0
0.6
1.7
0.7
0.6
0.5
0.8
0.6
1.8
0.6
1.0
1.0
1.8
0.8
1.7
1.2
0.8
2.3
0.8
0.7
1.1
1.1
1.9
0.6
2.1
0.7
2.1
0.8
1.0
2.3
1.1
0.8
1.5
1.1
2.4
0.7
1.1
0.7
0.6
1.1
0.8
1.1
7.7
1.3
4.0
1.2
0.9
0.8
1.0
1.4
0.8
1.5
1.9
1.6
1.2
1.3
1.0
1.6
1.4
1.3
3.0
0.6
5.3
3.0
1.7
1.0
1.7
1.0
0.6
2.0
1.2
1.6
2.5
1.5
1.7
0.8
0.7
2.0
1.1
1.2
1.2
1.0
1.1
1.1
1.3
1.3
1.2
0.6
0.8
0.6
1.0
1.1
2.1
0.9
2.2
0.6
1.9
1.7
1.3
1.1
1.1
0.7
3.4
1.2
0.7
0.9
0.9
1.1
1.7
1.1
0.7
1.1
3.0
1.0
1.5
1.1
1.8
0.8
1.7
0.9
1.2
0.8
17.6
5.1
1.3
1.1
1.1
1.2
1.4
4.5
0.6
1.2
1.8
1.1
1.0
0.9
2.0
1.4
0.7
1.1
0.9
1.0
1.3
1.4
1.2
1.0
1.0
1.2
1.0
0.9
1.0
0.9
1.3
0.8
1.7
0.6
1.1
1.7
1.0
1.0
1.1
0.9
1.5
0.6
0.7
0.9
0.8
1.3
1.1
1.0
0.4
1.0
5.0
0.9
1.0
1.1
1.0
1.1
0.9
0.8
*No peptide.
†Values are expressed as stimulation index scores. Significant stimulation index scores are in boldface underlined type.
‡Values are expressed as cpm.
same patient, whereas only 3 natural variants interacted
with both T cells and antibodies. This suggests that mimotopes may favor the phenomenon called T-B reciprocity as outlined by Shirai et al.18 Indeed, the investigators
suggested that helper T cells specific for the HVR1 region
itself are the most efficient at helping Ab production to
this region.
Because 3 positions of mimotopes (aa 21, 22, and 24)
were shown previously to be crucially involved in antibody recognition,12,14 their role in T-cell activation was
investigated. We synthesized 2 analogs of mimotopes 876
and 455 by substituting amino acids at position 21, 22,
and 24, as reported in Fig. 2. Mimotopes 876 and 455,
and the analogs 875 and 879, were used to stimulate
PBMCs of 3 patients responsive to mimotope 876 and
not to mimotope 455. A representative experiment performed on PBMCs of an HLA-DRB1*01/07 individual is
reported in Fig. 2. It is evident that substitutions at residues 21, 22, and 24 converted an immunogenic mimotope into a nonimmunogenic one and vice versa. The
same phenomenon was observed in the other 2 patients
tested (data not shown). The crucial role of these positions in T-cell activation derives from the use of a CD4⫹
T-cell line elicited by 455 (see Table 6) that lost the reactivity to 455 after substitutions of residues 21, 22, and 24.
Characterization of T-Cell Lines Elicited From PBMCs of Infected and Healthy Individuals by Natural
and Mimotopic Sequences of HVR1. Having established that PBMCs of chronically infected individuals recognized both mimotopes and natural variants, we verified
whether CD4⫹ T cells specific for natural variants could
be recalled or immunized in vitro by mimotopes. To this
aim, we elicited T-cell lines from PBMCs of patients 3, 4,
and 17 using mimotopes and natural variants to which
they were responsive (876, 877, 294, 295, and 299), as
shown in Table 4. The cells of the healthy individuals
(donors A, L, and GA) were primed in vitro by using
dendritic cells pulsed with 10 different mimotopes. The
T-cell lines obtained, all of the CD4 phenotypes, are
listed in Table 6. Antigenic specificity was determined as
previously reported.7 A typical experiment is reported in
Fig. 3, in which we showed that A296/2 and A877/3
reacted to the carboxy-terminal part of MAP (Fig. 3A).
The analysis of HLA restriction was included in the experiment (Fig. 3B). We also analyzed the composition of
the TCR repertoire of T-cell lines listed in Table 6 by
using spectratyping, a novel investigative tool that measures the heterogeneous length of the TCR ␤ chain
CDR3 (BV-CDR3), a random-coiled region contacting
the epitope residues of the antigenic peptide. Therefore,
the complexity of CDR3 heterogeneity reflects the complexity of the clonal repertoire elicited by HCV peptide
and mimotope. The results reported in Fig. 4A, show that
3 of 25 BV families were present in lines A296/2 and
A296/3 from a healthy individual, whereas 10 had been
expanded by mimotope 877 (A877/3). This data indicate
that mimotope 877 was able to stimulate and expand in
vitro a more complex repertoire of lymphocytes compared
with that elicited by the natural variants. We observed the
same wider usage of BV regions in T-cell lines raised with
658
FRASCA ET AL.
HEPATOLOGY, September 2003
Table 5. Homology Between Mimotope Sequences and HVR1
Natural Variant Sequences
Name
320
290
294
295
298
299
440
290
294
295
298
299
441
290
294
295
298
299
304
444
290
294
295
298
299
304
445
294
295
298
299
455
294
295
298
299
876
290
294
295
298
299
877
290
294
295
298
299
Amino Acid Sequence
1
10
20
27
P
P
P
P
QTTTTGGQVSHATAGLTGLFSLGPQQK
N-YV---SAGR-V--FA--LQP-AK-N
G---V-SA--ST-YRFA-M--Q-A--N
N-H-V--TEGF--QR--S--A---S-K-HV--MVAGKNAHT-SSI-TS--S-N
G-HV---K-AYT-Q-F-SF--R--S-1
10
20
27
P
P
P
P
QTTVVGGSQSHTVRGLTSLFSPGASQN
N-Y-T---AGRA-A-FAG-LQ---K-G--T--SAV-S-TYRFAGM--Q--Q-N-HT---TEGFATQR-----AL-P--K
K-H-T-MVAGKNAHT-S-I-TS-P--G-H-T--KVAY-TQ-F--F--R-P--K
1
10
20
27
P
P
P
P
QTHTTGGVVGHATSGLTSLFSPGPSQK
N-YV---SA-R-VA-FAG-LQ--AK-N
G-T-V-SA-SST-YRFAGM--Q-AQ-N
N---V--TE-F--QR-----AL----K--V--M-A-KNAHT-S-I-TS----N
G--V---K-AYT-Q-F--F--R----E--V---NA-RT-A--VG-LT--AK-N
1
10
20
27
P
P
P
P
QTTTTGGSASHAVSSLTGLFSPGSKQN
N-YV-----GR--AGFA--LQ--A--G---V-SAV-STTYRFA-M--Q-AQ-N-H-V--TEGF-TQR--S--AL-PS-K
K-HV--MV-GKNAHT-SSI-TS-PS-G-HV---KVAYTTQGF-SF--R-PS-K
E-HV---N-GRTTAG-V--LT--A--1
10
20
27
P
P
P
P
QTTVTGGQASHTTSSLTGLFSPGASQK
G--TV-SAV-S--YRFA-M--Q--Q-N
N-HTV--TEGFA-QR--S--AL-P--K-H---MV-GKNAHT-SSI-TS-P--N
G-H----KVAY--QGF-SF--R-P--1
10
20
27
P
P
P
P
QTHTTGGQAGHQAHSLTGLFSPGAKQN
G-T-V-SAVSSTTYRFA-M--Q--Q-N---V--TE-FATQR--S--AL-PS-K
K--V--MV--KN--T-SSI-TS-PS-G--V---KVAYTTQGF-SF--R-PS-K
1
10
20
27
P
P
P
P
TTRTTGGSASRQTSRLVSLFRQGPQQN
N-YV-----G-AVAGFAG-LQP-AK-G-T-V-SAV-ST-Y-FAGM-S--A--N-H-V--TEGFA-Q--T---AL--S-K
K-HV--MV-GKNAHT-S-I-TS--S-G-HV---KVAYT-QGFT-F-SR--S-K
1
10
20
27
P
P
P
P
TTHTTGGSASHQTSRLVSLFSPGAQQN
N-YV-----GRAVAGFAG-LQ---K-G-T-V-SAV-ST-Y-FAGM--Q----N---V--TEGFA-Q--T---AL-PS-K
K--V--MV-GKNAHT-S-I-TS-PS-G--V---KVAYT-QGFT-F--R-PS-K
% Identity
41%
48%
56%
30%
52%
48%
44%
44%
37%
48%
41%
33%
67%
48%
63%
48%
56%
41%
41%
33%
33%
48%
44%
44%
37%
52%
37%
44%
48%
37%
Fig. 1. Reactivity of 12 mimotopes and natural variants with sera and
PBMC from 5 HCV infected individuals. B ( ) and T ( ) cell reactivity
was measured by ELISA and proliferation assay. Empty white boxes refer
to not significant reactivity. For each serum, binding of antibodies to
mimotope and natural variants is reported as absorbance/cutoff ratio.
Proliferative responses of PBMC are expressed as stimulation index.
mimotope 877 from 2 patients (Fig. 4B and C). Indeed, a
larger expansion of BV clones by mimotope 877 was evident in T-cell lines FI877 and FA877: 12 and 9 of 25 BV
families had been expanded, respectively. In contrast, the
complexity of TCR repertoires expressed by the T-cell
lines FI295, FI299, FA294, FA295, and FA299 stimulated with natural sequences was more limited. The comparative analysis of the results from Fig. 4A-C revealed an
overlap in BV-CDR3 usage: BV families responding to
natural epitopes represent a subgroup of the wider TCR
repertoire elicited by mimotope 877. It is worth noticing
that the majority of these BV families also show a strong
similarity in the length of their CDR3 regions, suggesting
a strong analogy between the response elicited by mimotope and by natural HCV epitopes (Fig. 4, arrows).
41%
44%
48%
41%
37%
44%
48%
48%
41%
41%
NOTE. Boldface type refers to mimotope sequence. Dashes indicate identity of
mimotope residues with those of natural variants.
Fig. 2. Stimulatory activities of 876 and 455 mimotopes and the
corresponding analogs (879 and 875, respectively). PBMCs from patient
17 and T-cell line GA455 (see Table 6), both cells expressing HLADRB1*01/07, were activated with 10 ␮g of each MAP. The results are
representative of 3 independent experiments. The amino acids encompassing positions 15 to 27 are indicated and those corresponding to the
replaced positions are displayed in boxes.
HEPATOLOGY, Vol. 38, No. 3, 2003
FRASCA ET AL.
Table 6. HVR1- and Mimotope-Specific T-Cell Lines Elicited
From HCVⴙ and Healthy Individuals
Subject
HCV-Positive Patient
3
4
17
Healthy Donor
A
HLA DRB1 Typing
01/07
09/11
01/07
04/11
L
01/11
GA
01/07
T-Cell Line
Peptide
Specificity
FA294
FA295
FA299
FA877
FI295
FI299
FI877
TO876
294(nv)
295(nv)
299(nv)
877(mi)
295(nv)
299(nv)
877(mi)
876(mi)
A296/2
A296/3
A877/3
L295
L320
GA320
GA440
GA455
296(nv)
296(nv)
877(mi)
295(nv)
320(mi)
320(mi)
440(mi)
455(mi)
Abbreviations: nv, natural variant; mi, mimotope.
Analysis of Cross-Reactivity of T-Cell Lines Elicited
From PBMCs of Infected and Normal Individuals by
Natural and Mimotopic Sequences. We and other researchers have shown previously that a certain degree of
cross-reactivity is a peculiar feature of HVR1-specific
helper T cells7,18 isolated from infected hosts. We wondered whether the same characteristic would be found in
the T cells recalled or immunized in vitro with mimotopes. Therefore, we evaluated their cross-reactivity with
natural variants and compared their degree of cross-reactivity with that of T cells stimulated by natural sequences.
To this aim, the T-cell lines FI877 and FI295 from a
patient and GA440, A296/2, A296/3, A877/3, and L295
from 3 normal subjects (Table 6) were activated with
irradiated autologous EBV-B pulsed with a panel of
HVR1 natural variants at the same concentration in proliferation assays. The results of representative proliferative
responses (Fig. 5A-C) clearly suggest that mimotopes are
effective in recalling and priming in vitro helper T cells
recognizing natural sequences. Worthy of note is that although almost all lines presented a certain degree of crossreactivity, FI877 reacted with a higher number of
sequences (86%) than FI295 (33%, Fig. 5A), as did
A877/3 (85%) compared with A296/2 (0%) and A296/3
(32%) (Fig. 5C). The degree of cross-reactivity of the
T-cell line GA440 was 71% (Fig. 5B) whereas L295 did
not show any cross-reactivity (data not shown).
Occurrence of TCR-Antagonism in Mimotope-Activated Lines. Having identified mimotopes (877 and
440) as able to induce helper T cells with a particularly
high degree of cross-reactivity, we wondered whether we
659
had found a way to overcome TCR antagonism exerted by
natural HVR1 variants on HVR1-specific helper cells of
infected persons.9 We selected as antagonists 4 natural
variants (268, 298, 280, and 277) that did not stimulate
line A877/3 nor lines A296/2 and A296/3 (Fig. 5C). Both
natural variant 296 (Fig. 6A) and mimotope 877 (Fig. 6B)
were used as agonists for line A877/3, whereas the agonist
used with lines A296/2 and A296/3 was variant 296 (Fig.
6C and D). Although 3 variants acted as strong antagonists (277, 280, and 298), no significant difference in
susceptibility to TCR antagonism could be evidenced,
probably reflecting the different assortment of their TCR
repertoires. This means that antagonistic phenomena can
still occur in mimotope-activated lines despite their larger
TCR repertoire. However, it is evident from Fig. 5C that
many variants, although stimulatory for the mimotopeprimed T-cell line A877/3, were not stimulatory for natural variant–primed T cells (A296/2 and A296/3). It thus
seemed likely that those variants that stimulated 877/3
and not A296/2 and A296/3, could function as antagonists on these latter 2. Twenty-two variants were tested
with line A296/2 and 15 with A296/3 (Fig. 6E and F),
whereas 296 peptide was used as agonist. We found that 7
Fig. 3. T-cell epitope mapping and HLA restriction of T-cell lines
A296/2 and A877/3 primed in vitro with natural variant 296 and
mimotope 877, respectively. (A) T-cell epitopes of A296/2 and A877/3.
T-cell lines were determined by using peptides encompassing the whole
sequence (296 and 877) and the C-terminal 13 aa (296 C-term and
877 C-term). A296/3: F, 296; E, 296 C-tem; A877/3: F, 877; E, 877
C-tem. (B) HLA restriction was evaluated by using as APC autologous
(Autol) or partially matched homozygous EBV-B pulsed with 296 or 877
peptides.
660
FRASCA ET AL.
HEPATOLOGY, September 2003
of 22 and 9 of 15 variants acted as strong antagonists for
lines A296/2 and A296/3, respectively. A statistical evaluation of all these data show that, given a panel of 26
HVR1 variants, 12 (46%) and 10 (38%) variants acted as
TCR antagonists for the T-cell lines A296/3 and A296/2
(Fig. 6C-F), whereas only 3 strong antagonists (Fig. 6A)
were identified for line A877/3 (11.5%).
Discussion
Fig. 4. TCR repertoires of short T-cell lines elicited by HVR1 natural
variants and mimotopes. (A) T-cell lines A296/2, A296/3, and A877/3
derived from a healthy individual; (B, C) T-cell lines FA294, FA295, FA299,
and FA877 from patients 4 and T-cell lines FI295, FI299, and FI877 from
patient 3. Reverse-transcription PCR analysis of 25 BV families was performed on RNA isolated from T-cell lines. (A-C) The BV families identified are
reported as white boxes while gray boxes refer to the lack of PCR products.
(B, C) BV-CDR3 (white boxes) heterogeneity length profiles using PCR
products separated on DNA sequencing polyacrylamide gel by using an
automated ABI PRISM 377 apparatus (Applied Biosystems, Foster City, CA).
Band intensity was evaluated with Gene Scan software (Applied Biosystems)
and was converted to peaks. Arrows indicate CDR3 products with similar
molecular weight. (D) Comparative analysis of TCR repertoire reported in B
and C. The whole TCR repertoires elicited by 877 mimotope are reported as
gray circles. The smaller circles represent TCR repertoire elicited by 294,
295, and 299 peptides. Overlapping areas are proportional to the BV usage
overlapping. Not overlapping areas refer to different BV usage.
Combinatorial peptide libraries expressing a large collection of peptide sequences that mimic both linear and
conformational B-cell epitopes already have indicated a
feasible strategy to produce immunogens for inducing
anti-HVR1 cross-reacting humoral immune responses.12-15
However, our data provide evidence that when T and B
epitopes coexist within the same antigenic sequence,
mimics of T-cell epitopes can be obtained by selecting for
B-cell epitopes. We found that mimotopes developed to
mimic HVR1 B-cell epitopes also mimic HVR1-helper
T-cell epitopes. It is widely accepted that the more diverse
the clonal immune response in an infected individual, the
fewer possibilities for viral escape through variation of the
epitope sequence. Hence, HVR1 mimics that increase the
probability of multiple viral variants being recognized by
both antibodies and CD4⫹ T cells would appear to be an
important goal.
We have identified a degenerate consensus representative of HVR1 residues that is more frequently recognized
not only by B-cell receptors,12 but also by TCRs. This
latter conclusion was indicated by our data on PBMCs of
infected patients, shown to react with our synthetic
epitopes in primary proliferation assays with a frequency
of 65% (Table 4). Because we have shown previously that
the frequency of anti-HVR1 T-cell responses is significantly higher in patients who recovered after interferon
alfa therapy (45%) than in those who did not (15%),7 we
can claim that mimotopes are recognized with a higher
frequency than natural variants. Moreover, the evidence
that mimotopes were recognized and cross-reacted with
Abs and T cells from subjects probably infected with different HCV quasispecies supports the hypothesis that
they can behave as antigenic mimics of HVR1 determinants generated in the natural course of infection.
Table 2 shows how we obtained suitable helper T-cell
epitopes by selecting B-cell epitopes, by a visual comparison of the C-terminal sequences of the mimotopes.
Amino acidic positions 16, 19, 20, 23, and 26 are constant in these peptides, while positions 14, 15, 17, 18, 21,
22, 24, and 25 vary only slightly. The amino acids 18, 21,
22, and 24 in the C-terminal part of HVR1 mimotopes
have been identified previously as crucial for antibody
binding both in humans and animals,12,14 whereas posi-
HEPATOLOGY, Vol. 38, No. 3, 2003
FRASCA ET AL.
661
Fig 5. Cross-reactivity analysis of
the T-cell lines elicited with HCV natural variants 295 and 296 (FI 295,
A296/2, and A296/3) or mimotope
877 (FI 877 and A877/3) and 440
(GA440). Proliferative responses,
measured as [3H]-thymidine incorporation, were assessed by incubation of 2 ⫻ 104 T cells with 4 ⫻ 104
autologous B-cell lines pulsed with
30 ␮g/mL of each MAP for 72
hours. Horizontal lines define the
cpm corresponding to 3 times the
control values.
tions 16, 19, and 21 could represent the P1, P4, and P6
anchor motifs for binding to the most common DR alleles19-21 as previously indicated.7,9 This means that 3 residues (21, 22, and 24) may be involved in both T- and
B-cell activation. This hypothesis is further corroborated
by T-cell reactivity to mimotopes 876 and 455 being
profoundly modified by changing aa 22 and 24 (Fig. 2).
The amino acids phenylalanine, glycine, and glutamine at
positions 20, 23, and 26, respectively, may correspond to
the solvent-exposed residues P5, P8, and P11 of peptide/
major histocompatibility complex complex, important
for the TCR binding process.22 Because in each of our
Ab-selected mimotopes these 3 residues as well as the
major putative major histocompatibility complex anchors
are constant, we may have identified a sequence that is
representative of the minimal requirements for activating
HVR1-specific T cells, and that favors their cross-reactivity.
These mimotopes express a level of cross-reactivity
never observed in natural HVR1 variants.7 Indeed, mimotope-induced CD4⫹ T cells recognized between 71%
and 86% of 27 HVR1 variants extracted from natural
virus isolates, whereas the percentage of cross-recognition
of lines primed with natural variants was at the most 33%.
Of particular interest is how this broad specificity is
achieved with mimotope 877. A comparison between the
number of HVR1 sequences recognized by 877-specific
lines (FI877 and A877/3) (Fig. 5) and the number of
clonal expansions present in their TCR repertoire (Fig. 4),
reveals that the cross-reactive nature of each specific TCR
is not increased by using this peptide as immunogen. In-
stead, this broad specificity is probably due to the expansion of a larger panel of specific clones. This suggests that
(at least) mimotope 877 can amplify a more complex
TCR repertoire than that elicited by HVR1 natural variants. This is an important issue because reagents to amplify helper T-cell responses are required for the
development of vaccines against highly mutant pathogens, such as HCV.23 Because this phenomenon occurred
not only in healthy but also in infected patients, mimotopes could be used for both prophylactic and therapeutic
vaccines. Evidence of the complexity of T-cell repertoire
evoked from primary T cells moreover suggests that priming CD4⫹ T cells to mimotopes can result in priming to
several virus variants.
One possible consequence of the broader specificity of
immune recognition due to the heterogenicity of the mimotope-induced T-cell repertoire could be that multiple
HVR1 variants are less likely to function as TCR antagonists for mimotope-induced T cells. This hypothesis derives from our evidence that identifying natural variants
that act as antagonist peptides was more frequent when
antagonistic assays were performed using low cross-reactive HVR1-specific T-cell repertoires. This frequency
dropped when highly cross-reactive and multispecific repertoires, such as those induced by mimotopes, were used.
According to one in vivo model,24 abolishing activation of
helper T cells by TCR antagonist peptides also may abolish T-cell help to B cells specific to the same or closely
related variants. We suggest that opportune combinations
of the mimotopes described here may be considered to
fight these immune evasion phenomena.
662
FRASCA ET AL.
HEPATOLOGY, September 2003
viding blood of HLA-typed normal subjects and Croce
Rossa Italiana for buffy coats. The authors thank Janet
Clench for critically reading the manuscript.
References
Fig. 6. Natural HVR1 variants exert different degrees of TCR antagonism on activation of CD4⫹ T cells primed with either HVR1 natural
variants or mimotopes. Autologous APCs prepulsed overnight at 37°C
with the agonist variant were plated out (4 ⫻ 104 cell/well) and HVR1
variants were added at increasing concentrations 5 hours before adding
1.5 ⫻ 104 responding cells. Mimotope-induced T-cell line 877/3 was
pulsed either with the (B) cross-recognized variant 296 or with (A) 877,
whereas natural variant–induced T-cell lines (C and E) A296/2 and (D
and F) A296/3 were pulsed with 296. The added natural variants,
chosen as reported in the Results section, were either unable to stimulate
(A-D) mimotope- and natural variant–induced T-cell lines or (E and F)
stimulatory for the mimotope-primed T-cell line and not stimulatory for
natural variant–primed T cells. Data are reported as percent of inhibition
of proliferative response in the absence of antagonist.
There is strong evidence that HVR1 varies under selective pressure from the immune system,1,8,25 suggesting
that both cellular and humoral immune response directed
at this region could be used to select for escape mutants in
infected patients. This further indicates that these responses play an important role in reducing the initial viral
load. Harnessing this initial response could be an appropriate strategy to fight virus spread, and for this reason we
believe that vaccinating against a variable target such as
HVR1 is a rational approach.26 Mimotopes of this HCV
region could be effective in a vaccine because they would
pre-activate (or reinforce) a protective response that
would then lead to virus neutralization.
Acknowledgment: The authors are grateful to the Avis
(Bergamo) (Italian association of blood donors) for pro-
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A, et al. The outcome of acute hepatitis C predicted by the evolution of the
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3. Zibert A, Schreier E, Roggendorf M. Antibodies in human sera specific to
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4. Farci P, Shimoda A, Wong D, Cabezon T, De Gioannis D, Strazzera A,
Shimizu Y, et al. Prevention of hepatitis C infection in chimpanzees by
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