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*
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037;
Medizinische Klinik II, Klinikum Grosshadern and Institute for Immunology, University of Munich, Munich, Germany; and
Epimmune Inc., San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 154 HCV-infected patients at Scripps Clinic in La
Jolla were screened with class I HLA serotyping analysis. All 154
patients were persistently infected by HCV as defined by the following
criteria: HCV Ab detected by a second generation Ortho HCV ELISA test
system (Ortho Diagnostics, Raritan, NJ), presence of HCV RNA by RT-PCR
(National Genetics Institute, Los Angeles, CA) or branched DNA assay
(Chiron, Emeryville, CA), elevated serum alanine aminotransferase
(sALT) activity for >6 mo, liver histology consistent with chronic
hepatitis C, and exclusion of other causes of chronic liver disease.
Twenty-seven patients were further selected based on their HLA type and
the availability of PBMC for CTL analysis. The HLA distribution was as
follows: HLA-A3 (n = 12); HLA-B7 (n =
8); and HLA-B35, (n = 8). We did not analyze other HLA
alleles (e.g., HLA-A31, -A33, -A68, -B51, -B53, -B55, -B56, and -B67)
because patients with those HLA alleles were not as common or readily
available. Patient C10 was included in both HLA-A3 subgroup as well as
HLA-B7 subgroup. None of the patients had other known causes of chronic
liver disease (e.g., chronic HBV, autoimmune hepatitis,
hemochromatosis) or HIV coinfection. Healthy blood donors (normal ALT,
no history of liver disease, hepatitis B surface Ag negative, and no
detectable Abs to HBV, HCV, HIV) were also included for each HLA
subtype (10 A3, 10 B7, 10 B35) as normal controls. HLA typing of PBMC
by complement-dependent microcytotoxicity was performed with Terasaki
HLA-typing trays (One Lambda, Canoga Park, CA) according to the
manufacturer’s instructions, as previously described (18). The
clinical and virological characteristics of the patients with chronic
hepatitis C are shown in Table I
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. The diagnosis of acute hepatitis C
was based on the following criteria: acute onset of disease (malaise,
jaundice); rise in sALT at least 20 times the upper limit of normal;
presence of HCV RNA at least in acute phase serum samples; de novo
seroconversion to HCV Abs; and absence of other causes of hepatitis.
Resolution of hepatitis was defined as loss of viral RNA and
normalization of serum transaminases within 6 mo of onset of acute
hepatitis. Chronic infection was defined as persistent viremia and
abnormal transaminases beyond 6 mo.
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, Patients A1, A2, A5, and A6 spontaneously
resolved their viremia, and their sALT activity returned to normal.
Patient A7 was viremic for 4 mo but cleared HCV RNA and resolved her
hepatitis immediately after IFN therapy. In contrast, patients A3 and
A4 became persistently infected. The clinical/virological outcome for
Patient A8 has not been determined due to the short follow-up interval
at this time (<6 mo). Peripheral blood mononuclear cells
PBMC from patients and normal donors were separated on Ficoll-Histopaque density gradient (Sigma, St. Louis, MO), washed three times in HBSS (Life Technologies, Grand Island, NY), and used for culture directly or cryopreserved in media containing 80% FCS (Life Technologies), 10% DMSO (Sigma), and 10% RPMI 1640 (Life Technologies).
Synthetic peptides
The amino acid sequences of the peptides (Table III) were derived from the published HCV
genome sequences containing known HLA-A3 or -B7 supertype-binding
motifs (20, 21). The peptides were synthesized at Cytel (San
Diego, CA) and purified to 95% homogeneity by reverse phase HPLC or
were purchased as crude material from Chiron Mimotopes (Clayton,
Australia) as previously described (20, 21, 24). Peptide aliquots were
dissolved in 100% DMSO at 4–20 mg/ml for binding. For use in cell
culture, peptides were reconstituted at 20 mg/ml in DMSO and further
diluted to 1 mg/ml with RPMI 1640.
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The HLA-binding affinity of peptides was determined by measuring
their ability to competitively inhibit the binding of a radiolabeled
standard probe peptide to purified detergent-solubilized class I MHC
molecules, as previously described (20, 21, 24). Briefly, purified
class I molecules were incubated for 2 days at room temperature with
varying concentrations of competitor peptides, 5–10 nM concentrations
of the labeled peptide, 1 µM human ß2-microglobulin
(Scripps Laboratories, San Diego, CA), and a mixture of protease
inhibitors. After incubation, class I peptide complexes were separated
from free peptide by size exclusion gel filtration chromatography on a
TSK2000 column (7.8 mm x 15 cm) (TosoHaas, Montgomeryville, PA).
The concentration of peptide yielding 50% inhibition of the binding of
the radiolabeled probe peptide was calculated. The HLA-binding
affinities of individual peptides to various HLA subtype molecules are
shown in Table III.
Stimulation of PBMC with synthetic peptides
Cryopreserved PBMC (rapidly thawed at 37°C and washed three times in cold HBSS) or freshly isolated PBMC were resuspended in RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 µg/ml), HEPES (10 mM) (Life Technologies), and 10% heat-inactivated human AB serum at 4 x 106 cells/ml. To expand peripheral blood CTL specific for the HCV-derived peptides, PBMC from the chronically infected patients studied in La Jolla were stimulated with the peptides in 24-well plates (10 µg of peptide/ml, 4 x 106 PBMC/ml/well) using a previously described macrowell technique (4, 18). PBMC from HLA-A3-positive patients and donors were stimulated with the panel of eight A3 peptides. PBMC from HLA-B7 or -B35-positive patients and donors were stimulated with the single B7 peptide. Cultures were restimulated on days 7 and 14 with autologous irradiated (3000 rads) PBMC (1 x 106 cells/well) and peptide (10 µg/ml), and on days 3, 10, and 18 with 20 U/ml human rIL-2 (Hoffmann-La Roche, Nutley, NJ) in fresh media as described previously (4, 15, 18).
The CTL responses of the acute and chronic HCV patients evaluated at the University of Munich were studied using the 96-well microwell technique, as we have previously described (12, 13). Briefly, PBMC were stimulated in replicate cultures with each peptide in 96-well U-bottom plates (10 µg of peptide/ml, 4 x 105 PBMC/200 µl/well), and restimulated on days 7 and 14 with autologous irradiated (3000 rad) PBMC (1 x 105 cells/well) and peptide (10 µg/ml). On days 3, 10, and 18, 20 U/ml human rIL-2 (Hoffmann-La Roche) were added.
Target cell lines
Allogeneic EBV-transformed B-lymphoblastoid cell lines expressing HLA-A3, -B7, or -B35 were either purchased from the American Society for Histocompatibility and Immunogenetics (Boston, MA) or established from our own pool of HLA-matched donors as previously described (25). HLA-A3-restricted CTL activity was analyzed using target cell line positive for HLA-A3, -A2, -B45, -B71 or cell lines positive for HLA-A3, -B7. Analysis of HLA-B7-restricted CTL activity was performed using cell lines positive for HLA-A2, -B7, or HLA-A3, -B7. HLA-B35-restricted CTL activity was analyzed using cell lines positive for A31, A33, and B35.
Cytotoxicity assay
Cultures were tested for peptide-specific cytolytic activity on day 21 in a standard 4-h 51Cr release assay using round bottom 96-well plates containing 3000 target cells/well. Allogeneic HLA-matched EBV-transformed B cell lines (described above) were pulsed overnight with peptide (10 µg/ml) labeled for 1 h with 51Cr (0.2 mCi), and used as target cells in these assays, as previously described (4, 13, 18). Percent cytotoxicity was calculated using the formula: 100 x [(experimental release - spontaneous release)/(maximum release - spontaneous release)]. Maximum release was determined with 10% Triton X-100 (Mallinckrodt, Paris, KY). Spontaneous release was always <30%. The cutoff value for a positive response was determined as 12%, which was more than 3 SDs above the mean cytotoxicity detected for each peptide in the uninfected donors. To compare the strength of the CTL response in patients with chronic or resolved hepatitis C, the CTL response index for each peptide (CRI-P) was calculated by totaling % cytotoxicities for all 8 replicate wells using the 96-well microwell cultures, as previously described (12). CRI-P values greater than or equal to the 3 SD + mean CRI-P observed in normal uninfected donors was considered positive response. For the panel of eight A3 peptides, individual CRI-P values were added and expressed as total CTL response index to reflect the overall strength of the CTL response to these peptides, as previously described (12).
Anti-CD4 and anti-CD8 blocking assay
The contribution of CD4 and CD8 T cells to the CTL activity was determined by incubating effector cells with anti-CD8 or anti-CD4 Ab as previously described (12). Briefly, the effectors were preincubated with 10 µg/ml anti-CD4 or anti-CD8 (Becton Dickinson, San Jose, CA) for 1 h at 4°C before addition of target cells, and tested in duplicates against the peptide-pulsed, 51Cr-labeled targets in the standard 4-h CTL assay as described above.
Statistical analysis
Nonparametric Wilcoxon two-sample rank test was used to compare the sALT activities or the number of HLA-A3 epitope peptide recognized in patients receiving IFN and in patients not receiving IFN.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As shown in Table IV, the frequency
of HLA-A2, -A3, and -B7 supertype alleles present in the 158
chronic HCV patients (154 from La Jolla, 4 from Munich) is comparable
with the known frequencies of these alleles in the general population
(26, 27). The variations from the expected frequency (e.g., twofold
increase in the HLA-A3 and -B7 subtypes or twofold reduction in
HLA-A11, -A31, and -B51 among the patients) is consistent with the
frequency observed among Caucasians and most likely reflects the
prevalent ethnicity of the local patient population. These results
suggest that the presence of these class I alleles is not related to
persistent HCV infection. The relative frequency of A3 and B7
superfamily alleles in these patients as well as the general population
suggests that identification of HCV CTL epitopes restricted by these
supertypes will expand the pool of potential epitope vaccine recipients
to most of the world population.
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Potential HLA-A3 supertype-restricted CTL epitopes were identified
by scanning the predicted amino acid sequence of complete polyproteins
from 14 different HCV isolates for the presence of 9- and 10-mer
sequences containing the HLA-A3 supermotif main anchor specificity
(AILMVST in position 2 and R or K at the carboxy terminus). The
candidate sequences identified were also evaluated using a customized
algorithm, which takes into account the presence of positive or
deleterious secondary anchor residues (21). Algorithm-identified
sequences were then assessed for conservancy, and 27 sequences in which
100% of the residues were conserved in 75% or more of the 14 isolates
scanned were synthesized. When the 27 corresponding peptides were
tested for binding to HLA-A3 and -A11, the 2 most prevalent A3
supertype alleles, 15 peptides were identified that bound A3 and/or A11
with affinities of 500 nM or less. These 15 binders were next tested
for cross-reactivity to the other common A3 supertype alleles (A3101,
A3301, and A6801). Seven of the 15 peptides were found to bind at least
3 of the 5 A3 supertype alleles tested with 50% inhibitory
concentration (IC50) 
500 nM. In a separate analysis, it
was noted that an additional peptide (NS3 1267) carrying a G2-K9 motif
was also capable of binding three A3 supertype alleles. Thus, this
additional peptide was included in the study described herein.
Potential HLA-B7 supertype-restricted CTL epitopes were identified by
scanning the same 14 HCV isolates for the presence of 9- and 10-mer
sequences containing the broad B7 supertype motif (Pin position 2 and
AILMVFW or Y at the carboxy terminus) (20). After evaluation for
conservancy, as described above, 35 peptides were synthesized and
tested for binding to HLA-B0702, the most common B7 supertype allele.
Thirteen peptides bound B0702 with IC50 500 nM. These
peptides were then tested for binding to other common B7 supertype
alleles (B3501, B51, B5301, and B5401). One peptide, Core 169,
was capable of binding to three or more of the five B7 supertype
alleles tested. In summary, eight A3-supertype and one B7 supertype
candidate CTL epitopes were identified (Table III). Each of these
peptides are degenerate supertype binders and are derived from
conserved regions of the HCV genome.
CTL response to HCV-derived candidate A3 and B7 CTL epitope peptides in chronic HCV patients
To examine the immunogenicity of the 8 HCV-derived A3 supermotif
peptides, PBMC from 12 HLA-A3 positive individuals with chronic HCV
infection and 10 HLA-matched healthy uninfected blood donors were
cultured for 3 wk in vitro with the 8 A3 supermotif peptides using the
24-macrowell technique. Little to no CTL activity against these
peptides was observed among the 10 normal controls, as shown at the
bottom of Fig. 1. In contrast, CTL
responses to 1 or more of the A3 peptides were observed in 7 of 12
(58%) patients (mean, 1.5 peptides/patient; range, 0–5
peptides/patient). A positive CTL response (Fig. 1, 
) was observed
in 18 of 96 possible instances (19%). All peptides were immunogenic in
at least 1 patient (mean, 2 patients/peptide; range, 1–3
patients/peptide), and responses were observed in patients infected by
HCV subtype 1a as well as 1b, 2b, or 3a. Interestingly, the NS4 1863
peptide was recognized in 4 of 12 (33%) patients, whereas the NS4 1864
peptide was immunogenic in only a single patient despite similar
HLA-binding affinity and a single N-terminal amino acid difference. Two
HLA-A11-positive chronic HCV patients were also tested but did not show
CTL activity to the same panel of eight A3 HCV peptides (data not
shown).
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). Furthermore,
compared with the patients without ongoing IFN therapy (patients C1,
12, 3, 4, 6, 8, 10, and 11), samples obtained from patients with
concomitant IFN therapy (patients C2, 5, and 9) displayed CTL
responsiveness to more peptides (mean 3.3 peptides vs 0.9 peptides per
patient, p = 0.025) and lower sALT activity (43 U/L vs
122 U/L, p = 0.028). Since all patients with concurrent
IFN therapy exhibited low to normal sALT activity (Fig. 2, unfilled
diamonds), it is possible that IFN therapy may be responsible for the
apparent inverse relationship between CTL and sALT activity. Future
studies with additional patients are needed to establish the biological
and clinical relevance of this observation. Also, since the viremia
levels on the dates of CTL activity were not analyzed, we could not
determine the relationship between CTL responsiveness to these epitopes
and viral titer.
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. Using a cutoff of 12% as
described in Materials and Methods, a CTL response to the B7
peptide was detected in one (13%) of the eight B7-positive patients,
and in two (25%) of the eight B35-positive patients. The CTL activity
at varying E:T ratios is shown in Fig. 4.
The CTL activity could be blocked by anti-CD8 Ab but not
anti-CD4 Ab, consistent with the expectation that CTL activity was
mediated by CD8-positive T cells. The mean sALT activity was lower
among patients exhibiting B7 peptide-specific CTL response than in
patients without B7 peptide-specific CTL response (66 vs 101), although
this difference was not statistically significant.
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Next, we compared the CTL responses of patients with acute and
chronic hepatitis C from Munich, using the semiquantitative microwell
technique previously described (12). The CTL responses of group of
HLA-A3- and -B7-positive patients to the panel of A3 and B7 peptides
are shown in Figs. 5–7, expressed as
CRI-P described in Materials and Methods. CRI-P values >3
SD above the mean normal donor CTL response for each peptide are
indicated by hatched bars. The time points at which the patients were
analyzed are shown in Table II as months after onset of acute
hepatitis. Both patients with acute self-limited hepatitis C and
patients with chronic HCV infection responded to one or more peptides,
although the level of CTL activity was generally low. While there was a
tendency for the B7-restricted CTL response to be stronger and more
frequent in the patients who resolved the infection as shown in Fig. 6 and Table II, these differences were
relatively small and of questionable significance.
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, Patient A1 responded to six A3
peptides at 2 mo during which time she was viremic but displayed normal
sALT activity. In contrast, as the virus became undetectable by PCR,
the response decreased to only 3 A3 peptides at 4 mo and 2 A3 peptides
at 9 mo. In contrast, the response to B7 peptide (Core 169) gradually
increased over this observation period. Patient A2 showed similar
fluctuations although he was studied later, at 12 and 15 mo after the
onset of acute hepatitis and viral clearance.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we identify nine new HLA-restricted CTL epitopes, thereby expanding the known HCV CTL epitope repertoire that can contribute to the development of a broad spectrum CTL epitope vaccine relevant to the general population. Indeed, considering all members of the HLA-A2, -A3, and -B7 superfamilies, the current set of epitopes together with the previously described HLA-A2-restricted epitopes will cover >80% of the world population. Identification of multiple epitopes is of potential importance in development of immunotherapy of HCV infection since multispecific responses have been shown to correlate with a better clinical outcome (12). Similar observations have been made in the case of HIV infection (28, 29, 30, 31). Therapeutic augmentation of CTL response has already been reported in human malignancies such as melanoma (32, 33) and B cell lymphomas (34, 35). Furthermore, a synthetic peptide vaccine derived from the HLA-A2-restricted HBV core 18–27 CTL epitope was found to be immunogenic in HLA-A2-positive donors (36), and a Phase II trial of the same vaccine in HLA-A2-positive patients with chronic hepatitis B is currently underway. Our study also supports the usefulness of the HLA-binding supermotif and HLA-binding affinities to predict potential CTL epitopes, as has already been reported for HBV (22) and other conditions (2, 4, 37).
The HLA-A3-restricted CTL epitopes are derived from both structural and nonstructural regions of HCV and are relatively conserved (75–100% conservation). Focusing the immune response against highly conserved epitopes might be of particular importance in the case of the highly variable hepatitis C virus.
Each peptide was immunogenic in one or more patients studied (range,
8–33%; mean, 19%). The most frequently immunogenic epitope was NS4
1863 (GVAGALVAFK) which was recognized in 4 of 12 patients (33%).
Interestingly, the NS4 1864 peptide (VAGALVAFK), which lacks the
N-terminal glycine residue of NS4 1863, was recognized by only one
patient (patient C9), who also showed response to NS4 1863. It is
possible that the additional N-terminal G may influence the processing
and transport of the peptide since the HLA-binding affinity of the two
peptides is quite comparable. Alternatively, the N-terminal glycine may
interact with the TCR and the MHC-peptide complex. It is important to
note that, because they are highly conserved, the CTL responses to
these genotype 1a-derived peptides were observed in patients infected
with viral strains other than genotype 1a. For example, patient C5 who
was infected with HCV subtype 2b responded to Core 51, E1 290, and NS4
1863. This is consistent with high amino acid sequence conservation of
the peptides, as shown on Table III, and CTL cross-reactivity between
the viral subtypes as we previously reported (12, 18).
There was a correlation between the number of the A3 epitopes recognized and sALT activity among the HLA-A3-positive chronic HCV patients, compatible with a potential curative role of the CTL response as described in hepatitis B (38, 39). A similar inverse relationship between CTL responsiveness and sALT activity has been previously demonstrated in chronic HCV patients using a panel of 10 HLA-A2-restricted HCV CTL epitope peptides (12) but not in another study looking at the intrahepatic CTL (40). However, the patients with low to normal sALT activity and with greater CTL epitope reactivity were also undergoing IFN therapy, which could enhance CTL activity due to increased class I HLA expression. It is also possible that the HCV-specific CTL no longer home to the liver and are more frequently detected in circulation as hepatitis and viral load diminish during IFN therapy. However, we could not test this hypothesis, since we did not study the intrahepatic HCV-specific CTL response or the corresponding viral titers in these patients.
Among HLA-A3-positive patients with recent acute hepatitis C, neither
the magnitude of the CTL response nor the total number of epitopes
recognized correlated with the outcome of their infection. On the other
hand, patients with spontaneous HCV clearance produced a stronger CTL
response to the HLA-B7 peptide than those with chronic hepatitis,
suggesting that the CTL response to this peptide may be particularly
important in viral clearance and disease resolution. Interestingly, as
shown in Fig. 6, CTL responsiveness to the B7 peptide (Core 169)
appeared to increase over time in the two patients who clear HCV
(patients A6, A9) while CTL responsiveness to the 8 A3 peptides became
weaker and more narrowly focused over time. However, given the low
number of patients with acute hepatitis C and generally weak CTL
response, the significance of these observations is uncertain at this
time.
In conclusion, using a motif search and HLA-binding affinity protocol, we have identified nine new HLA-A3 supertype-restricted and HLA-B7 supertype-restricted CTL epitopes in structural and nonstructural proteins that are recognized by both acute and chronic HCV patients. We also demonstrated a provocative inverse relationship between sALT activity and the number of CTL epitopes recognized by these patients, compatible either with the notion that the CTLs exert a protective effect in HCV infection or with a pleiotropic effect of IFN therapy. While this very interesting observation deserves further investigation, collectively, the current results demonstrate the feasibility of developing a broad spectrum therapeutic CTL vaccine for the treatment of chronic HCV infection that should cover most infected patients, irrespective of genotype or ethnicity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Francis V. Chisari, SBR-10, Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.
3 Abbreviations used in this paper: HCV, hepatitis C virus; sALT, serum alanine aminotransferase; HBV, hepatitis B virus; CRI-P, CTL response index for peptide; IC50, 50% inhibitory concentration.
Received for publication June 16, 1998. Accepted for publication September 24, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
negative responses. The total number of peptides recognized in each
patient is shown on the right column marked # CTL responses. The number
of patients responding to each peptide is shown at the bottom.
, data points from
patients receiving IFN therapy; and 
, data points from patients not
receiving IFN therapy. *, Patients C3 and C7 were excluded from the
analysis for lack of sALT measurement or because the PBMC were pooled
from two different dates with varying sALT activities,
respectively.
, Positive responses in
patients; 
