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The Blood Research Institute, The Blood Center, Milwaukee, WI 53201
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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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Extensive structural and functional studies on the HCV nonstructural 3
protein (NS3) have revealed that NS3 is a protein of 
70 kDa in size
and possesses three known catalytic activities consisting of a serine
protease in the first 180 amino acids followed by a nucleotide
triphosphatase-dependent RNA helicase in the C-terminal region
(11, 12, 13, 14, 15). Therefore, NS3 is critical to HCV infection and replication.
Immune responses to HCV are poorly understood. Despite the fact that strong Ab responses are observed in most patients, up to 90% of Ab-positive patients are persistently viremic. Studies on cellular immunity to HCV demonstrate that specific CD4 (helper) and CD8 (cytotoxic) T cells can be found in peripheral blood or liver from infected individuals (16, 17, 18). Why such T cells are ineffective at eliminating HCV in vivo is unknown. One hypothesis is that escape mutants arise due to hypermutability in the HCV RNA genome (19). A similar mechanism has been proposed to explain persistence of human immunodeficiency virus and hepatitis B virus (20).
We have seen that certain epitopes of HCV NS3, identified using overlapping synthetic peptides, caused secretion of IL-2, but not IL-10, and others that stimulated IL-10 release but not IL-2 (21). We have also observed extensive sequence variation in the HCV NS3 region throughout the infective course of a chronic HCV patient, and most nonsynonymous mutations were found to be clustered within specific regions of NS3. All such variable regions occurred in epitopes recognized by host T cells.5 Because it is thought that escape variants may arise in response to immune selection, we wanted to determine whether T cell epitopes that stimulated release of different cytokines might be subject to different selective pressures and, if so, what the functional implications might be. In a patient infected over the course of 2 years, we examined sequence variations in one IL-2-producing epitope and one IL-10-producing epitope. Significant variations were observed in both epitopes, and functional studies revealed that synthetic peptides corresponding to these epitope variants either failed to stimulate or elicited weak T cell proliferation, suggesting that mutations in the HCV genome have the potential for generating escape variants with significant functional consequences for CD4 T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood samples were collected from a patient (P.B3019) with chronic HCV at about 12 (P.B3019.1), 22 (PB.3019.2), 28 (P.B3019.3), and 34 (P.B3019.4) mo after infection. The presence of HCV-specific Abs in the patient’s serum was determined by second and third generations ELISA testing using diagnostic kits provided by Abbott Laboratories (Irving, TX) and used by The Blood Center in its routine screening of prospective blood donors. Viremia was also evaluated using a previously published set of nested primers that amplify a conserved segment of the 5' noncoding region of the HCV RNA genome (22). Other than Ab and viremia, this patient has no other symptoms. Blood was collected in acid citrate dextrose anticoagulant, centrifuged at 400 x g for 15 min, and divided into plasma and buffy coat fractions. After isolation of PBMC over Lymphocyte Separation Medium (Organon Teknika, Durham, NC), plasma and PBMC were stored at -70°C or in liquid nitrogen, respectively.
T cell proliferation assays
PBMC from samples P.B3019.1, P.B3019.2, P.B3019.3, and P.B3019.4 were resuspended at a concentration of 1 x 106/ml in RPMI 1640 tissue culture medium containing 25 mM HEPES, 2.0 mM L-glutamine, 1.0 mM sodium-pyruvate, 10 U/ml sodium-heparin, 100 U/ml penicillin, 100 µg/ml streptomycin, 5.0 µg/ml gentamicin sulfate, and 10% pooled human plasma. To 96-well round-bottom plates, 100 µl aliquots of PBMC were added to Ags at varying dilutions also in 100 µl 10% pooled human plasma tissue culture medium. Medium without Ag was used as a negative control. Cultures were incubated at 37°C in 5% humidified CO2 for 6 days, pulsed overnight with 1.0 µCi/well [3H]TdR, and harvested onto glass fiber filters. Radioactive label incorporation was measured by gas scintillation spectroscopy by counting for 4 min; results are represented as the mean ± SEM of triplicate cultures.
Cytokine analysis
To measure secreted cytokines, PBMC from P.B3019.1 and P.B3019.3
were cultured in the presence of synthetic peptides as in the T cell
proliferation assays described above. Results from P.B3019.2 have been
published elsewhere (21). Supernatants were characterized in duplicate
for secreted IFN-
, IL-2, and IL-10 using a commercial Ag-capture
human IL-2 ELISA kit from Endogen (Boston, MA) and human IFN-
and IL-10 ELISA sets from PharMingen (San Diego, CA).
Experimental values were compared with a standard curve derived using
recombinant cytokines. Negative controls consisted of background levels
derived from cultures of PBMC in the presence of tissue culture medium
alone.
RNA extraction and synthesis of cDNA
HCV genomic RNA was isolated from 1 ml each of P.B3019.1 and P.B3019.3 sera using RNAZol B (Tel-Test, Friendswood, TX). cDNA was synthesized using reverse transcriptase from Moloney’s murine leukemia virus (Life Technologies, Gaithersburg, MD) and random hexadeoxynucleotide primers (Pharmacia Biotech, Piscataway, NJ).
Amplification of HCV cDNA by PCR
Specific primers were designed to amplify the HCV NS3 region based on previously reported HCV sequence information. Primers NS3-3A (5'-CGGACCTTTACCTGGTCACG-3') and NS3-2M (5'-CGCCCTCCCAAAATCCAAGATGG-3') were used for the first PCR amplification in which HCV cDNA was subjected to 35 cycles of amplification in a buffer with 10 mM Tris, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.02% NP40, and 5% DMSO. Each cycle consisted of 30 s at 94°C, 60 s at 55°C, and 60 s at 72°C with an initial denaturation (94°C, 5 min) and a final extension step (72°C, 7 min). Then, 2 µl of the products from the first PCR was used for the second PCR amplification with the same conditions using primers NS3-3C (5'-CAAGTTCCTTGCCGACGCCGG-3') and NS3-2M. The amplified fragments were 790 bp in length and encompassed nucleotides 813-1602 of the NS3 region.
Cloning and sequencing
PCR products were cloned using a T-vector cloning kit (Invitrogen, San Diego, CA). Ligation and transformation were performed essentially according to the manufacturer’s instructions. Recombinant clones were screened and selected by specific PCR amplification of the inserted fragment. Plasmid DNA was prepared from the selected clones with a Wizard Plus miniprep DNA purification kit (Promega, Madison, WI) according to the standard protocol. Fifteen independent clones for each sample of P.B3019.1 and P.B3019.3 were sequenced in both directions by dye-termination cycle sequencing using an automated DNA sequencer (373A, Applied Biosystems, Foster City, CA). The sequencing results were analyzed using the ABI Gene Works software (IntelliGenetics, Mountain View, CA). Plasmid DNA from the Hutchinsion strain (1a) of HCV (23) was diluted to 10-14 g/ml then amplified, cloned, and sequenced as a control for polymerase errors. This concentration of plasmid DNA produced PCR amplification bands with intensities comparable to those obtained from viral cDNA under essentially identical conditions. The Taq error rate under such conditions was calculated as [no. of sporadic changes ÷ (no. of clones x sequence length x PCR cycles)] according to Smith et al. (24).
Peptide synthesis
Peptides with variant sequences of NS3358–375 and
NS3505–521 were synthesized locally using F-moc chemistry
and purified by HPLC. Peptide powder was dissolved in a drop of DMSO
and adjusted to 1 mg/ml with RPMI 1640 tissue culture medium.
Peptide was used at indicated concentration to stimulate PBMC in T cell
proliferation or cytokine assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous studies have reported that two synthetic peptides,
NS3358–375 and NS3505–521, respectively
corresponding to residues 358–375 and 505–521 of HCV strain 1a NS3,
could be preferentially recognized by peripheral blood T cells from
donor P.B3019 (21). To know if there were any differences of T cell
recognition between early and later stage of infection, we analyzed T
cell proliferative responses to NS3358–375 and
NS3505–521 at about 12 (P.B3019.1), 22 (PB.3019.2), 28
(P.B3019.3), and 34 (P.B3019.4) mo after infection with HCV.
Proliferative results are shown in Fig. 1
. Peptides NS3358–375
stimulated strong dose-dependent proliferation of T cells from
P.B3019.1 to P.B3019.4, while the response to peptide
NS3505–521 was definite but comparably weak over the
period from which these results were obtained. In contrast, these
peptides did not stimulate proliferation of cells from the normal
individual P1115.
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To determine whether any differences in cytokine secretion existed
in the early or later stages of infection, we analyzed IFN-, IL-2,
and IL-10 secreted in response to the same peptides above, with B3019.1
and B3019.3 by cytokine-specific ELISA. The results are shown in
Fig. 2. Consistent with our previous
results (21), NS3358–375 could stimulate strong
IL-2 and IFN-, but not significant IL-10. In contrast,
NS3505–521 could stimulate neither significant IL-2 nor
IFN-, while it could stimulate IL-10 secretion by PBMC from
P.B3019.3.
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To determine whether mutations were accumulating in HCV sequences in response to immune selection and to understand their impact on immune recognition, cloning and sequencing analysis of PCR products from specific epitope regions of NS3 were performed using cDNA templates synthesized from RNA isolates of P.B3019.1 and P.B3019.3.
Of 30 sequenced clones, 11 (37%) had nucleotide mutations in
NS3358–375 producing 9 amino acid changes, and 5 (17%)
had mutations in NS3505–521 causing 4 residue
substitutions (Table I).
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The amino acid changes in the regions encoding NS3358–375
and NS3505–521 as well as their flanking regions are shown
in Fig. 3. More mutations were observed
in epitope NS3358–375 throughout the whole infective
course. In the early bleed P.B3019.1, there were both synonymous and
nonsynonymous mutations and the ratio of synonymous/nonsynonymous was
4/6, whereas in the later bleed P.B3019.3, all mutations were
nonsynonymous (5/5). In contrast, for the IL-10-inducing epitope
NS3505–521, fewer mutations were observed and the majority
occurred in the earlier isolate (Fig. 3 and Table I
). Thus, for the
IL-2-producing epitope NS3358–375, immune selection would
seem to be stronger and persists longer than that for the
IL-10-producing epitope NS3505–521 for which selective
pressure may weaken with time.
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To distinguish actual viral mutations from possible sporadic nucleotide
substitutions caused by Taq misincorporation errors, we
calculated the error rate using a known plasmid template (0.01 pg) from
which amplified bands were comparable to those obtained from viral
cDNA. Even with 70 cycles of amplification, we calculated a polymerase
error rate of 2.16 x 10-5, which was well within the
usual Taq error range of 0.2–2 x 10-4
reported by others (25, 26). Thus, we discount the possibility of
underestimating the number of artifacts due to saturation reached after
only a few PCR cycles. The expected numbers of sporadic amino acid
substitutions were calculated and compared with the actually observed
nonsynonymous mutations in both peptide regions and flanking regions as
shown in Table II and Fig. 3. In both
cases, those regions encoding epitopes NS3358–375 and
NS3505–521 contained significantly increased numbers
of nonsynonymous mutations.
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According to the variant sequences shown in Fig. 3, peptides were
synthesized and used to stimulate T cells in proliferation analysis. Of
the seven NS3358–375 variants tested, three, H369R, S370P,
and K371E, showed dramatically decreased T cell proliferation, and the
K373R variant caused a threefold shift in the dose-response curve, but
the level of response was comparable to wild-type,
NS3358–375, at high peptide concentrations (Fig. 4a). Of the three
NS3505–521 variants tested, L513P stimulated no T cell
proliferation, while the response to V511A was weak (Fig. 4b). Taken together, these results indicate that residues
369, 370, 371, and 513 play critical roles in either MHC binding or
contact with the T cell antigenic receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our present study, we have examined mutations in a region of
the NS3 Ag that contains two functionally distinct epitopes,
NS3358–375 and NS3505–521. We have shown that
NS3358–375 preferentially stimulates IL-2 and IFN-,
whereas NS3505–521 stimulates IL-10 production. Coincident
with this functional difference, the localized mutation rate in these
two NS3 epitopes is very different. The region corresponding to
NS3358–375 underwent a consistently higher
frequency of changes in both the early and later isolates and most of
the mutations produced amino acid changes. In contrast, nonsynonymous
changes in the nucleotides encoding NS3505–521 were
frequent in the early isolate, but fewer were seen in the clones from
the later sample. This observation is consistent with the possibility
that NS3358–375 and NS3505–521 are under
different intensities of selection pressure as exerted by CD4 T cells.
We cannot at this time rule out an effect by CD8 T cells. We raise the
question as to whether it is merely coincidental that selection
intensity, as measured by mutational frequencies, correlated with the
functional nature of the epitope, which presumes that stimulation of
IL-2 and IFN- is more conducive to viral elimination (29).
It is important to distinguish viral mutations arising from
immune selection from those that may be artifactual (24, 30, 31). We
observed in two regions, one encompassing the functionally important
DExH box and Switch region and another covering the RNA unwinding
region of the HCV helicase, that the observed nonsynonymous mutation
frequency was lower than that expected from sporadic amino acid
substitutions, supporting a negative selection model in these
functionally critical regions. In contrast, for the two variable
regions containing NS3358–375 and NS3505–521,
which are located neither near nor in the known functionally
important portions of HCV helicase, the observed nonsynonymous mutation
rate was significantly higher than expected, supporting a positive or
over-dominant selection model for mutations in these epitopes (Table II
and Fig. 3). Taken together, our observations support the hypothesis
that nonsynonymous mutations in the regions encoding peptides
NS3358–375 and NS3505–521 represent natural
changes to the HCV RNA genome rather than artifactual PCR errors.
We were able to examine whether nonsynonymous mutations in the NS3358–375 and NS3505–521 epitopes affect CD4 T cell recognition. Clearly, a large minority of the amino acids can be considered loss-of-recognition mutations as seen in our studies with the synthetic peptide variants. Loss of recognition could occur through decreased binding to MHC molecules or through disruption of critical TCR contact sites. A third possibility includes a shift in cytokine production such that the ability to stimulate IL-2 was lost while maintaining the ability to stimulate production of other cytokines, which is currently under investigation. Obviously, none of these is mutually exclusive. Furthermore, we would predict that after having elucidated which of the preceding mechanisms is operative, we should see emergence of viral isolates that would produce similar functional effects as we continue with longitudinal studies in this patient. While we did see an attenuation in the response to NS3505–521 over time, perhaps reflective of a developing anergic state, we observed a more intense response to wild-type NS3358–375 in PBMC derived later in the infection. The fact that we see such a vigorous recall response to NS3358–375 wild-type sequence probably reflects intense selection on this epitope, and thus we would expect to see that escape mutants should continue to emerge with time.
In addition to loss of recognition, mechanisms by which the T cell anti-viral response can be suppressed would include production of inhibitory cytokines. As an example, IL-10 is known to down-regulate essential costimulatory molecules such as CD80 on the surfaces of APCs with the result that transmembrane signaling via CD28 is impaired, which in turn leads to a failure to stabilize the IL-2 message induced by TCR ligation (32). Thus, viral mutation of the NS3505–521 epitope would not be driven as intensively due to stimulation of IL-10 by this and other epitopes and perhaps would abate with time. Inconsistent with this hypothesis is the fact that IL-2 production in response to NS3358–375 is virtually shut down when the epitope is presented in the context of the intact NS3 Ag (33). Until we know whether the cytokine dysregulation that we observe in vitro differs from that which occurs in vivo, we cannot distinguish between these two alternatives.
The production of IL-10 is not a unique feature of HCV. EBV, for example, encodes an IL-10 homologue (34). Small RNA viruses like HCV lack the genomic capacity to carry their own immunomodulatory cytokines, but perhaps they have found ways to exploit this niche within the host environment by tolerating high mutational loads that allow IL-10-stimulating epitopes to evolve through extended host-viral interactions. In this regard, it is interesting to note that NS3358–375 and NS3505–521 are not localized within any known functional region of the NS3 helicase and thus may represent "expendable" regions of the Ag that can be allowed to mutate into forms that elicit cytokines conducive to virus survival. The fact that the NS3358–375 region is relatively conserved among diverse HCV and hepatitis G virus species, yet differs within a given patient, may imply that the virus has evolved a means by which to ensure this capacity.
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Acknowledgments |
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Footnotes |
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2 The nucleotide sequence data reported in this paper can be found in the DDBJ, EMBL, and GenBank nucleotide sequence databases with accession numbers AF035122 through AF035150.
3 Address correspondence and reprint requests to Dr. David D. Eckels, The Blood Research Institute, The Blood Center, P.O. Box 2178, Milwaukee, WI 53201-2178. E-mail address:
4 Abbreviations used in this paper: HCV, hepatitis C virus; NS, nonstructural.
5 H. Wang, T. H. Bian, and D. D. Eckels. Sequence heterogeneity in the nonstructural 3(NS3) gene of the hepatitis C virus genome: evidence for positive and negative selection. Submitted for publication.
Received for publication September 21, 1998. Accepted for publication January 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene. Proc. Natl. Acad. Sci. USA 84:4254.-2a therapy in cryoglobulinemia associated with hepatitis C virus. N. Eng. J. Med. 330:751. therapy: relationship to genotypes of hepatitis C virus. Hepatology 16:293.[Medline]
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