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Reemergence of Hepatitis C Virus after
8.5 Years in a Patient with Hypogammaglobulinemia: Evidence for an
Occult Viral Reservoir
The Journal of
Infectious Diseases
Sept 15, 2005;192:1088-1092
William M. Lee,1 Julie E. Polson,1 D. Spencer Carney,1 Bogachan Sahin,2
and Michael Gale, Jr.2
1Department of Internal Medicine, Division of Digestive and Liver
Diseases, and 2Department of Microbiology, University of Texas
Southwestern Medical Center, Dallas
The question of whether viruses persist after apparent clearance of
infection remains unanswered. Here, we describe a patient with
hypogammaglobulinemia whose acute hepatitis C virus (HCV) infection
appeared to resolve after receipt of interferon therapy, relapse
immediately, and then clear spontaneously-only to relapse after receipt
of corticosteroid therapy, and clear again, 8.5 years later. Sequencing
indicated that the viruses detected during each relapse were virtually
identical, with the hypervariable region 1 of E2 appearing to be
monoclonal, which is typical of patients with hypogammaglobulinemia.
Nonstructural 5A sequences exhibited quasispecies diversity initially
but, after 8.5 years, had become monoclonal. The prolonged period of
negativity for HCV RNA followed by relapse suggests that HCV may persist
in apparent sustained viral responders.
Discussion.
Humoral immunity appears to play little role in HCV clearance, possibly
because of immune escape by way of rapid mutation of the HVR1. Viral
clearance is associated with a broad and vigorous T cell response and is
likely influenced by intracellular antiviral defenses [8, 9]. Innate and
IFN-induced antiviral pathways may determine viral clearance by
interfering with viral replication and translation and by enhancing
cell-mediated immunity [10]. Examination of the response to HCV
infection in patients with hypogammaglobulinemia provides an opportunity
to study these responses.
Following an episode of severe active hepatitis (with remarkably high
aminotransferase levels), our patient had complete resolution of
infection after discontinuation of IFN therapy. Resolution-as evidenced
by long-term normalization of aminotransferase levels and clearance of
the virus for 8.5 years-then was followed by relapse with a nearly
identical viral species. The initial resolution of infection after a
striking disease flare appears to have resulted from strong
intracellular and/or cell-mediated immune processes that were
independent of the humoral immune response.
As was expected, when we sequenced multiple clones from the time of
initial relapse and from the time of the second relapse 8.5 years later,
we found virtually no evidence of heterogeneity in the HVR1 of the viral
envelope [3, 11]. This finding is consistent with previous evidence
indicating that HVR1 diversity results from the selection of escape
variants in response to humoral immune pressure but contrasts with the
level of quasispecies variation within NS5A at the time of the initial
relapse. Higher numbers of amino acid mutations in the ISDR, compared
with the previously described HCV genotype 1a viral sequence, correlate
with increased sensitivity of HCV to the intracellular antiviral
response and to IFN in general [4, 7]. The viral evolution to a single
NS5A sequence at the time of the second relapse could have resulted from
intracellular immune pressure and selection of a single persistent
variant. The documentation of viral mutation in response to this host
pressure in the absence of antibody argues that the immune selection of
viral quasispecies that are resistant to intracellular defenses and/or T
cell immunity may be responsible for viral persistence in this patient.
Acute hepatitis C responds to IFN, and successful viral clearance in
patients with hypogammaglobulinemia has been reported [12]; spontaneous
viral clearance without treatment is rare [13]. The role of IFN in our
patient's disease course is unclear. Our patient experienced an
immediate relapse after 5 months of IFN therapy, which was followed by
viral clearance for 8.5 years. The very-delayed relapse of infection
after >8 years may have resulted from transient immunosuppression due to
repeated corticosteroid use. However, other factors, including abnormal
immunity and cytokine signaling associated with hypogammaglobulinemia,
could also have effected this relapse pattern [14]. That the phenomenon
did not represent reinfection was proved by the fact that only a single
amino acid difference in the NS5A region was found over the 8.5-year
period. We interpret the minimal shift in quasispecies diversity and the
repeatedly negative serum HCV RNA PCR assays to represent a low level of
viral replication during this long quiescent period. Although considered
to be a sustained viral responder, our patient continued to have a
reservoir of low-replicating virus that was held in check but not
eradicated by her immune system until the corticosteroid-induced immune
suppression led to the relapse. Recent studies in immunocompetent
patients support the presence of such a reservoir [15]. An HCV reservoir
that requires continued innate or T cell immune surveillance to prevent
disease activity even years after the infection appeared to have
resolved may exist in at least some sustained viral responders.
Patients with hypogammaglobulinemia who become infected with hepatitis C
virus (HCV) tend to have severe disease that may progress to cirrhosis
and liver failure after only a few years [1, 2]. Responses to interferon
(IFN) therapy have varied in these patients.
In patients with chronic HCV infection, the hypervariable region 1
(HVR1) of the viral E2 envelope glycoprotein exhibits a range of
quasispecies variation that is considered to be the result of host
humoral immune pressure that leads to viral adaptation and
antibody-escape variants. By contrast, the sequence diversity of the
nonstructural (NS) 5A protein-coding region has been shown to be
associated with antiviral pressure from IFN defenses of the infected
cell, such that quasispecies variation in the protein kinase R-binding
domain (PKR-BD) and the included IFN sensitivity determining region (ISDR)
of NS5A may influence intracellular defenses. These processes likely
play a role in viral persistence by facilitating immune evasion and
regulation. Studies of patients with hypogammaglobulinemia have
demonstrated little quasispecies variation in the HVR1 over periods of
up to 8 years of infection [3]. This lack of rapid sequence variation is
attributed to a lack of humoral immune selection pressure, but an effect
on the NS5A sequence of HCV in such patients has not been addressed.
Here, we describe the course of HCV infection in a patient with subclass
3 IgG deficiency who, in 1994, developed acute HCV infection and was
treated with IFN-2b
for 5 months. Immediately after treatment, the patient experienced a
brief, self-limited relapse. She then experienced a sustained virologic
remission that lasted for 8.5 years, only to experience another brief
relapse before the infection remitted again in 2003. We explore the
virologic features of this unique infection pattern and report on the
level of sequence heterogeneity within the HVR1 of the E2 protein and
the PKR-BD of the NS5A protein during the 2 separate relapses.
Case patient, materials, and methods.
A 23-year-old white woman with recurrent sinusitis and bronchitis/asthma
received a diagnosis of subclass 3 IgG immunodeficiency in late 1991 and
began receiving treatment with intravenous immunoglobulin (IVIG) at
2-week intervals (20-25 g each dose). The results of her liver-function
tests were entirely normal. In early March 1994, she began to develop
nonspecific symptoms of fatigue and anorexia around the time her
physician was notified of the contamination of units of Gammagard
(Baxter Healthcare) by HCV, and screening for infection was suggested.
Her aminotransferase levels were 217/347 (aspartate aminotransferase
[AST]/alanine aminotransferase [ALT]) IU/L, increasing to peak values of
1850/2020 IU/L 1 month later (figure 1A). A polymerase chain reaction (PCR)
assay for HCV RNA was initially positive, but then a quantitative PCR
assay was negative (Roche Amplicor RT PCR Assay [limit of detection,
<1000 copies/mL]). After 5 weeks, her aminotransferase levels had
decreased to 54/281 IU/L. Her total bilirubin level remained normal.
Because of a concern that her infection would persist and be aggressive
in this setting, treatment with IFN-2b
(Intron A; 3 million units 3 times/week; Schering-Plough) was initiated
on 25 April 1994. Her aminotransferase levels remained normal or near
normal throughout treatment, HCV RNA was undetectable on 1 occasion
during therapy, and the medication was discontinued at her request on 13
September 1994. Two days later, she developed fatigue, myalgias,
anorexia, nausea, and vomiting, and she was hospitalized 5 days after
discontinuation with aminotransferase levels of 8078/8531 IU/L (figure
1A). She was repeatedly positive for HCV RNA by PCR assays during this
period, with the titers decreasing in parallel with her aminotransferase
levels. Other causes of her high aminotransferase levels, such as
acetaminophen ingestion, were excluded. Her total bilirubin level peaked
at 4.2 mg/dL 3 days later, and her symptoms resolved rapidly, with her
aminotransferase levels returning to normal within 3 weeks without
further treatment. She continued IVIG treatment over the next decade
with no evidence of ongoing HCV infection, having consistently normal
aminotransferase levels and negative results of PCR assays documented in
1994, 1995, 1996, and 2000. However, in early March 2003, she complained
of symptoms that were "like my previous hepatitis." During the previous
6 months, she had been receiving intravenous methylprednisolone (125 mg)
with each dose of IVIG as well as intermittent prednisone therapy for
asthmatic episodes. On physical examination, there were no
abnormalities. Her aminotransferase levels were elevated (1200/1085 IU/L),
and she was again positive for HCV RNA (figure 1B); quantitative PCR
indicated an HCV RNA titer of 267,000 IU/L. The genotype of the
infecting virus was 1a. One week later, her HCV RNA titer had decreased
to 15,200 IU/L. All corticosteroid therapy was discontinued, and, over
the next 2 months, her HCV RNA titer again became undetectable without
further treatment. Her aminotransferase levels returned to normal, and
she has remained negative for HCV RNA during repeated testing (6
occasions) for 18 months. Liver biopsy was not performed.
Serum samples stored at -80C were available from the 1994 treatment
period and all later time points. HCV RNA was quantified in serum
samples from 1994 by use of the Roche Amplicor RT PCR Assay (limit of
detection, <1000 copies/mL). Negative assays were confirmed by use of
more-sensitive assays (limit of detection, <200 copies/mL) after 1996
and, most recently, by a Roche Amplicor assay (limit of detection, <50
IU/L).
For sequence analysis, HCV RNA was amplified by reverse-transcription
PCR (RT-PCR). Total RNA was isolated from 500
L
of the patient's serum by use of Trizol (Gibco), in accordance with the
manufacturer's protocol. RNA products were precipitated in ethanol and
were dissolved in 10 L
of 0.1% diethylpyrocarbonate-treated H2O containing 1.0 U of RNAse
inhibitor/L
of suspension (Gibco) in a final concentration of 1 mmol/L
dithiothreitol. Resuspended RNA was stored at -80C until use. Ten
microliters of RNA was used for RT in the presence of 4.5 pmol of mixed
hexamer primers and 200 U of Maloney murine leukemia virus reverse
transcriptase, and reaction mixtures were incubated for 1 h at 37C.
cDNA products were stored frozen until use. Nested PCRs were conducted
on 2 L
of the cDNA product with oligonucleotide primer pairs that targeted the
HVR1 of E2 or the PKR-BD and flanking variable region of NS5A [4]. For
the HVR1, primary PCR products were amplified as described elsewhere
[5], but with 50 pmol of the primer pair 5-ggtgctcactggggagtcctg-3
(sense primer encoding nt 1389-1409 of HCV genotype 1a) [6] and 5-cattgcagttcagggccgtgcta-3
(antisense primer encoding nt 1610-1633). The primer pair 5-tgacgtccatgctcactgat-3
(sense primer encoding nt 6854-6873) and 5-gctcggccaaggcagtagat-3
(antisense primer encoding nt 7360-7380) was used for primary
amplification of NS5A sequences. Primary PCRs were conducted with 30
rounds of amplification at an annealing temperature of 54C. Two
microliters of primary PCR product was used to program the nested PCRs
containing the HVR1 oligonucleotide primer pair 5-tccatggtggggaactggggc-3
(sense primer encoding nt 1428-1447) and 5-tgccaactgccgttggtgtt-3
(antisense primer encoding nt 1575-1604) or the NS5A oligonucleotide
primer pair 5-cctcccatataacagcagag-3
(sense primer encoding nt 6875-6894) and 5-gattcggtgaggaccaccgt-3
(antisense primer encoding nt 7336-7354). Nested PCRs were conducted
with 30 rounds of amplification at an annealing temperature of 52C.
All PCRs were conducted by use of the Advantage HF 2
proofreading-polymerase system (Clontech). The specificity of each PCR
was controlled by conducting parallel reactions with HCV genotype 1a
cDNA [6] and non-reverse-transcribed RNA products as positive and
negative controls, respectively. PCR products were analyzed by agarose
gel electrophoresis, were purified by gel extraction, and were cloned
into the pCR2.1 vector by use of the TA cloning system (Invitrogen).
Escherichia coli transformants that contained the cloned PCR products
were selected on amplification medium. Plasmid DNA was isolated from 10
different clones of HVR1 or NS5A sequences, which represented 5 PCRs
each. The nucleotide sequence of each clone was determined by use of
plasmid-encoded primer sequences and an ABI automated sequencer.
Nucleotide and deduced amino acid sequences were compared with the H77
HCV genotype 1a reference sequence [6] and analyzed by use of Vector NTI
software (InforMax).
Results.
Quasispecies diversity was assessed by analysis of 2 coding regions, the
HVR1 of E2 and the PKR-BD of NS5A, which includes the ISDR [5, 7]. Ten
independent clones, isolated by RT-PCR, were sequenced for each serum
sample. At the time of the patient's initial relapse in 1994, HVR1
sequences were identical among the clones and were separated into 2
quasispecies populations by virtue of a single nucleotide substitution
encoding a KM
mutation at HCV polyprotein amino acid position 370, which is outside
the HVR1. In contrast, we identified 5 NS5A quasispecies variants, each
harboring mutations within the PKR-BD and the ISDR, as well as 1
sequence with an additional mutation in a previously described variable
region, termed "V4," that flanks the PKR-BD [7]. Sequence analysis was
also performed for the virus associated with the patient's relapse in
2003, and these sequences were compared with those associated with her
previous infection. With the exception of a PQ
reversion at position 2235 in the NS5A coding region, the sequences of
the HVR1 and the NS5A coding region of 10 independent clones at this
time point were identical to the dominant sequences of the previous
isolates.
References
1. Rossi G, Tucci A, Cariani E, Ravaggi A, Rossini A, Radaeli E.
Outbreak of hepatitis C virus infection in patients with hematologic
disorders treated with intravenous immunoglobulins: different prognosis
according to the immune status. Blood 1997; 90:1309-14. First citation
in article | PubMed
2. Bjoro K, Froland SS, Yun Z, Samdal HH, Haaland T. Hepatitis C
infection in patients with primary hypogammaglobulinemia after treatment
with contaminated immune globulin. N Engl J Med 1994; 331:1607-11. First
citation in article | PubMed | CrossRef
3. Booth JC, Kumar U, Webster D, Monjardino J, Thomas HC.
Comparison of the rate of sequence variation in the hypervariable region
of E2/NS1 region of hepatitis C virus in normal and
hypogammaglobulinemic patients. Hepatology 1998; 27:223-7. First
citation in article | PubMed | CrossRef
4. Gale M Jr, Blakely CM, Kwieciszewski B, et al. Control of PKR
protein kinase by hepatitis C virus nonstructural 5A protein: molecular
mechanisms of kinase regulation. Mol Cell Biol 1998; 18:5208-18. First
citation in article | PubMed
5. Gretch DR, Polyak SJ, Wilson JJ, Carithers RL Jr, Perkins JD,
Corey L. Tracking hepatitis C virus quasispecies major and minor
variants in symptomatic and asymptomatic liver transplant recipients. J
Virol 1996; 70:7622-31. First citation in article | PubMed
6. Kolykhalov AA, Agapov EV, Blight KJ, Mihalik K, Feinstone SM,
Rice CM. Transmission of hepatitis C by intrahepatic inoculation with
transcribed RNA. Science 1997; 277:570-4. First citation in article |
PubMed | CrossRef
7. Enomoto N, Sakuma I, Asahina Y, et al. Mutations in the
nonstructural protein 5A gene and response to interferon in patients
with chronic hepatitis C virus 1b infection. N Engl J Med 1996;
334:77-81. First citation in article | PubMed | CrossRef
8. Chang KM. Immunopathogenesis of hepatitis C virus infection.
Clin Liver Dis 2003; 7:89-105. First citation in article | PubMed |
CrossRef
9. Pavio N, Lai MM. The hepatitis C virus persistence: how to evade
the immune system? J Biosci 2003; 28:287-304. First citation in article
| PubMed
10. De Francesco R. Molecular virology of the hepatitis C virus. J
Hepatol 1999; 31(Suppl 1):47-53. First citation in article | PubMed |
CrossRef
11. Li Y, Zhang T, Ho C, Orange JS, Douglas SD, Ho WZ. Natural
killer cells inhibit hepatitis C virus expression. J Leukoc Biol 2004;
76:1171-9. First citation in article | PubMed | CrossRef
12. Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV.
Determinants of viral clearance and persistence during acute hepatitis C
virus infection. J Exp Med 2001; 194:1395-406. First citation in article
| PubMed | CrossRef
13. Kumar U, Monjardino J, Thomas HC. Hypervariable region of
hepatitis C virus envelope glycoprotein (E2/NS1) in an
agammaglobulinemic patient. Gastroenterology 1994; 106:1072-5. First
citation in article | PubMed
14. Ma CS, Hare NJ, Nichols KE, et al. Impaired humoral immunity in
X-linked lymphoproliferative disease is associated with defective IL-10
production by CD4+ T cells. J Clin Invest 2005; 115:1049-59. First
citation in article | PubMed | CrossRef
15. Radkowski M, Gallegos-Orozco JF, Jablonska J, et al.
Persistence of hepatitis C virus in patients successfully treated for
chronic hepatitis C. Hepatology 2005; 41:106-14. First citation in
article | PubMed | CrossRef
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