Cellular immune responses to HCV core increase and
HCV RNA levels decrease during successful
Janine Rohrbach,1Nicola Robinson,2Gillian Harcourt,2Emma Hammond,3
Silvana Gaudieri,3,4Meri Gorgievski,5Amalio Telenti,6Olivia Keiser,7
Huldrych F Gu ¨nthard,8Bernhard Hirschel,9Matthias Hoffmann,10Enos Bernasconi,11
Manuel Battegay,12Hansjakob Furrer,1Paul Klenerman,2Andri Rauch,1,3
the Swiss HIV Cohort Study
Background Hepatitis C virus (HCV) infection is a major
cause of morbidity in HIV infected individuals. Coinfection
with HIV is associated with diminished HCV-specific
immune responses and higher HCV RNA levels.
Aims To investigate whether long-term combination
antiretroviral therapy (cART) restores HCV-specific T cell
responses and improves the control of HCV replication.
Methods T cell responses were evaluated longitudinally
in 80 HIV/HCV coinfected individuals by ex vivo
interferon-g-ELISpot responses to HCV core peptides,
that predominantly stimulate CD4+T cells. HCV RNA
levels were assessed by real-time PCR in 114 individuals.
Results The proportion of individuals with detectable T
cell responses to HCV core peptides was 19% before
starting cART, 24% in the first year on cART and
increased significantly to 45% and 49% after 33 and
70 months on cART (p¼0.001). HCV-specific immune
responses increased in individuals with chronic (+31%)
and spontaneously cleared HCV infection (+30%).
Median HCV RNA levels before starting cART were 6.5
log10IU/ml. During long-term cART, median HCV-RNA
levels slightly decreased compared to pre-cART levels
(?0.3 log10 IU/ml, p¼0.02).
Conclusions Successful cART is associated with
increasing cellular immune responses to HCV core
peptides and with a slight long-term decrease in HCV
RNA levels. These findings are in line with the favourable
clinical effects of cART on the natural history of hepatitis
C and with the current recommendation to start cART
earlier in HCV/HIV coinfected individuals.
Hepatitis C is a major cause of morbidity and death
in HIV infected individuals.1In the Swiss HIV
Cohort Study (SHCS), 33% of HIV infected indi-
viduals are coinfected with the hepatitis C virus
(HCV).2Coinfection with HIV accelerates the
progression to liver cirrhosis3and is associated
with higher HCV RNA levels,4 5particularly in
individuals with low CD4+Tcell counts.6Cellular
immune responses, crucial for the control of HCV
infection,7are severely diminished in HIV infected
individuals; HCV-specific CD8+and CD4+T cell
responses are weak in chronic hepatitis C and are
further impaired in HIV coinfected individuals.8e10
The loss of cellular immune responses to HCV is
particularly evident in individuals with low CD4+
Tcell counts.11 12
reduces liver-related mortality in HIV/HCV coin-
fected individuals.13 14Potential favourable effects
of cART on the course of hepatitis C include
a reduction in immune activation and an increase in
cellular immune responses. It is unclear to what
extent a successful cART restores HCV-specific
immune responses. Previous studies suggested that
HCV RNA levels increase in the first 3e6 months
Significance of this study
What is already known about this subject?
< Coinfection with HIV accelerates the progression
of liver fibrosis in individuals with chronic
< Coinfection with HIV is associated with dimin-
ished hepatitis C specific T cell responses and
with higher hepatitis C virus (HCV) RNA levels.
What are the new findings?
< Cellular immune responses to HCV core peptides
increase significantly during successful combi-
nation antiretroviral therapy (cART).
< Immune responses to HCV core peptides during
long term cART are comparable to those in HCV
< HCV RNA levels slightly decrease long term in
individuals with immune recovery through
a successful cART.
How might it impact on clinical practice in the
< cART can improve the immunological and viral
control of HCV infection.
< This finding is in line with the current recom-
mendation to start cART earlier in HIV/HCV
coinfected individuals to reduce liver-related
morbidity in HIV/HCV coinfected individuals.
< An earlier start of cART might also improve the
response to HCV therapy, as lower HCV RNA
levels are an important predictor of a sustained
See commentary, p 1167
<Additional figures and tables
are published online only. To
view these files please visit the
journal online (http://gut.bmj.
For numbered affiliations see
end of article.
Andri Rauch, Klinik und Poliklinik
fu ¨r Infektiologie, University
Hospital Berne and University of
Berne, Inselspital PKT2B, 3010
The members of the Swiss HIV
Cohort Study are: M Battegay,
E Bernasconi, J Bo ¨ni,
HC Bucher, Ph Bu ¨rgisser,
A Calmy, S Cattacin,
M Cavassini, R Dubs, M Egger,
L Elzi, P Erb, M Fischer, M Flepp,
A Fontana, P Francioli (President
of the SHCS, Centre Hospitalier
Universitaire Vaudois, CH-1011,
Lausanne), H Furrer (Chairman
of the Clinical and Laboratory
Committee), C Fux,
M Gorgievski, H Gu ¨nthard
(Chairman of the Scientific
Board), H Hirsch, B Hirschel,
I Ho ¨sli, Ch Kahlert, L Kaiser,
U Karrer, C Kind, Th Klimkait,
B Ledergerber, G Martinetti,
B Martinez, N Mu ¨ller, D Nadal,
M Opravil, F Paccaud,
G Pantaleo, A Rauch,
S Regenass, M Rickenbach
(Head of Data Center), C Rudin
(Chairman of the Mother & Child
Substudy), P Schmid,
D Schultze, J Schu ¨pbach,
R Speck, P Taffe ´, P Tarr,
A Telenti, A Trkola, P Vernazza,
R Weber, S Yerly.
Revised 31 March 2010
Accepted 13 April 2010
Published Online First
26 July 2010
1252 Gut 2010;59:1252e1258. doi:10.1136/gut.2009.205971
group.bmj.com on August 26, 2010 - Published by gut.bmj.comDownloaded from
after the start of cART.15 16However, it is unclear whether
immune restoration through cART improves the control of HCV
replication long term.17
In this study, we longitudinally investigated the impact of
HIV and of a successful cART on HCV-specific T cell responses
and on HCV RNA levels.
Participants and study design
Study participants were included from the Swiss HIV Cohort
Study (SHCS), a prospective multicentre study carried out at
seven major Swiss hospitals and their local affiliated centres.18 19
Written informed consent, including for genetic testing, was
mandatory for inclusion, and the study was approved by all local
ethical committees. Demographic and clinical characteristics
were extracted from clinical databases.
HCV-specific T cell responses and HCV RNA levels were
assessed longitudinally during untreated HIV infection and
during successful cART. Successful cART was defined as HIV-
RNA levels below 400 copies/ml after 6 months from cARTstart
and thereafter. Follow-up was censored at the first virological
failure (HIV-RNA above 1000 cp/ml). Low level viral replication
(HIV-RNA 400e1000 cp/ml) post-cART was present in 2% of
measurements. In these instances, follow-up was not censored
as these events were not considered immunologically relevant.
SHCS participants fulfilling the following criteria were
included: (i) anti-HCV seropositivity (using ELISA and confirmed
by immunoblot or recombinant immunoblot assay (RIBA)) and
detectable HCV RNA, assessed by quantitative or qualitative
assays; and (ii) availability of frozen peripheral blood mono-
nuclear cells (PBMCs) and/or plasma samples before commence-
ment of cART (median 0.8 (IQR 4 to 0.2) months pre-cART) and
after more than 2 years of successful cART (median 33 (IQR 30 to
36) months on cART). In some individuals, analyses were
performed additionally at enrolment, during the first year and
after more than 4 years on successful cART. We restricted our
analyses to individuals infected with HCV-genotype 1 (n¼73) or
3 (n¼46). For cellular analyses we also included individuals with
spontaneous HCV clearance (n¼22) of unknown HCV genotype
(genotyping was not possible because clearance had already
occurred when entering the SHCS). Spontaneous HCV clearance
was defined as HCV seropositivity and undetectable HCV-RNA.
All analyses were before HCV therapy. Table 1 shows the
characteristics of the study participants. Figure 1 shows the
number of individuals with available viable cells and/or plasma
samples at each time-point, and the median CD4+Tcell counts
and HIV-RNA levels at these time-points.
Peptides and proteins
Weusedthesame experimental approachasin previousstudiesto
investigate the influence of cARTon HCV-specific Tcell immu-
peptides are reproducibly detectable in a reasonable fraction of
patients, that these are dominated by CD4+Tcell responses, and
that they are rarely associated with virus escape.9 20e22Core
10) that cover amino acids 1e191 and were pooled to a final
concentration of 10 mg/ml of each peptide. To further investigate
whether the change in immune responses to cART was only
restricted to core peptides, we included the recombinant HCV
non-structural (NS) proteins NS3, NS4 and NS5. These proteins
have been shown to induce immune responses especially during
acute HCV infection or in individuals with spontaneous HCV
clearance.23 24The recombinant HCV genotype 1 proteins NS3,
NS4 and NS5 were pooled to a final concentration of 1 mg/ml for
each antigen in the ELISpot assay. We used pooled recombinant
NS proteins instead of overlapping peptides, as these predomi-
nantly stimulate CD4+T cell responses and to maximise the
concentration of 1 mg/ml.
ELISpot assay for interferon g secretion
ELISpot assays were performed as previously described.9PBMCs
(100000 per well) were used and plates were read with an AID
plate reader. Each sample was tested in duplicate against HCV
antigens, HIV-p24, EpsteineBarr virus (EBV) and cytomegalo-
virus (CMV) antigens. Phytohaemagglutinin (PHA) was used as
Chronic HCV infection
Cleared HCV infection
Age, median (IQR)
*Number of individuals with $1 viable peripheral blood mononuclear
cells and/or plasma sample.
HCV, hepatitis C virus; MSM, men who have sex with men.
Median CD4+T cell counts and HIV-RNA levels at the different study
time-points are shown. The table below the graph indicates the number
of individuals with available viable peripheral blood mononuclear cells for
cellular assays, and with plasma samples for the measurement of
hepatitis C virus (HCV) RNA levels. cART, combination antiretroviral
Study population, CD4+T cell counts and HIV-RNA levels.
Gut 2010;59:1252e1258. doi:10.1136/gut.2009.2059711253
group.bmj.com on August 26, 2010 - Published by gut.bmj.comDownloaded from
a positive control. Samples without any detectable PHA
response were excluded. A test was considered positive if the
probability of a spot appearing in the test well was significantly
different (p#0.05) from the probability of a spot appearing in
the control well (background), assuming a binominal distribu-
tion for each test antigen (Excel BINOMDISTstatistics program,
Microsoft). The mean number of spot forming units (SFU) in
control wells was subtracted from the mean SFU number in the
test wells to give a final reading.
HCV RNA measurement
For the preparation of RNA from plasma samples we used the
EasyMag Magnetic extraction kit according to the manufac-
turer’s instructions (Biomérieux, Geneva, Switzerland). In brief
200 ml of plasma was incubated with 2 ml lysis buffer containing
guanidinthiocyanate and 2.5 ml carrier RNA (1 mg/ml) for 10 min
at room temperature. After adding 550 ml NucliSens easyMAG
magnetic silica and incubation for another 10 min at room
temperature, several wash steps (using NucliSens easyMAG
extraction buffer 1 and 2) were performed, and finally the
purified RNAwas eluted with 110 ml NucliSens extraction buffer
3. HCV RNA standards were obtained from AcroMetrix (Opti-
QuantHCV RNA quantification
Netherlands) and extracted according to the same procedure as
described above, together with clinical samples.
Viral load assay
Based on the method previously published by Castelain et al,25
a real-time RT-PCR assay using TaqMan (fluorescence-based
real-time PCR) and minor groove binding (MGB) probes was
designed for quantitative determination of HCV RNA in clinical
samples. The specific reverse transcription of HCV was
performed in 20 ml reaction mixture containing 10.5 ml of eluted
RNA and reverse transcription reagents including MultiScribe
TM reverse transcriptase, dNTP(2.5 mM), MgCl2 (2.5 mM),
103 buffer with RNAsin (20 U/ml), and random hexamers
(Applied Biosystems, Rotkreuz, Switzerland). The products
were hybridised by incubating at 258C for 10 min, incubated at
488C for 30 min for reverse transcription, followed by heating to
958C for 5 min to deactivate the reverse transcriptase. To
increase specificity, a second round of reverse transcription at
a higher temperature and subsequent cDNA amplification was
performed using 96-well plates in duplicate reactions requiring
6 ml of cDNA template in 20 ml reactions containing rTH DNA
polymerase (2.5 U/ml), AmpErase UNG, dATP, dGTP, dCTP,
dUTP and a 53 buffer containing a passive reference (6-
carboxyrhodamine labelled with ROX) (EZ-RT PCR kit, Applied
Biosystems, Rotkreuz, Switzerland) forward primer (500 nM)
HCV-S1 (nucleotides: 127 to 145; 59-TCCCGGGAGAGCC-
ATAGTG-39), reverse primer (1 mM) HCV-AS2 (nucleotides:
202 to 185;59-TCCAAGAAAGGACCCRGT-39)
TaqMan minor groove binding (MGB) probe labelled with
The ABI 7900 HT real-time PCR detection system (Applied
Biosystems) was used for analysis. Thermal cycling conditions
were designed as follows: 508C for 2 min, for contamination
control with AmpErase UNG, followed by 608C for 30 min for
continued reverse transcription with rTH DNA polymerase,
then 958C for 15 sec and 608C for 60 sec. Fluorescent measure-
ments were recorded during each annealing step. At the end of
each PCR run, data were automatically analysed by the system
and amplification plots were generated. The HCV copy number
was determined by reading from the standard curve (OptiQuant
HCV RNA quantification panel, AcroMetrix, Netherlands, range
50 to 5 Mio IU/ml). Mean cycle threshold (CT) values for the
main range of HCV RNA levels (5 to 7 log10IU/ml) were 28.5
(SD60.5), 25.2 (SD60.5) and 23.5 (SD60.1). To avoid inter-
assay variability, all samples from one patient were measured
within the same assay. The standard transcripts, the controls
and all study samples were run in duplicate. Replicate samples
varying by more than 5% of their quantification cycle were
repeated. To assess inter-assay variability, we included in each
run plasma from one individual with a known HCV RNA level
(5.0 log10IU/ml, assessed by the Roche Cobas assay). RNA levels
from this individual measured by our in-house assay were
comparable and showed low inter-assay variability over 15 runs
(mean quantity 5.1 (SD60.25) log10IU/ml). In addition, control
plasma samples with a wide range of HCV RNA levels provided
by Roche Diagnostics, Switzerland, were included in each run
and also showed low inter-assay variability in both the low and
high range (mean quantity 2.1 (SD60.3) and 5.1 (SD60.2)
obtained from AcroMetrix (OptiQuant HCV RNA quantifica-
tion panel, AcroMetrix, Netherlands) and distilled water were
included as non-template controls in every run. All negative
controls were always amplified after CT values of 40.
Cross-sectional comparisons were performed using a robust
variance estimation model for cluster-correlated data to consider
data points representing repeated measurements.26Longitudinal
analyses were done by using the non-parametric paired
Wilcoxon signed rank test. Statistical analyses were performed
in STATA V.10.0 software and the figures were drawn using
Graphpad Prism 5.01 software.
HCV-specific T cell responses to HCV core peptides
We quantified longitudinally HCV-specific T cell responses to
HCV core peptides by ex vivo interferon g (IFNg) ELISpot
assays in untreated and treated HIV infection. In cross-sectional
analyses, the proportion of individuals with detectable HCV-
specific immune responses to HCV core peptides was signifi-
cantly higher during successful cART compared to untreated
HIV infection (figure 2A). During untreated HIV infection there
was no significant change in HCV-specific immune responses
(13% (2/16) vs 19% (12/64), p¼0.54). However, during
successful cART, the proportion of individuals with detectable T
cell responses increased significantly: 24% (12/50) during the
first year, 45% (29/64) after more than 3 years and 49% (18/37)
after more than 5 years of successful cART (p¼0.001). Accord-
ingly, the frequency of spot forming cells was significantly
higher after initiation of HIV therapy (figure 2B). The increase in
responses to HCV core peptides on cART was observed for
chronic and spontaneously cleared HCV infection (figure 2D)
and for both HCV genotypes (figure 2C). This is in line with the
high sequence similarity of the HCV core region with 94%
identity between genotypes 1 and 3.27
Neither CD4+Tcell counts at the time of the experiments nor
nadir CD4+T cell counts correlated significantly with the
presence or absence of HCV-specific Tcell responses (p>0.1 for
all comparisons). At baseline, the relative CD4+Tcell count was
higher in individuals with detectable HCV-specific T cell
responses to HCV core peptides, compared to those without
detectable responses (22% vs 15%, p¼0.05), while there was no
1254 Gut 2010;59:1252e1258. doi:10.1136/gut.2009.205971
group.bmj.com on August 26, 2010 - Published by gut.bmj.comDownloaded from
statistically significant difference with regard to absolute CD4+
Tcell counts. There was no significant correlation of relative or
absolute CD4+Tcell counts with HCV-specific Tcell responses
after starting cART.
In a subgroup of 54 individuals (supplementary table 1) with
available viable PBMCs 0.8 months before starting HIV therapy
and after a median of 33 months on successful cART, we
assessed longitudinally the evolution of HCV-specific T cell
responses. The proportion of individuals with detectable T cell
responses and the frequency of IFNg producing Tcells increased
significantly during cART (22% vs 44%, p¼0.02) (supplementary
figure 1). Emergence of detectable T cell responses was much
more frequent than loss of responses during cART (31% vs 9%).
Individuals with increases in immune responses had lower
CD4+Tcell counts at baseline compared to individuals without
detectable T cell responses to HCV core peptides pre- and on-
cART (150 vs 225 CD4+T cells/ml; p¼0.03). However, this
difference was driven by two individuals with increases in Tcell
responses who started cART with CD4+T cell counts below
5 cells/ml, and the relevance of this difference remains uncertain.
For the remaining characteristics, there were no significant
differences among the four groups.
T cell responses to further peptides
In contrast to the responses to HCV core peptides, there was
no significant change in the response to recombinant HCV
NS3-5 proteins. From the 48 individuals with at least one
response to HCV core peptides, 11 (23%) also responded to
proportion of individuals with detectable ELISpot responses to HCV core peptides increased significantly during successful cART (A). Peak frequency of
interferon-g producing T cells per million peripheral blood mononuclear cells recognising HCV-core peptides significantly increased after initiation of
a successful cART (B). The increase in responses to HCV core peptides on cART was observed for chronic (C) and spontaneously cleared HCV infection
(D) and for both HCV genotypes (C). Only p-values <0.05 are shown. SFC, spot forming cells.
T cell ELISpot responses to hepatitis C virus (HCV) core peptides before and on successful combination antiretroviral therapy (cART). The
detectable ELISpot responses to
hepatitis C virus (HCV) core peptides
and to different control peptides.
Arrows denote the time-point of start of
combination antiretroviral therapy
(cART). ND, no data.
Proportion of individuals with
Gut 2010;59:1252e1258. doi:10.1136/gut.2009.205971 1255
group.bmj.com on August 26, 2010 - Published by gut.bmj.com Downloaded from
NS3-5 peptides. Conversely, 11 of 12 (92%) individuals with
responses to NS3-5 proteins also responded to core peptides.
This is in line with previous findings that responses to core
proteins are detected more frequently than responses to NS
proteins.9 23Although some NS3-5 epitopes are shared between
genotype 1 and 3 sequences,28e30other epitopes are clearly
genotype-specific.29 31Therefore, it is likely that some genotype
3 specific responses to the NS proteins were missed. Responses
to HIV-p24 decreased, while responses to CMV peptides
increased significantly during cART (p¼0.001) (figure 3).
Twenty-eight of the 48 (58%) individuals with responses to
HCV core peptides also responded to CMV peptides.
HCV RNA levels
HCV-RNA levels were evaluated in 119 HIV/HCV coinfected
individuals. In cross-sectional analysis, median HCV RNA levels
during untreated HIV infection were 6.3 (IQR 5.6e6.8) log10IU/
ml at enrolment and 6.5 (IQR 5.9e7.0) log10IU/ml just before
starting cART. During the first year on successful cART, median
HCV-RNA levels slightly increased to 6.6 (IQR 6.0e7.2)
log10IU/ml. During long-term cART, median HCV-RNA levels
decreased to 6.4 (IQR 5.9e6.9) and 6.2 (IQR 5.6e6.7) log10IU/
ml after 3 and 5 years, respectively (figure 4A). HCV clearance
was not observed despite long-term cART.
In individuals with longitudinal samples during untreated
HIV infection, the median change in HCV-RNA levels was +0.4
log10IU/ml. In the first year on successful cART, there was
a slight increase of HCV-RNA levels (+0.1 log10IU/ml). During
long-term cART, median HCV-RNA levels slightly decreased
compared to pre-cART levels (?0.3 log10IU/ml, p¼0.02; figure 4B).
HCV RNA levels slightly decreased on successful cART in both
HCV-genotype 1 (?0.2 log10 IU/ml) and HCV-genotype 3
infected individuals (?0.3 log10IU/ml). The increase in absolute
CD4+T cell counts on successful cART was not significantly
associated with the change in HCV RNA levels (p¼0.1).
In the subgroup of individuals with increasing HCV-specific
immune responses a more marked decline of HCV-RNA levels
on cART, we observed compared to individuals without any
detectable ELISpot responses to HCV-core peptides (figure 5). In
individuals with increasing HCV-specific T cell responses to
HCV-core peptides, the median HCV RNA change from baseline
was ?0.5 and ?0.8 log10IU/ml. For individuals without T cell
responses, the median change from baseline after 3 and 5 years
of successful cART was ?0.2 and ?0.1 log10IU/ml. However,
these differences were not statistically significant.
Coinfection with HIV substantially diminishes the immuno-
logical control of HCV. Previous studies have shown that HCV-
specific immune responses are reduced in HIV infected individ-
uals.8 9 11Furthermore, the high HCV RNA levels observed in
HIV infected individuals indicate a loss of control of HCV
replication.1Here, we show that successful cART partially
restores T cell responses to HCV-core peptides. The proportion
of individuals with detectable immune responses on long-term
cART (49%) was very similar to that observed by Harcourt et al
in HCV monoinfected individuals (52%) using the same exper-
imental approach.9A successful cART also increased cellular
immune response to HCV core peptides in individuals that
spontaneously cleared HCV infection. This finding is in line
with the observation that cellular immunity is maintained long
term in individuals who spontaneously clear infection.32
in addition to previous experiments using flow cytometry,22 33
were fromCD4+Tcells. Therefore, themeasured increasein Tcell
responses largely represents CD4+T cell reactivity. High sensi-
tivity techniques to isolate T cell subsets would have required
larger cell numbers and ideally fresh samples, that were not
available due to the long-term follow-up in this study.
The absence of a significant increase in responses to HCV
NS3-5 proteins in our HIV/HCV coinfected patients is in
accordance with previous studies where these responses were
not detectable in the majority of HCV monoinfected individ-
uals.9Non-structural HCV proteins are highly polymorphic and
the consensus sequence that is used to generate the peptide
libraries contains in many instances escaped variants that are
poorly immunogenic.31 34 35Viral adaptation to T cell pressure
through mutational escape is more likely in polymorphic
proteins, which might explain why T cell responses to NS3-5
peptides are more difficult to detect compared to responses to
the conserved core peptides. Because we used different methods
(core peptides versus non-structural proteins), the responses to
core and NS are not directly comparable. However, a comparison
of the relative importance of immune responses to different
peptides and proteins was beyond the scope of this study.
Instead, we used an optimised, reproducible and well studied
approach to investigate the influence of cART on the evolution
of T cell responses. Future studies are underway and will aim
to assess the relationship between responses to structural and
non-structural gene products in more detail.
Concurrently with the increase of cellular immune responses
during cART, we observed a slight but significant decrease in
HCV RNA levels. This effect was particularly evident in a small
hepatitis C virus (HCV) RNA levels. Median HCV RNA levels at the
different study time-points are shown in (A) in cross-sectional analysis.
The change in HCV RNA levels within individuals in untreated and treated
HIV infection is outlined in (B). Only p-values #0.1 are shown.
Impact of combination antiretroviral therapy (cART) on
1256 Gut 2010;59:1252e1258. doi:10.1136/gut.2009.205971
group.bmj.com on August 26, 2010 - Published by gut.bmj.comDownloaded from
subgroup of individuals with increasing HCV specific immune
responses. The temporal association of increasing HCV specific
T cell responses and decreasing HCV RNA levels might poten-
tially indicate that immune reconstitution through successful
cART improves the control of HCV replication, although it is
difficult to show cause and effect in this case. A transient
increase in HCV RNA levels during the first year of cART has
been observed previously (reviewed in Cooper and Cameron17).
Suggested mechanisms for such an increase include increased
hepatocyte lysis through restored T cell immunity, and
a decrease in interferon levels through treatment of HIV.15
Clearance of HCV infection during cART has been reported in
only a few cases.36e41We did not observe HCV clearance despite
a substantial increase in Tcell responses on cART. This suggests
that in most cases, immunological HCV clearance cannot be
achieved by restoring Tcell responses through successful cART.
Our study has strengths and limitations. A main strength is
the long-term follow-up and the longitudinal setting that largely
avoids confounding through clinical and demographic charac-
teristics.Additionally, we could
responses and HCV RNA levels in the same cohort and therefore
simultaneously evaluate the impact of HIV and cART on T cell
responses and viral loads. Through the restriction to successful
and uninterrupted cART, we could avoid biases through treat-
ment failures and interruptions. A limitation of the study is the
restriction of cellular experiments through the use of stored
PBMCs. Due to the limited number of frozen PBMCs, we could
not assess CD8+or regulatory T cell responses, or perform an
in-depth analysis of HLA-restricted immune responses. In addition
viable cells and/or plasma were not available for all participants
at all study time-points. We acknowledge that we might have
missed many HCV specific Tcell responses that could have been
detected after in vivo culture, as shown recently.24However, we
explicitly wanted to assess the influence of an increased immune
pressure in-vivo and therefore avoided additional in-vitro stim-
ulation that can strongly influence Tcell responses, as shown by
Schnuriger et al.24Additional culturing could have masked the
main study question about the effects of cARTon HCV-specific
T cell reactivity. Ex-vivo ELISpot responses probably underesti-
mate the total number of HCV-specific cells that can be gener-
ated after culturing. However, this does not alter the main
conclusion that cARTcan increase HCV-specific Tcell responses.
As HCV specific T cell responses were not detectable in most
individuals, we could not reliably correlate changes in the
frequency of T cell responses with HCV RNA levels and estab-
lish possible cause and effect.
Our findings are in line with the beneficial clinical effects of
cART on the natural course of hepatitis C in HIV infected
individuals13 14and with the current recommendation to start
cARTearlier in HCV/HIV coinfected individuals.42 43However,
it remains unclear to what extent the increases in cellular
immune responses during cART result in beneficial effects on
liver disease progression. Other favourable effects of cART on
liver disease include a reduction in immune activation and in
bacterial translocation. Although one study found that higher
hepatitis C RNA levels were associated with increased mortality
from end stage liver disease,44most reports did not find a corre-
lation between HCV RNA levels and liver disease.45 46It is
therefore unlikely that the modest effect of cART on HCV
viraemia results in large clinical effects. However, as viral load is
an important predictor of the response to HCV therapy,47
a reduction in HCV RNA levels through cART might result in
better response rates to HCV therapy. Future studies should
clarify whether starting cART early in the course of HIV infec-
tion would lower the risk of progression to end-stage liver
disease and improve HCV treatment responses.
1Division of Infectious Diseases, University Hospital Berne and University of Berne,
2Nuffield Department of Clinical Medicine, Oxford University, UK
3Centre for Clinical Immunology and Biomedical Statistics, Royal Perth Hospital and
Murdoch University, Perth, Australia
4Centre for Forensic Sciences and School of Anatomy and Human Biology, University
of Western Australia, Perth, Australia
5Institute for Infectious Diseases, University of Berne, Switzerland
6Institute of Microbiology, University Hospital Centre and University of Lausanne,
7Institute of Social and Preventive Medicine, University of Berne, Switzerland
8Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich,
University of Zurich, Switzerland
9Department of Infectious Diseases, Geneva University Hospital, Switzerland
10Division of Infectious Diseases, Kantonsspital St Gallen, Switzerland
11Division of Infectious Diseases, Ospedale Regionale di Lugano, Switzerland
12Division of Infectious Diseases and Hospital Epidemiology, University Hospital Basel,
Acknowledgements We would like to thank all the participating patients, the
physicians and the study nurses of all clinical centres for excellent patient care, and
the SHCS core laboratories for their invaluable processing, storage and retrieval of
the samples. We also thank the virology lab technicians from the Institute of
Infectious Diseases, University of Berne.
Funding This study has been financed in the framework of the Swiss HIV Cohort
Study, supported by the Swiss National Science Foundation (SNF grants
#3345-062041 and #324730-116862), the Wellcome Trust and the James Martin
School for 21st Century Oxford, the NIHR Biomedical Research Centre Program Oxford
and the National Health and Medical Research Council Program, Western Australia.
(HCV) RNA levels by HCV-specific
immune responses. The change in HCV
RNA levels on combination antiretroviral
therapy (cART) is shown for the
subgroup of individuals with increases
in HCV-specific cellular immune
responses during cART and for those
without immune responses pre- and on-
Change in hepatitis C virus
Gut 2010;59:1252e1258. doi:10.1136/gut.2009.205971 1257
group.bmj.com on August 26, 2010 - Published by gut.bmj.com Downloaded from
The study sponsors had no role in the study design, data collection, analysis,
interpretation of data and writing of this manuscript.
Competing interests None.
Ethics approval This study was performed in the framework of the Swiss HIV Cohort
Study (www.shcs.ch). Written informed consent, including for genetic testing, was
mandatory for inclusion, and the study was approved by all local ethical committees.
Provenance and peer review Not commissioned; externally peer reviewed.
Sulkowski MS, Thomas DL. Hepatitis C in the HIV-Infected Person. Ann Intern Med
Rauch A, Rickenbach M, Weber R, et al. Unsafe sex and increased incidence of
hepatitis C virus infection among HIV-infected men who have sex with men: the
Swiss HIV Cohort Study. Clin Infect Dis 2005;41:395e402.
Graham CS, Baden LR, Yu E, et al. Influence of human immunodeficiency virus
infection on the course of hepatitis C virus infection: a meta-analysis. Clin Infect Dis
Thomas DL, Astemborski J, Vlahov D, et al. Determinants of the quantity of hepatitis
C virus RNA. J Infect Dis 2000;181:844e51.
Thomas DL. The challenge of hepatitis C in the HIV-infected person. Annu Rev Med
Rauch A, Gaudieri S, Evison J, et al. Low current and nadir CD4+ T cell counts are
associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study.
Antivir Ther 2008;13:455e60.
Klenerman P, Lucas M, Barnes E, et al. Immunity to hepatitis C virus: stunned but
not defeated. Microbes Infect 2002;4:57e65.
Kim AY, Schulze zur Wiesch J, Kuntzen T, et al. Impaired hepatitis C virus-specific T
cell responses and recurrent hepatitis C virus in HIV coinfection. PLoS Med
Harcourt G, Gomperts E, Donfield S, et al. Diminished frequency of hepatitis C virus
specific interferon gamma secreting CD4+ T cells in human immunodeficiency virus/
hepatitis C virus coinfected patients. Gut 2006;55:1484e7.
Lauer GM, Barnes E, Lucas M, et al. High resolution analysis of cellular immune
responses in resolved and persistent hepatitis C virus infection. Gastroenterology
Kim AY, Lauer GM, Ouchi K, et al. The magnitude and breadth of hepatitis C virus-
specific CD8+ T cells depend on absolute CD4+ T cell count in individuals
coinfected with HIV-1. Blood 2005;105:1170e8.
Dutoit V, Ciuffreda D, Comte D, et al. Differences in HCV-specific T cell responses
between chronic HCV infection and HIV/HCV co-infection. Eur J Immunol
Thein HH, Yi Q, Dore GJ, et al. Natural history of hepatitis C virus infection in HIV-
infected individuals and the impact of HIV in the era of highly active antiretroviral
therapy: a meta-analysis. AIDS 2008;22:1979e91.
Qurishi N, Kreuzberg C, Luchters G, et al. Effect of antiretroviral therapy on liver-
related mortality in patients with HIV and hepatitis C virus coinfection. Lancet
Braitstein P, Palepu A, Dieterich D, et al. Special considerations in the initiation and
management of antiretroviral therapy in individuals coinfected with HIV and hepatitis
C. AIDS 2004;18:2221e34.
Rutschmann OT, Negro F, Hirschel B, et al. Impact of treatment with human
immunodeficiency virus (HIV) protease inhibitors on hepatitis C viremia in patients
coinfected with HIV. J Infect Dis 1998;177:783e5.
Cooper CL, Cameron DW. Review of the effect of highly active antiretroviral therapy
on hepatitis C virus (HCV) RNA levels in human immunodeficiency virus and HCV
coinfection. Clin Infect Dis 2002;35:873e9.
Ledergerber B, Egger M, Opravil M, et al. Clinical progression and virological failure
on highly active antiretroviral therapy in HIV-1 patients: a prospective cohort study.
Swiss HIV Cohort Study. Lancet 1999;353:863e8.
Schoeni-Affolter FRM, Furrer HJ, Rudin C, et al. COHORT PROFILE: The Swiss HIV
Cohort Study. Int J Epidemiol 2009:1e11.
Fleming VM, Harcourt G, Barnes E, et al. Virologic footprint of CD4+ T cell
responses during chronic HCV infection. J Gen Virol 2010:1396e406.
Semmo N, Day CL, Ward SM, et al. Preferential loss of IL-2-secreting CD4+ T
helper cells in chronic HCV infection. Hepatology 2005;41:1019e28.
Semmo N, Krashias G, Willberg C, et al. Analysis of the relationship between
cytokine secretion and proliferative capacity in hepatitis C virus infection.
J Viral Hepat 2007;14:492e502.
Danta M, Semmo N, Fabris P, et al. Impact of HIV on host-virus interactions during
early hepatitis C virus infection. J Infect Dis 2008;197:1558e66.
Schnuriger A, Dominguez S, Guiguet M, et al. Acute hepatitis C in HIV-infected
patients: rare spontaneous clearance correlates with weak memory CD4 T cell
responses to hepatitis C virus. AIDS 2009;23:2079e89.
Castelain S, Descamps V, Thibault V, et al. TaqMan amplification system
with an internal positive control for HCV RNA quantitation. J Clin Virol
Williams RL. A note on robust variance estimation for cluster-correlated data.
Simmonds P. Genetic diversity and evolution of hepatitis C viruse15 years on.
J Gen Virol 2004;85:3173e88.
Neumann-Haefelin C, Frick DN, Wang JJ, et al. Analysis of the evolutionary
forces in an immunodominant CD8 epitope in hepatitis C virus at a population level.
J Virol 2008;82:3438e51.
Neumann-Haefelin C, Timm J, Schmidt J, et al. Protective effect of human
leukocyte antigen B27 in hepatitis C virus infection requires the presence of
a genotype-specific immunodominant CD8+ T cell epitope. Hepatology
Thimme R, Neumann-Haefelin C, Boettler T, et al. Adaptive immune responses to
hepatitis C virus: from viral immunobiology to a vaccine. Biol Chem
Rauch A, James I, Pfafferott K, et al. Divergent adaptation of hepatitis C virus
genotypes 1 and 3 to human leukocyte antigen-restricted immune pressure.
Fitzmaurice K, Klenerman P. Cellular immunity and acute hepatitis C infection. Curr
Pharm Des 2008;14:1666e77.
Barnes E, Ward SM, Kasprowicz VO, et al. Ultra-sensitive class I tetramer analysis
reveals previously undetectable populations of antiviral CD8+ T cells. Eur J Immunol
Gaudieri S, Rauch A, Park LP, et al. Evidence of viral adaptation to HLA class
I-restricted immune pressure in chronic hepatitis C virus infection. J Virol
Timm J, Li B, Daniels MG, et al. Human leukocyte antigen-associated sequence
polymorphisms in hepatitis C virus reveal reproducible immune responses and
constraints on viral evolution. Hepatology 2007;46:339e49.
Falconer K, Gonzalez VD, Reichard O, et al. Spontaneous HCV clearance in HCV/HIV-1
coinfection associated with normalized CD4 counts, low level of chronic immune
activation and high level of T cell function. J Clin Virol 2008;41:160e3.
Fialaire P, Payan C, Vitour D, et al. Sustained disappearance of hepatitis C viremia in
patients receiving protease inhibitor treatment for human immunodeficiency virus
infection. J Infect Dis 1999;180:574e5.
Torti C, Barnes E, Quiros-Roldan E, et al. Suppression of hepatitis C virus replication
is maintained long term following HAART therapy, in an individual with HCV/HIV co-
infection. Antivir Ther 2004;9:139e42.
Weissbrich B, Langmann P, Schubert J, et al. Resolution of HCV infection in a HIV-
infected patient under HAART after several hepatitis flare-ups. Eur J Med Res
Zeitoun JD, Mallet V, Chaix ML, et al. Stable recovery from HCV in HIV-HCV co-
infection under antiretroviral therapy. J Clin Virol 2007;40:71e3.
Yokozaki S, Takamatsu J, Nakano I, et al. Immunologic dynamics in hemophiliac
patients infected with hepatitis C virus and human immunodeficiency virus: influence
of antiretroviral therapy. Blood 2000;96:4293e9.
Hammer SM, Eron JJ Jr, Reiss P, et al. Antiretroviral treatment of adult HIV
infection: 2008 recommendations of the International AIDS Society-USA panel.
Clumeck N, Pozniak A, Raffi F. European AIDS Clinical Society (EACS) guidelines for
the clinical management and treatment of HIV-infected adults. HIV Med
Hisada M, Chatterjee N, Kalaylioglu Z, et al. Hepatitis C virus load and
survival among injection drug users in the United States. Hepatology
De Moliner L, Pontisso P, De Salvo GL, et al. Serum and liver HCV RNA levels in
patients with chronic hepatitis C: correlation with clinical and histological features.
Lagging LM, Garcia CE, Westin J, et al. Comparison of serum hepatitis C virus RNA
and core antigen concentrations and determination of whether levels are associated
with liver histology or affected by specimen storage time. J Clin Microbiol
Kau A, Vermehren J, Sarrazin C. Treatment predictors of a sustained virologic
response in hepatitis B and C. J Hepatol 2008;49:634e51.
1258Gut 2010;59:1252e1258. doi:10.1136/gut.2009.205971
group.bmj.com on August 26, 2010 - Published by gut.bmj.comDownloaded from