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Hepatology
Volume 72, Issue 5 p. 1541-1555
Original Article
Open Access
CHARACTERIZING HEPATITIS C VIRUS–SPECIFIC CD4+ T CELLS FOLLOWING VIRAL-VECTORED
VACCINATION, DIRECTLY ACTING ANTIVIRALS, AND SPONTANEOUS VIRAL CURE
Felicity Hartnell,
Corresponding Author
Felicity Hartnell
* felicityhartnell@gmail.com
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Joint first and last authors.
Address Correspondence and Reprint Requests to:
Felicity Hartnell, M.B.B.S.
Peter Medawar Building for Pathogen Research, University of Oxford
South Parks Road
Oxford, OX1 3SY, United Kingdom
E-mail: felicityhartnell@gmail.com
Tel.: (+44) 1865 281231
Search for more papers by this author
Ilaria Esposito,
Ilaria Esposito
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Joint first and last authors.Search for more papers by this author
Leo Swadling,
Leo Swadling
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
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Anthony Brown,
Anthony Brown
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
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Chansavath Phetsouphanh,
Chansavath Phetsouphanh
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
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Catherine de Lara,
Catherine de Lara
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
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Chiara Gentile,
Chiara Gentile
CEINGE–Advanced Biotechnologies, Naples, Italy
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Bethany Turner,
Bethany Turner
Jenner Vaccine Trials, Nuffield Department of Medicine, University of Oxford,
Oxford, United Kingdom
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Lucy Dorrell,
Lucy Dorrell
Jenner Vaccine Trials, Nuffield Department of Medicine, University of Oxford,
Oxford, United Kingdom
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Stefania Capone,
Stefania Capone
Reithera Srl, Rome, Italy
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Antonella Folgori,
Antonella Folgori
Reithera Srl, Rome, Italy
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Eleanor Barnes,
Eleanor Barnes
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Jenner Vaccine Trials, Nuffield Department of Medicine, University of Oxford,
Oxford, United Kingdom
NIHR Biomedical Research Centre Oxford, John Radcliffe Hospital, Oxford, United
Kingdom
Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford, United
Kingdom
Joint first and last authors.Search for more papers by this author
Paul Klenerman,
Paul Klenerman
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Jenner Vaccine Trials, Nuffield Department of Medicine, University of Oxford,
Oxford, United Kingdom
NIHR Biomedical Research Centre Oxford, John Radcliffe Hospital, Oxford, United
Kingdom
Translational Gastroenterology Unit, John Radcliffe Hospital, Oxford, United
Kingdom
Joint first and last authors.Search for more papers by this author
Felicity Hartnell,
Corresponding Author
Felicity Hartnell
* felicityhartnell@gmail.com
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Joint first and last authors.
Address Correspondence and Reprint Requests to:
Felicity Hartnell, M.B.B.S.
Peter Medawar Building for Pathogen Research, University of Oxford
South Parks Road
Oxford, OX1 3SY, United Kingdom
E-mail: felicityhartnell@gmail.com
Tel.: (+44) 1865 281231
Search for more papers by this author
Ilaria Esposito,
Ilaria Esposito
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Joint first and last authors.Search for more papers by this author
Leo Swadling,
Leo Swadling
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Search for more papers by this author
Anthony Brown,
Anthony Brown
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Search for more papers by this author
Chansavath Phetsouphanh,
Chansavath Phetsouphanh
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Search for more papers by this author
Catherine de Lara,
Catherine de Lara
Peter Medawar Building for Pathogen Research, University of Oxford, Oxford,
United Kingdom
Search for more papers by this author
Chiara Gentile,
Chiara Gentile
CEINGE–Advanced Biotechnologies, Naples, Italy
Search for more papers by this author
Bethany Turner,
Bethany Turner
Jenner Vaccine Trials, Nuffield Department of Medicine, University of Oxford,
Oxford, United Kingdom
Search for more papers by this author
Lucy Dorrell,
Lucy Dorrell
Jenner Vaccine Trials, Nuffield Department of Medicine, University of Oxford,
Oxford, United Kingdom
Search for more papers by this author
Stefania Capone,
Stefania Capone
Reithera Srl, Rome, Italy
Search for more papers by this author
… See all authors
First published: 03 February 2020
https://doi.org/10.1002/hep.31160
Citations: 9
Potential conflict of interest: Dr. Dorrell received grants from Vaccitech.
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ABSTRACT
BACKGROUND AND AIMS
Induction of functional helper CD4+ T cells is the hallmark of a protective
immune response against hepatitis C virus (HCV), associated with spontaneous
viral clearance. Heterologous prime/boost viral vectored vaccination has
demonstrated induction of broad and polyfunctional HCV-specific CD8+ T cells in
healthy volunteers; however, much less is known about CD4+ T-cell subsets
following vaccination.
APPROACH AND RESULTS
We analyzed HCV-specific CD4+ T-cell populations using major histocompatibility
complex class II tetramers in volunteers undergoing HCV vaccination with
recombinant HCV adenoviral/modified vaccinia Ankara viral vectors.
Peptide-specific T-cell responses were tracked over time, and functional
(proliferation and cytokine secretion) and phenotypic (cell surface and
intranuclear) markers were assessed using flow cytometry. These were compared to
CD4+ responses in 10 human leukocyte antigen–matched persons with HCV
spontaneous resolution and 21 chronically infected patients treated with
directly acting antiviral (DAA) therapy. Vaccination induced tetramer-positive
CD4+ T cells that were highest 1-4 weeks after boosting (mean, 0.06%). Similar
frequencies were obtained for those tracked following spontaneous resolution of
disease (mean, 0.04%). In addition, the cell-surface phenotype (CD28, CD127)
memory subset markers and intranuclear transcription factors, as well as
functional capacity of peptide-specific CD4+ T-cell responses characterized
after vaccination, are comparable to those following spontaneous viral
resolution. In contrast, helper responses in chronic infection were infrequently
detected and poorly functional and did not consistently recover following HCV
cure.
CONCLUSIONS
Helper CD4+ T-cell phenotype and function following HCV viral vectored
vaccination resembles “protective memory” that is observed following spontaneous
clearance of HCV. DAA cure does not promote resurrection of exhausted CD4+
T-cell memory in chronic infection.
ABBREVIATIONS
aa amino acids CCR7 C-C chemokine receptor type 7 ChAd3 chimpanzee adenovirus
DAA directly acting antiviral DMSO dimethyl sulfoxide Eomes Eomesodermin EOT end
of treatment FACS fluorescence-activated cell sorting HCV hepatitis C virus HLA
human leukocyte antigen ICS intracellular cytokine staining IFN-γ
interferon-gamma IL interleukin MHC major histocompatibility complex MVA
modified vaccinia Ankara NS nonstructural PBMCs peripheral blood mononuclear
cells SR spontaneous viral resolution T-bet T-box TF TBX21 Tcm central memory T
cells Tem effector memory T cells Temra effector memory retinoic acid–positive
cells TFs transcription factors TNF-α tumor necrosis factor alpha Tscm stem
memory T cells
Hepatitis C virus (HCV) is a global pathogen infecting approximately 71 million
people with an estimated 1.75 million new cases annually.(1) Following primary
infection, most people develop chronic liver disease, which may result in
decompensated liver cirrhosis and hepatocellular carcinoma (HCC).(2) Despite the
introduction of highly efficacious directly acting antiviral (DAA) agents, there
remains a strong rationale for preventative HCV vaccination. It is estimated
that only 20% of people living with HCV have been diagnosed, largely
attributable to the clinically silent nature of the virus, and many present only
once liver fibrosis is established.(1) Of those diagnosed, only 7.4% of patients
have had treatment, in part because of the financial barriers of accessing
DAAs.(1) Finally, those who are treated remain at risk of reinfection, providing
a rationale for a preventative approach.
A T-cell–mediated vaccine may be an ideal candidate for a preventative HCV
strategy. Both CD4+ and CD8+ T cells have been shown to play a crucial role in
immune control against HCV. This was first demonstrated in chimpanzee challenge
studies, where it was shown that following previous successful spontaneous viral
resolution (SR), antibody-mediated CD4+(3) or CD8+(3) T-cell depletion led to
prolonged viraemia after HCV reinfection. In particular, early robust T-helper 1
CD4+ T-cell responses are thought to be critical in HCV clearance.(4, 5) It is
hypothesized that functional CD4+ T cells prime an effective CD8+ T-cell
response against the virus,(6) and the absence of this early priming can lead to
an exhausted/dysfunctional immune response observed in chronic HCV infection.(5)
We have previously shown that a heterologous prime-boost strategy, using
chimpanzee adenovirus (ChAd3) and modified vaccinia Ankara (MVA) virus encoding
the nonstructural (NS) region of HCV, induces robust HCV-specific T-cell
responses against a broad range of HCV epitopes in healthy volunteers.(7, 8)
However, these vaccine studies, as well as other studies assessing
antigen-specific T cells in natural HCV infection, have largely focused on the
behavior of HCV-specific CD8+ T cells, and there is a paucity of data assessing
the behavior of HCV-specific CD4+ T cells following both vaccination and
infection. The reasons for this include very small population numbers (typically
0.001%-0.100%) of CD4+ T cells(9) and limited tools with which to assess these.
Proliferation or stimulation assays are often used; however, these do not allow
for accurate assessment of ex vivo phenotype and function. In this study, we
have used a panel of HCV-specific major histocompatibility complex (MHC) class
II tetramers on large populations of peripheral blood mononuclear cells (PBMCs),
in addition to intracellular cytokine staining (ICS), to identify and perform a
comprehensive ex vivo phenotypic analysis on HCV-specific CD4+ T cells following
viral vectored vaccination. We have compared these with the “gold-standard” CD4+
responses observed following SR given that, ideally, vaccination would aim to
recapitulate these events. Finally, we have interrogated CD4+ T-cell behavior
following DAA treatment given that this group is a target group for vaccination
and restoration of CD4+ T-cell function following DAA cure may determine immune
response to subsequent vaccination.
MATERIALS AND METHODS
RECRUITMENT OF SUBJECTS
DAA PATIENTS
Patients with chronic HCV infection (n = 29), including 21 receiving DAA therapy
were identified at the John Radcliffe Hospital (Oxford, UK) and recruited
following written informed consent. The study protocol conformed to the ethical
guidelines of the 1975 Declaration of Helsinki as reflected in a prior approval
by the Oxford Biomedical Research Centre (REC 09/H0604/20). Inclusion criteria
included a negative HCV viral load at the end of treatment with human leukocyte
antigen (HLA) matching the tetramer panel. Additional clinical data were
collected (Supporting Fig. S1A,B).
VACCINE TRIALS
Healthy volunteers were recruited at the Churchill Hospital, Oxford into two
separate vaccine studies both trialing identical HCV candidate vector vaccines
(Endura CT 2009-018260-10(7) and 2014-000730-30(10)). All volunteers received
intramuscular vaccination with experimental vaccines ChAd3-NSmut1 (ChAd3) and
MVA-NSmut (MVA). Ten volunteers were selected on the basis of a positive
enzyme-linked immunospot response, a matching HLA type for the tetramer panel,
and availability of stored vaccine sample (Supporting Fig. S1C,D).
SPONTANEOUS HCV RESOLUTION
Individuals with SR were identified from the John Radcliffe Hospital, Oxford
defined as HCV-antibody positive, HCV-PCR negative. All were treatment naïve,
with HLA matching the tetramer panel, and recruited following written informed
consent. Where possible, information was gathered about the date of HCV
transmission and clearance (Supporting Fig. S1E).
VACCINES
Development of ChAd3 and MVA vectors have been described.(7, 11, 12) Both
vectors encoded the NS3-5b region of HCV genotype 1b BK strain (1,985 amino
acids [aa]). Development of the HCV immunogen has also been described.(13)
PEPTIDES, ANTIGENS, AND TETRAMERS
A panel of MHC class II tetramers was donated from the National Institutes of
Health Core Tetramer Facility (Atlanta, GA) and Proimmune (Oxford, UK). A total
of 11 tetramers were used in the study (Supporting Fig. S2A). Peptides matching
the MHC class II tetramer sequence were obtained from Mimotopes. HCV genotype-1a
(H77, Mimotopes Wirral, UK) and genotype-1b (J4 strain [structural regions] and
BK strains [NS regions]) peptides spanning the entire HCV genome were used for
vaccinated volunteers and DAA patients (obtained from BEI Resources, Manassas,
VA). Each peptide was between 15 and 18 aa in length (overlapping by 11 aa) and
arranged in pools representing HCV viral proteins.
CELL LINES
Short-term cell lines were used for tetramer and ICS analyses. PBMCs (2-3 × 106)
were stimulated with peptide and costimulatory purified mouse antihuman antibody
CD28 (1 µg/mL; BD Biosciences) in 1 mL of RH10 at 37°C. Media were changed and
recombinant interleukin (IL)-2 (50 U/mL; Roche) was added at days 3, 7, and 10.
Cells were harvested on day 13 and left to rest overnight in RH10 before ICS and
tetramer staining assays.
MHC CLASS II TETRAMER, CELL-SURFACE MARKER, AND INTRANUCLEAR STAINING
MHC class II tetramer staining was performed on cultured and ex vivo PBMCs.
Tetramers were based on immunodominant HCV epitopes described within the NS
region.(14-17) Cultured cells were rested overnight before staining, and
approximately 2 × 105 PBMCs were used per tetramer. For ex vivo tetramer
staining, 6-8 × 106 cells were thawed using RH10 medium with DNase (0.01 mg/mL)
before counting (Guava Personal Cell Analysis system). Tetramers were
centrifuged for 5 minutes at 14,000g at 4°C before staining. Cells were stained
with fixable live/dead dye for 5 minutes followed by tetramer staining
(phycoerythrin labeled) for 60 minutes at 37°C (1 µg/100 µL) in 50 µL of RH10.
Following these, cells were stained with the surface marker panel for
30 minutes. Additionally, for intranuclear staining, cells were then fixed (1%
paraformaldehyde), permeabilized (10× perm buffer; eBioscience), and then
stained with internal antibody cocktail for 60 minutes (see Supporting Fig. S3
for a full list of antibodies and fluorochromes).
A positive tetramer response was defined as a discrete cluster of cells and
>0.004% tetramer+/CD4+ T cells. This cutoff was determined after an analysis of
tetramer+ CD4+ cells in healthy individuals. In addition, following vaccination,
a positive tetramer+ CD4+ cloud was required to be 3× baseline (prevaccination).
Furthermore, each tetramer was trialed with HLA-matched and -mismatched persons
who had not been exposed to either HCV infection or HCV peptides. All tetramers
demonstrated clean staining with low background (Supporting Fig. S2B-G).
INTRACELLULAR CYTOKINE STAINS
ICS was performed following in vitro expansion using short-term cell lines.
Before staining, cells were left to rest overnight in 1 mL of RH10 and 37°C.
Cells were then plated at 1-5 × 105 PBMCs/well in 96-well U-bottomed plates.
PBMCs were stimulated using individual peptides matching the tetramer sequence
or pooled peptides F+G+H (matching NS3-4) plus unstimulated (controlled for
dimethyl sulfoxide [DMSO]) and phorbol myristate acetate/ionomycin (50 and
500 ng/mL, respectively). Brefeldin A (10 µg/mL) was added after 1 hour, and
cells were stimulated overnight at 37°C. Cells were then stained with fixable
live/dead dye, fixed (1% paraformaldehyde), permeabilized (eBiosciences 10× perm
buffer), and stained with the antibody panel (Supporting Fig. S3).
All fluorescence-activated cell sorting (FACS) data were analyzed by an LSRII
flow cytometer (BD Biosciences, Franklin Lakes, NJ). Data were collected with BD
FACS DIVA software (BD Biosciences, San Jose, CA) and analyzed with TreeStar
FlowJo software (FlowJo, LLC, Ashland, OR).
STATISTICAL ANALYSIS
GraphPad Prism software (version 7; GraphPad Software Inc., La Jolla, CA) was
used for all statistical analysis. Nonparametric or parametric two-tailed tests
were used, based on distribution of the population: Paired t tests were used for
comparisons for matched samples and unpaired t tests for unrelated sample
comparisons (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). Only
statistically significant results were reported in the figures. Unless stated
otherwise, all values will be shown as mean population with 95% confidence
interval range.
RESULTS
HCV HETEROLOGOUS PRIME-BOOST VACCINATION WITH CHAD3 AND MVA-NS INDUCES
HCV-SPECIFIC MHC CLASS II TETRAMER+ CD4+ T CELLS
All vaccinated volunteers chosen for tetramer analysis received priming
vaccination with ChAd3-NSmut1 (dose, 2 × 1010 plaque-forming units), and 8 of 10
volunteers received boosting vaccination with MVA-NSmut at week 8 (dose, 2 × 108
viral particles [vp]). The remaining 2 volunteers (HCV003347 and HCV003374)
received boosting with MVA-NSmut at week 8 at doses of 2 × 107 and 2 × 106 vp,
respectively. Some volunteers also received either an additional round of
ChAd3/MVA vaccination or single additional MVA vaccination as part of their
trial protocol. Characteristics of vaccine volunteers can be found in Supporting
Fig. S1C.
In these volunteers, MHC class II tetramer-positive populations were assessed
over time; at baseline before vaccination, after prime vaccination, after boost
vaccination, and at the end of study. Examples plots are shown in Fig. 1A for 2
vaccinated volunteers at different time points.
Fig. 1
Open in figure viewerPowerPoint
HCV-specific MHC class II tetramer+ CD4+ T cells following viral vectored
vaccination. (A) Example FACS plot of staining with tetramers 14
HCV-NS4B1806-1818 in 2 vaccinated healthy volunteers over the study course.
Gating is on live CD3+ cells. Values indicate the percentage of CD4+ T cells
binding the tetramer. (B) Percentage of tetramer+ CD4+ cells after ex vivo
staining in vaccinated healthy volunteers at different time points of the
vaccination regimen. Controls are represented by healthy volunteers stained with
mismatched MHC class II tetramer. Error bars represent the SEM.
The highest frequency of HCV-specific T cells was observed following boosting
vaccination with MVA (mean, 0.062% [0.023-0.10] of CD4+ T cells). The population
was significantly larger compared with other trial time points. The control
population, healthy HLA-mismatched PBMCs not exposed to HCV virus or
vaccination, was comparable to baseline staining (Fig. 1B).
ROBUST AND DURABLE EX VIVO TETRAMER-POSITIVE CD4+ T-CELL POPULATIONS ARE
DETECTABLE ALSO IN SR, BUT NOT IN CHRONIC HCV, PATIENTS FOLLOWING DAA THERAPY
We then sought to compare the HCV tetramer-specific CD4+ T-cell population
induced following vaccination to a natural model of HCV clearance (SR) as well
as to cured HCV infection with DAA therapy. Given the widely published data
suggesting negligible or absent HCV-specific CD4+ T cells in chronic
infection,(15, 18) we were interested whether these populations recover
following DAA-mediated viral clearance.
Ten samples from volunteers with SR and 21 patients receiving DAA therapy were
selected (where HLA matched available tetramers). Samples were taken between 0
and 52 weeks before commencing treatment (pre-DAA) and, on average, 6 weeks
following treatment completion (range, 0-26 weeks; post-DAA).
MHC class II tetramer+ CD4+ T cells were detectable following SR (mean, 0.036%
[0.0-0.08] of total CD4+ T cells; Fig. 2B,C). This population was comparable in
magnitude to the vaccine groups, in particular at the final trial time point
(mean, 0.027% [0.017-0.038]). There were few detectable CD4+ tetramer-positive T
cells following ex vivo staining in the DAA-treated patient groups at either
time point (Fig. 2A,C).
Fig. 2
Open in figure viewerPowerPoint
Assessment of ex vivo tetramer+ CD4+ T cells pre-DAA and post-DAA treatment,
following vaccination and in SR volunteers. (A,B) FACS example plot of staining
with tetramers 14 HCV-NS4B1806-1818, 24 HCV-NS31535-1551 or pool of tetramers 17
HCV-NS31582-1597, tetramer 18 HCV-NS31411-1425, and tetramer 19 HCV-NS31535-1551
in 2 DAA patients pretherapy and posttherapy and in 2 SRs. Gating is on live
CD3+ cells. Values indicate the percentage of CD4+ T cells binding the tetramer.
(C) Percentage of tetramer+ CD4+ cells after ex vivo staining at the end of
study of vaccinated healthy volunteers, DAA pretherapy and posttherapy, and in
SR persons. Error bars represent the SEM. Only statistical differences are
shown.
HCV VACCINATION INDUCES HIGH LEVELS OF LONG-LIVED MEMORY (CD127) AND
COSTIMULATORY (CD28) MARKERS IN EX VIVO HCV-SPECIFIC CD4+ T CELLS ANALOGOUS TO
SPONTANEOUS RESOLUTION
We assessed the phenotypic profile of ex vivo HCV-specific tetramer+ CD4+ T
cells in chronic HCV patients, vaccinated volunteers, and SR persons. Following
staining with MHC class II tetramers, PBMCs were stained with a surface panel
containing costimulatory and memory markers (gating strategy showed in
Supporting Fig. S4).
CD28 (a critical costimulatory molecule for T-cell activation) was highly
expressed at all time points after vaccination, with peak expression postboost
(mean, 96.6% [94.3-98.9]). CD28 expression was significantly lower in patients
with chronic HCV (mean, 81.4% [71.1-91.8]; Fig. 3A).
Fig. 3
Open in figure viewerPowerPoint
Analysis of costimulatory and memory cell-surface markers in chronic HCV,
vaccinated, and SR volunteers. (A,B) Thawed PBMCs were costained with MHC class
II tetramers and anti-CD127 and -CD28 antibodies in chronic HCV patients,
vaccinated volunteers at different trial time points, and in SR persons. (C)
Proportion of tetramer+ cells expressing Tcm, Tem, Temra, and Tscm phenotype in
the same cohorts. All tetramer staining and phenotyping were performed ex vivo.
Error bars represent the SEM. Only statistical differences are shown.
CD127 (IL7R) expression of tetramer+ T cells increased during the course of the
trial and reached peak expression at end of treatment (EOT) time point (mean,
74.3% [64.8-83.8]). CD127 expression at EOT was comparable to that observed in
SR (mean, 71.05% [60.02-82.1]), and both these groups demonstrated significantly
higher expression compared to both prime and boost vaccination and chronic HCV
patients (Fig. 3B).
Memory subsets were further assessed as follows: stem memory T cells (Tscm;
CCR7+/CD45RA+), effector memory T cells (Tem; CCR7−/CD45RA−); central memory T
cells (Tcm; CCR7+/CD45RA−); and effector memory retinoic acid–positive cells
(Temra; CCR7−/CD45RA+).
Central and effector memory subsets were the most predominant at all vaccine
time points as well as in SR (Fig. 3C). The percentage of Tcm cells was similar
between these groups, ranging from 29.9% (20.3-39.1) to 38% (28.15-47.7) of
tetramer+ CD4+ T cells (prime and SR, respectively). Chronic HCV patients
comparatively had lower expression (mean, 23.8% [12.5-35]), significantly so
when compared to SR (P = 0.04).
Similarly, robust Tem populations were observed in all groups and was highest
following boosting vaccination (49.6% [33.8-65.6]), reducing significantly by
EOT (mean, 31.6% [20.9-42.4]; (P = 0.0075). Populations of Tscms were observed
to contract between prime and boost vaccination (mean, 22.8% [12.4-33.3] to
13.2% [4.5-21.8]; P = 0.01%) and then re-expand at the end of the trial (mean,
25.3% [17.2-33.5]; P = 0.005%).
Temras were the smallest population observed out of the memory marker subsets.
However, numbers increased significantly at the end of the trial (mean, 9.8%
[2.1-17.5]) compared with prime (mean, 6.1% [0.75-11.5]; P = 0.01) and boost
vaccination (mean, 4.6 [0.0-11.2]; P = 0.007; Fig. 3C).
T-BOX TF TBX21 AND EOMESODERMIN EXPRESSION FOLLOWING VACCINATION MIRRORS SR
Intranuclear phenotypic analysis was performed on two transcription factors
(TFs) important in T-cell activation and differentiation into effector and
memory cells: T-box TF TBX21 (T-bet) and Eomesodermin (Eomes). Robust T-bet
expression was demonstrated following prime and boost vaccination, but, however,
was reduced at the EOT (mean, 41.4% [15.9-55] of tetramer+ CD4+ T cells), which
reached significance when compared with T-bet staining following boosting
vaccination (mean, 61.5% [29.3-84.6]; P = 0.03). T-bet expression in SR was
lower than all other groups, reaching significance when compared with prime
(P = 0.04) and boost vaccination (P = 0.009; Fig. 4A). There are fewer published
studies assessing Eomes behavior in CD4+ T cells.(19, 20) Expression of Eomes
was low at all vaccine time points as well as in chronic HCV patients and in SR
individuals (Fig. 4B).
Fig. 4
Open in figure viewerPowerPoint
TF analysis in chronic HCV, vaccinated, and SR volunteers. (A,B) Percentage of
tetramer+ CD4+ T cells expressing T-bet and Eomes in chronic HCV patients,
vaccinated volunteers, and SR persons. (C) Costaining with class II tetramers
and anti-human T-bet and Eomes antibodies in the same cohorts. Error bars
represent the SEM. Only statistical differences are shown.
Furthermore, there is a growing body of evidence that changing levels of T-bet
and Eomes expression occur on the pathway to T-cell exhaustion resulting in a
final T-betlo/Eomeshi population.(21) This exhausted population was very low in
chronic HCV patients, and in each time point after the study, although it was
significantly higher at the EOT compared to SR (P = 0.0087; Fig. 4C).
In summary, expression of T-bet significantly decreased over the course of the
trial and was lowest in SR.
PROLIFERATIVE CAPACITY OF HCV TETRAMER-SPECIFIC CD4+ T CELLS FOLLOWING HCV
VACCINATION IS ROBUST, BUT LIMITED FOLLOWING DAA MEDIATED CURE
We sought to assess the proliferative capacity of HCV-specific CD4+ T cells
following DAA-mediated cure (n = 21) and compare with HCV vaccination (n = 5)
and SR (n = 10). Proliferative capacity of HCV-specific CD4+ T cells in all
groups was assessed using MHC class II tetramers following in vitro culture with
peptide corresponding to the tetramer and IL-2 for 2 weeks (Fig. 5A-D). Between
one and six tetramers were used with each sample (depending on HLA specificity),
with the highest-magnitude tetramer for each individual sample chosen for
analysis (Supporting Fig. S1D).
Fig. 5
Open in figure viewerPowerPoint
Proliferative capacity of CD4+ T cells in pre-DAA– and post-DAA–treated
patients, vaccinated, and SR volunteers. (A-C) Example FACS plots of tetramer+
CD4+ T cells after 14 days of culture with peptide matching tetramer sequence in
2 DAA patients pretreatment and posttreatment (A), 2 vaccinated volunteers at
boost and EOT (B), and 2 SR persons (C). Values indicate the percentage of CD4+
T cells binding tetramer. (D) Scatter plot with bar showing the percentage of
tetramer+ CD4+ T cells after culture in pre-DAA and post-DAA, vaccination, and
SR. Error bars represent the SEM. Only statistical differences are shown.
There was a robust population of HCV tetramer+ CD4+ T cells induced following
viral vectored vaccination (mean, 10.94% [0.0-26.6] at boost and 6.7% [0.0-14.6]
at EOT). Likewise, there was a comparable, though smaller, population in SR
(mean, 3.6% [0.6-6.6]; Fig. 5B-D). In comparison, there was a poor proliferative
response in the DAA patient cohorts (mean, 1.3% [0.0-3.1] and 0.8% [0.02-1.70]
for pre-DAA and post-DAA, respectively; Fig. 5A,D). There was no clear pattern
or trend between the pre-DAA and post-DAA treatment groups, with subgroup
analyses comparing patients who had both a 2-fold increase and 2-fold decrease
in tetramer+ populations showing no significant differences in phenotypic
characteristics (Supporting Fig. S5).
Single-epitope sequences that were contained within the tetramers were compared
following in vitro culture to assess the immunogenicity of each. The peptide
sequence contained in tetramer 14 (NS31806-1818) restricted to HLA DRB1*0101
was, by far, the most immunogenic across all four groups. In comparison, T-cell
populations binding tetramers 22 (NS2794-810) and 29 (NS5A1957-1975) were rarely
detectable (Supporting Fig. S6).
FUNCTIONALITY OF CD4+ T CELLS IN PRE-DAA AND POST-DAA TREATMENT, VACCINATED, AND
SR GROUPS
To evaluate the capacity of cytokine secretion following HCV vaccination, SR,
and following DAA treatment, ICS assays were performed following in vitro
expansion using peptide pools F-H (corresponding to NS3-4), CD28, and periodic
addition of IL-2. These peptide pools were chosen based on their immunogenicity
demonstrated in HCV vaccine trials to date(7, 8) as well as published data
identifying a number of immunogenic CD4+ epitopes in NS3-4 proteins.(22, 23)
Representative ICS plots are shown in Fig. 6A.
Fig. 6
Open in figure viewerPowerPoint
Functional capacity of CD4+ T cells in pre-DAA– and post-DAA–treated patients,
vaccinated, and SR volunteers. (A) Example FACS plots showing TNFα/IFN-γ after
intracellular cytokine staining are shown for CD4+ T cells stimulated with NS3-4
or DMSO control in DAA patients pretreatment and posttreatment, vaccine
volunteers (after boost vaccination), and SR. (B) Comparison of cytokine
production by CD4+ T cells pre-DAA (n = 14) and post-DAA treatment (n = 14),
after ChAd3/MVA vaccination (n = 5), and in SR (n = 7). PBMCs were cultured for
14 days with peptide matching NS3-4 (pools F+G+H), rested, and restimulated with
the same peptides overnight. Staining in DMSO wells was subtracted. Error bars
represent the SEM. Only statistical differences are shown.
Overall, there was poor cytokine production in the DAA patient cohort, with no
recovery of cytokine production observed following viral cure and no significant
differences between the two groups (Fig. 6B). In comparison, vigorous cytokine
production was observed for three of the four cytokines tested in both SR and
following vaccination. Production of tumor necrosis factor alpha (TNF-α),
interferon-gamma (IFN-γ), and macrophage inflammatory protein 1 beta (MIP-1β)
was significantly greater in these groups compared to both pre-DAA and post-DAA
treatment, peaking at 21.2% (10.18-32.20) for TNF-α production in SR. No
significant difference was observed between SR and vaccination in any measured
cytokine (Fig. 6B). We observed a strong correlation of cytokine production
between CD4+ and CD8+ T cells measured at the same time points after vaccination
(r = 0.73; P ≤ 0.0001; Supporting Fig. S7).
DISCUSSION
This research addresses two important areas of HCV research; the behavior of
viral-specific CD4+ T cells following HCV vaccination and the extent of immune
recovery following DAA-mediated HCV cure. CD4+ T cells are widely believed to
play a vital role in the early immune response to HCV infection. However,
HCV-specific CD4+ T cells have been challenging to study, largely because of
limited/absent populations and limited tools for assessing them ex vivo. The use
of MHC class II tetramers to predictably assess CD4+ responses has been limited
by the promiscuous epitope binding to MHC class II alleles(17) and a variable
length of amino acids optimal for binding to the MHC.(24) Here, we used a panel
MHC class II tetramers, ICS, and peptide-stimulated cell lines to perform a
detailed analysis of the functional and phenotypic behavior of CD4+ T cells
following HCV viral vectored vaccination in chronic HCV infection before and
following DAA-mediated HCV cure and in SR.
Using large input cell numbers (6-8 × 106 cells), we were able to detect
HCV-specific tetramer+ CD4+ T cells following ChAd3/MVA prime-boost viral
vectored vaccination in ex vivo analysis. Tetramer populations were identified
at every time point after vaccination and peaked following boosting vaccination,
comparable to the kinetics of CD8+ T cells following identical vaccination
schedules.(7) Within our panel of 10 tetramers, we observed variable peptide
sequence immunogenicity; however, epitopes restricted to NS3, notably DRB1*01
restricted sequence NS31806-1818, were the most immunogenic, supporting previous
publications.(15) Importantly, the size of the tetramer+ cell population at the
final trial time point, and thus the best predictor of the residual memory
population following vaccination, was comparable to that observed in SR.
Although the number of HCV-specific CD4+ T cells induced by vaccination is
undoubtedly important, the phenotypic and functional properties of CD4+ T cells
are likely to be significant determinants of a protective response. We
hypothesize that vaccine-induced CD4+ T cells that function in an analogous way
to CD4+ T cells following SR may be predictive of a protective response. We
analyzed a number of cell-surface markers associated with T-cell activation
(CD28) and memory differentiation (CD127, C-C chemokine receptor type 7 [CCR7],
and CD45RA) in chronic HCV patients, vaccinated volunteers, and SR individuals,
using tetramer+ populations in ex vivo CD4+ T cells.
A critical component of a successful vaccine is one that induces long-lived
memory cells capable of homeostatic proliferation,(25) with appropriate
costimulatory molecules. CD28 is a critical costimulatory molecule in T-cell
activation,(26) and CD4+CD28– T cells are widely observed to be present in
chronic viral infections, such as cytomegalovirus,(27) human immunodeficiency
virus,(28) and hepatitis B virus.(29) CD28 was highly expressed at all time
points after vaccination. Significantly lower levels were expressed in chronic
HCV patients compared to vaccinated and SR volunteers. CD127 (IL-7rα) is
strongly associated with T-memory-cell development. Using adoptive transfer
models in mice, Kaech et al. showed that expression of CD127 was required as a
precursor for functional memory-cell development.(30) IL7 (the ligand for CD127)
has been shown to play a critical role in maintenance of a polyclonal and
functionally diverse repertoire of human CD4(+) memory T cells in the absence of
ongoing antigen stimulation.(31) We have shown CD127 expression to be
progressively up-regulated in ex vivo HCV tetramer+ CD4+ T cells following HCV
vaccination, suggesting that this marker is associated with memory-cell
development. When compared to expression in natural HCV clearance, expression
was similar to the final trial time point (mean, 74.3% at EOT and 71% for the SR
group) and significantly lower in chronic HCV patients (mean 49%).
Much work has been directed at elucidating and characterizing T-cell memory
subsets with a focus on CD8+ T cells and a paucity of data assessing CD4+
T-memory subsets. Here, we provide a valuable insight into the behavior of these
cell populations in tetramer+ CD4+ T cells in chronic HCV patients, vaccinated
volunteers, and SR individuals. In all settings, Tcms and Tems were the
predominant memory subset induced, in keeping with published data in CD8+
populations. These two populations are perhaps the most widely studied, and both
contribute critical functions to a successful memory response—rapid
differentiation on antigen re-exposure (Tems) and long half-life and
proliferative capacity (Tcms). Tscms have been recently described as long-lived
multipotent memory cells.(32, 33) We show a robust population of Tscms in all
study groups. Notably, Tscm expression expanded significantly between boosting
and EOT (mean, 13.2%-25.3%), similar to previous observations in CD8+ T cells
following ChAd3/MVA prime boost.(7) CD45RA re-expression in long-lived memory
cells is important for the ability to self-renew, and to differentiate into
other memory subsets, whereas CCR7 re-expression is important for exposure to
circulating antigen—both critical components of memory cells. They have been
suggested to play a role in the persistence of latent HIV reservoirs in CD4+ T
cells of infected hosts,(34) but their role in CD4+ T cells in maintaining
protection following both viral clearance and vaccination—although presumed—has
been challenging to identify. We observed small populations of Temras in all
groups, consistent with previous observations in CD4+ T cells,(35) and,
similarly to Tscm, the expansion of this subset between boosting and EOT may
reflect an evolving CD4+ memory phenotype with time. The role of Temras in CD4+
T cells has not been fully elucidated, although they have been associated with
CX3C chemokine receptor 1 expression, suggesting a potential cytotoxic
phenotype.(36, 37)
TF analysis was performed on tetramer-gated ex vivo CD4+ T cells specific for
HCV. T-bet, a central regular in CD4+ Th1 differentiation and widely studied in
both CD4+ T and CD8+ subsets, was observed to decrease in expression over time
following vaccination, reflecting previous observations that T-bet expression
declines as CD4+ T cells gain a more memory-like phenotype.(20) The reduced
T-bet expression in SR (on average, sampled many years post-HCV exposure)
further supports this. Unlike its CD8+ counterpart, there is a lack of
literature assessing Eomes expression in CD4+ T cells following antigen
exposure. Knowledge to date is largely confined to assessing Eomes in bulk CD4+
phenotyping, with low expression observed, particularly in Tcms compared with
Tems and effector cells.(19, 20) We observed low levels of Eomes expression
throughout the vaccine trial and in SR, in keeping with published data.
Importantly, the comparable expression of both TFs following HCV
vaccination—particularly at the final trial time point—with SR indicates that
vaccination may induce a memory CD4+ T cell with a protective transcriptional
signature.
In the chronic HCV group, we observed the memory phenotype and TF expression of
HCV tetramer+ CD4+ T cells to be similar to all vaccine time points and SR, with
the exception of a significant decrease in Tcm in chronic HCV compared to SR. A
possible explanation for this similarity is selection bias, in that those CD4+ T
cells detectable in chronic infection are relatively functionally preserved,
while the most functionally exhausted are deleted.
The ability of antigen-specific memory T cells to rapidly expand and produce
cytokine upon re-exposure to antigen is a critical component of a functional and
protective cell-mediated response.(38-40) Following peptide-stimulated
short-term culture, we observed robust proliferation as well as effector
cytokine production after ChAd3/MVA vaccination, comparable to SR. In
particular, antiviral cytokines IFN-γ and TNF-α were readily produced following
ICS stimulation, which are well-defined correlates of highly effective T cells.
IL-2 production was attenuated; progressively diminishing IL-2 production has
previously been observed following T-cell activation through a negative feedback
loop.(41, 42) We hypothesize that poor IL-2 secretion was attributed to IL-2
down-regulation following activation and proliferation in cell lines. Overall,
these results mirror the behavior of HCV-specific CD8+ T cells following
vaccination,(7, 8) and there was a high correlation of cytokine production
between CD4+ and CD8+ T cells, suggesting a coordinated response to vaccination.
Similar phenotypic and functional correlations between peptide-specific CD4+ and
CD8+ T cells have been observed in highly effective viral vaccines, such as
yellow fever vaccine.(43) The fact that CD4+ vaccine-induced responses are
phenotypically and functionally analogous to that observed in natural HCV
clearance is suggestive of a protective immune response. Importantly, our
observations in SR, made on average many years or decades following exposure,
suggests that the phenotype observed is long-lived, as suggested by previous
studies.(44)
The other critical question addressed here is the state of the host immune
system after DAA-mediated HCV cure. T-cell exhaustion, or the hierarchical loss
of effector functions and eventual anergy and deletion,(45) is a well-described
process in chronic infection, including HCV.(46, 47) The new era of DAA
treatment gives a unique opportunity to interrogate immunological recovery
following cure of chronic viral infection. Using tetramer assays and ICS
following peptide stimulation, we were unable to detect an increase in
proliferative capacity or production of antiviral cytokines following
DAA-mediated HCV cure. There are a number of hypotheses to explain this. First,
more time may be needed for the host immune system to “reset” following cure and
functional ability to be restored. Second, antigen repriming with naïve thymic
emigrants may be required following viral cure to induce a functional immune
response. Third, all the patients in this cohort had liver cirrhosis, well
described to induce an immunosuppressive state in the host.(48) A final
hypothesis, however, is that the host HCV-specific immune response in chronic
HCV infection is terminally exhausted and unable to be reversed following cure.
Our observations contrast with a previous study demonstrating T-cell recovery
following IFN-free therapy(49); the study by Martin et al. assessed CD8 T cells
in treatment-naïve patients without cirrhosis, suggesting that the limited
recovery observed in our population may be attributable to previous or current
IFN treatment or presence of cirrhosis. However, HCV reinfection is known to
occur following DAA treatment in those with ongoing HCV exposure, including
patients without cirrhosis. These clinical data show that DAAs do not
consistently restore HCV-specific immune responses to protect against
reinfection. An important remaining question is whether HCV-specific immune
responses can be restored following DAA cure to a level that will enable
effective HCV-specific vaccination strategies. Clinical trials in progress
(NCT03688061) will address this question directly.
In summary, we provide a detailed analysis of phenotypic and functional
characteristics of HCV-specific CD4+ T cells. Following vector-based HCV
vaccination, HCV-specific CD4+ T cells are analogous to those in the
gold-standard setting of SR in both phenotypic and functional parameters,
suggesting the induction of effective and protective CD4+ T cells following
vaccination. Furthermore, we show minimal functional recovery of the same
population of cells following DAA-mediated cure. This work has wider
implications to both T-cell vaccine development and chronic viral infection.
Further studies assessing both in vivo responses to HCV vaccines and behavior of
CD4+ T cells following vaccination in DAA-mediated cure are required.
ACKNOWLEDGMENT
The vaccine trials were supported by funding from the EU Sixth (HEPACIVAC) and
Seventh (FP7/2007-2013) Framework Programmes. This research was supported by the
Wellcome Trust (WT109965MA to P.K.), NIHR Senior Fellowships (P.K. and E.J.B.),
NIH U19 I082360 (P.K.), and MRC (E.B.). The authors acknowledge the BRC Oxford
GI Biobank and BRC Oxford IBD Cohort in collecting and making available the
samples/data used in the generation of this publication. The research was also
funded/supported by the National Institute for Health Research (NIHR) Oxford
Biomedical Research Centre (BRC). The views expressed are those of the author(s)
and not necessarily those of the NHS, the NIHR. or the Department of Health. The
authors also acknowledge the hepatology nurses at the John Radcliffe Hospital,
Oxford, and the staff at the CCVTM, Oxford.
AUTHOR CONTRIBUTIONS
E.B., F.H., L.D., S.C., A.F. and P.K. contributed to conception and design.
F.H., I.E., L.S., A.B., C.P., C.L., C.G., B.T., J.K. contributed to acquisition
analysis. E.B., F.H., I.E. and P.K. contributed to the interpretation of the
data. E.B., F.H. and I.E. drafted the article, revised critically for important
intellectual content and worked for a final approval of version to be published.
SUPPORTING INFORMATION
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Author names in bold designate shared co-first authorship.
CITING LITERATURE
Volume72, Issue5
November 2020
Pages 1541-1555
* FIGURES
* REFERENCES
* RELATED
* INFORMATION
RECOMMENDED
* An immunomodulatory role for CD4+CD25+ regulatory T lymphocytes in hepatitis
C virus infection
Roniel Cabrera, Zhengkun Tu, Yiling Xu, Roberto J. Firpi, Hugo R. Rosen, Chen
Liu, David R. Nelson,
Hepatology
* Detection of functionally altered hepatitis C virus-specific CD4+ T cells in
acute and chronic hepatitis C
Axel Ulsenheimer, J. Tilman Gerlach, Norbert H. Gruener, Maria-Christina
Jung, Carl-Albrecht Schirren, Winfried Schraut, Reinhart Zachoval, Gerd R.
Pape, Helmut M. Diepolder M.D.,
Hepatology
* Hepatitis C virus infection after liver transplantation is associated with
lower levels of activated CD4+CD25+CD45RO+IL-7rαhigh T cells
Donatella Ciuffreda, Laura Codarri, Leo Buhler, Laure Vallotton, Emiliano
Giostra, Gilles Mentha, Philippe Morel, Giuseppe Pantaleo, Manuel Pascual,
Liver Transplantation
* A pan-genotype hepatitis C virus viral vector vaccine generates T cells and
neutralizing antibodies in mice
Timothy Donnison, Joey McGregor, Senthil Chinnakannan, Claire Hutchings, Rob
J. Center, Pantelis Poumbourios, Paul Klenerman, Heidi E. Drummer, Eleanor
Barnes,
Hepatology
* CD4+ Memory Stem T Cells Recognizing Citrullinated Epitopes Are Expanded in
Patients With Rheumatoid Arthritis and Sensitive to Tumor Necrosis Factor
Blockade
Beatrice C. Cianciotti, Eliana Ruggiero, Corrado Campochiaro, Giacomo
Oliveira, Zulma I. Magnani, Mattia Baldini, Matteo Doglio, Michela Tassara,
Angelo A. Manfredi, Elena Baldissera, Fabio Ciceri, Nicoletta Cieri, Chiara
Bonini,
Arthritis & Rheumatology
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