Publications by authors named "Patrick T F Kennedy"

39 Publications

Clinical and occupational health management of healthcare workers living with chronic hepatitis B: UK policy and international comparisons.

J Viral Hepat 2021 Mar 4. Epub 2021 Mar 4.

Barts Liver Centre, Immunobiology, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.

Hepatitis B virus (HBV) is a highly infectious bloodborne virus, which remains endemic in large geographic areas and represents a major global healthcare challenge. HBV transmission from healthcare workers, who perform exposure prone procedures (EPP), to patients is a recognized transmission risk, which varies widely globally. Although the risk is small in developed countries, it increases significantly in high-prevalent, low-resource countries, representing a major challenge to these healthcare systems and underlining the necessity for robust guidance to be in place. The HBV landscape has evolved as a result of global vaccination programs, implementation of standard precautions and the advent of new generation antiviral agents (3rd generation nucleos(t)ide analogues). In light of the progress in the field, the UK Advisory Panel for Healthcare Workers Infected with Bloodborne Viruses (UKAP) recently issued updated guidance, which essentially removes past barriers, restricting healthcare workers from performing EPPs solely on the basis of HBV DNA level, regardless of hepatitis B 'e' antigen and/or treatment status. Although the current recommendations remain conservative compared to those of other developed healthcare systems, UK practice is now in line with other high-income countries, while ensuring patient safety remains paramount, without unduly restricting HCWs from clinical practice. The current article presents the latest UKAP guidance, considers its implications for HCWs and compares it with the guidance from major international scientific societies and governing bodies.
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http://dx.doi.org/10.1111/jvh.13494DOI Listing
March 2021

Chronic hepatitis B: the demise of the 'inactive carrier' phase.

Hepatol Int 2021 Feb 27. Epub 2021 Feb 27.

Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India.

Chronic hepatitis B (CHB) remains a global healthcare burden. Although the recent developments in the field have led to a reduction in incidence, the morbidity and mortality including liver cirrhosis and hepatocellular carcinoma (HCC) remain a formidable challenge. Advances in understanding the immunopathogenesis of CHB have led to a recent change in clinical categorization. EASL introduced the term hepatitis B 'e' antigen (HBeAg)-negative chronic infection, to replace the historical term 'inactive carrier' disease phase, the commonest CHB phase. Although this disease phase is associated with a favorable prognosis, it is not a truly 'inactive' disease phase with no ostensible liver disease, as inferred by the previous anachronistic terminology, and the risk of spontaneous reactivation and the potential risk of disease progression and HCC development are not negligible. Likewise, the APASL also uses the term "Incidentally Detected Asymptomatic Hepatitis B surface antigen (HBsAg)-positive Subject (IDAHS)", comprising all HBsAg-positive subjects who are incidentally detected during routine tests, without any previous or present symptoms of liver disease. This entity includes HBV infection with varied stages of liver disease. Antiviral treatment is generally reserved for patients with active inflammation and/or at risk of disease progression and HCC development. HBsAg loss is considered an optimal treatment endpoint, and may also be achievable in HBeAg-negative chronic infection and IDAHS. In light of this, and the emerging novel HBV therapies, lowering the treatment threshold and a 'Treat All' approach should now be considered. In this review, we summarize the literature and guidance on HBeAg-negative chronic infection, and we make a concerted effort to present the reasons why the one-dimensional term 'inactive carrier' should be abandoned.
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http://dx.doi.org/10.1007/s12072-021-10137-2DOI Listing
February 2021

Whole exome HBV DNA integration is independent of the intrahepatic HBV reservoir in HBeAg-negative chronic hepatitis B.

Gut 2020 Dec 21. Epub 2020 Dec 21.

Barts Liver Cente, Immunobiology, Blizard Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK

Objective: The involvement of HBV DNA integration in promoting hepatocarcinogenesis and the extent to which the intrahepatic HBV reservoir modulates liver disease progression remains poorly understood. We examined the intrahepatic HBV reservoir, the occurrence of HBV DNA integration and its impact on the hepatocyte transcriptome in hepatitis B 'e' antigen (HBeAg)-negative chronic hepatitis B (CHB).

Design: Liver tissue from 84 HBeAg-negative patients with CHB with low (n=12), moderate (n=25) and high (n=47) serum HBV DNA was analysed. Covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) were evaluated by quantitative PCR, whole exome and transcriptome sequencing was performed by Illumina, and the burden of HBV DNA integrations was evaluated by digital droplet PCR.

Results: Patients with low and moderate serum HBV DNA displayed comparable intrahepatic cccDNA and pgRNA, significantly lower than in patients with high HBV DNA, while hepatitis B core-related antigen correlated strongly with the intrahepatic HBV reservoir, reflecting cccDNA quantity. Whole exome integration was detected in a significant number of patients (55.6%, 14.3% and 25% in high, moderate and low viraemic patients, respectively), at a frequency ranging from 0.5 to 157 integrations/1000 hepatocytes. Hepatitis B surface antigen >5000 IU/mL predicted integration within the exome and these integrations localised in genes involved in hepatocarcinogenesis, regulation of lipid/drug metabolism and antiviral/inflammatory responses. Transcript levels of specific genes, including the proto-oncogene hRAS, were higher in patients with HBV DNA integration, supporting an underlying oncogenic risk in patients with low-level to moderate-level viraemia.

Conclusions: HBV DNA integration occurs across all HBeAg-negative patients with CHB, including those with a limited HBV reservoir; localising in genes involved in carcinogenesis and altering the hepatocyte transcriptome.
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http://dx.doi.org/10.1136/gutjnl-2020-323300DOI Listing
December 2020

Prioritisation and the initiation of HCC surveillance in CHB patients: lessons to learn from the COVID-19 crisis.

Gut 2020 11 25;69(11):1907-1912. Epub 2020 May 25.

Barts Liver Centre, Immunobiology, Blizard Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK

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http://dx.doi.org/10.1136/gutjnl-2020-321627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295856PMC
November 2020

Effects of Hepatitis B Surface Antigen on Virus-Specific and Global T Cells in Patients With Chronic Hepatitis B Virus infection.

Gastroenterology 2020 08 14;159(2):652-664. Epub 2020 Apr 14.

Emerging Infectious Diseases Program, Duke-NUS Medical School, Singapore; Singapore Immunology Network, A∗STAR, Singapore. Electronic address:

Background & Aims: Chronic hepatitis B virus (HBV) infection is characterized by the presence of defective viral envelope proteins (hepatitis B surface antigen [HBsAg]) and the duration of infection-most patients acquire the infection at birth or during the first years of life. We investigated the effects of these factors on patients' lymphocyte and HBV-specific T-cell populations.

Methods: We collected blood samples and clinical data from 243 patients with HBV infection (3-75 years old) in the United Kingdom and China. We measured levels of HBV DNA, HBsAg, hepatitis B e antigen, and alanine aminotransferase; analyzed HBV genotypes; and isolated peripheral blood mononuclear cells (PBMCs). In PBMCs from 48 patients with varying levels of serum HBsAg, we measured 40 markers on nature killer and T cells by mass cytometry. PBMCs from 189 patients with chronic infection and 38 patients with resolved infections were incubated with HBV peptide libraries, and HBV-specific T cells were identified by interferon gamma enzyme-linked immune absorbent spot (ELISpot) assays or flow cytometry. We used multivariate linear regression and performed variable selection using the Akaike information criterion to identify covariates associated with HBV-specific responses of T cells.

Results: Although T- and natural killer cell phenotypes and functions did not change with level of serum HBsAg, numbers of HBs-specific T cells correlated with serum levels of HBsAg (r = 0.3367; P < .00001). After we performed the variable selection, the multivariate linear regression model identified patient age as the only factor significantly associated with numbers of HBs-specific T cells (P = .000115). In patients younger than 30 years, HBs-specific T cells constituted 28.26% of the total HBV-specific T cells; this value decreased to 7.14% in patients older than 30 years.

Conclusions: In an analysis of immune cells from patients with chronic HBV infection, we found that the duration of HBsAg exposure, rather than the quantity of HBsAg, was associated with the level of anti-HBV immune response. Although the presence of HBs-specific T cells might not be required for the clearance of HBV infection in all patients, strategies to restore anti-HBV immune responses should be considered in patients younger than 30 years.
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http://dx.doi.org/10.1053/j.gastro.2020.04.019DOI Listing
August 2020

Early treatment of chronic hepatitis B in children: Everything to play for?

J Hepatol 2020 04 14;72(4):802-803. Epub 2020 Feb 14.

Barts Liver Centre, Immunobiology, Blizard Institute, Barts and The London, School of Medicine & Dentistry, Queen Mary University of London, London, United Kingdom.

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http://dx.doi.org/10.1016/j.jhep.2019.12.007DOI Listing
April 2020

Hepatitis B reactivation: reducing the risk in patients with inflammatory bowel disease.

Gut 2020 11 30;69(11):2053-2054. Epub 2020 Jan 30.

Barts Liver Centre, Barts and The London School of Medicine and Dentistry, London, UK

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http://dx.doi.org/10.1136/gutjnl-2019-319480DOI Listing
November 2020

Emerging tools in the changing landscape of chronic hepatitis B management.

Expert Rev Anti Infect Ther 2019 12 27;17(12):943-955. Epub 2019 Nov 27.

Barts Liver Centre, Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK.

: The availability of a preventative vaccine, interferon, and nucleos(t)ide analogs have provided progress in the control of chronic hepatitis B (CHB). Despite this, it remains a major contributor to global morbidity and mortality. Developments in our understanding of the pathogenesis of CHB and the emergence of new therapies are paving the way, as we move toward HBV cure.: We performed bibliographical searches of online databases to review the literature regarding conventional disease phases of CHB. We provide the latest evidence challenging the perception of the natural history of CHB, noting that previously considered quiescent disease phases may not represent benign disease states devoid of progression. We explore the use of potential novel immunological and viral tools which should enhance disease stratification and management decisions in the coming years. Finally, we discuss the timing of treatment and how this could be initiated earlier to improve treatment outcomes, preventing sequelae of chronic infection.: The treatment paradigm in CHB is set to change with multiple novel agents in early phase clinical trials with the aim of a functional cure. An improved understanding of disease pathogenesis and the timing of treatment will be critical to the success of new therapies.
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http://dx.doi.org/10.1080/14787210.2019.1694906DOI Listing
December 2019

Dynamics and genomic landscape of CD8 T cells undergoing hepatic priming.

Nature 2019 10 2;574(7777):200-205. Epub 2019 Oct 2.

Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.

The responses of CD8 T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8 T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8 T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8 T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8 T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.
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http://dx.doi.org/10.1038/s41586-019-1620-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858885PMC
October 2019

HBV antiviral immunity: not all CD8 T cells are born equal.

Gut 2019 05 30;68(5):770-773. Epub 2019 Jan 30.

Liver Centre, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.

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http://dx.doi.org/10.1136/gutjnl-2018-317959DOI Listing
May 2019

Fine needle aspirates comprehensively sample intrahepatic immunity.

Gut 2019 08 28;68(8):1493-1503. Epub 2018 Nov 28.

Division of Infection and Immunity, Institute of Immunity and Transplantation, University College London, London, UK.

Objective: In order to refine new therapeutic strategies in the pipeline for HBV cure, evaluation of virological and immunological changes compartmentalised at the site of infection will be required. We therefore investigated if liver fine needle aspirates (FNAs) could comprehensively sample the local immune landscape in parallel with viable hepatocytes.

Design: Matched blood, liver biopsy and FNAs from 28 patients with HBV and 15 without viral infection were analysed using 16-colour multiparameter flow cytometry.

Results: The proportion of CD4 T, CD8 T, Mucosal Associated Invariant T cell (MAIT), Natural Killer (NK) and B cells identified by FNA correlated with that in liver biopsies from the same donors. Populations of Programmed Death-1 (PD-1)CD39 tissue-resident memory CD8 T cells (CD69CD103) and liver-resident NK cells (CXCR6T-betEomes), were identified by both FNA and liver biopsy, and not seen in the blood. Crucially, HBV-specific T cells could be identified by FNAs at similar frequencies to biopsies and enriched compared with blood. FNAs could simultaneously identify populations of myeloid cells and live hepatocytes expressing albumin, Scavenger Receptor class B type 1 (SR-B1), Programmed Death-Ligand 1 (PD-L1), whereas hepatocytes were poorly viable after the processing required for liver biopsies.

Conclusion: We demonstrate for the first time that FNAs identify a range of intrahepatic immune cells including locally resident sentinel HBV-specific T cells and NK cells, together with PD-L1-expressing hepatocytes. In addition, we provide a scoring tool to estimate the extent to which an individual FNA has reliably sampled intrahepatic populations rather than contaminating blood. The broad profiling achieved by this less invasive, rapid technique makes it suitable for longitudinal monitoring of the liver to optimise new therapies for HBV.
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http://dx.doi.org/10.1136/gutjnl-2018-317071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691856PMC
August 2019

The impact of currently licensed therapies on viral and immune responses in chronic hepatitis B: Considerations for future novel therapeutics.

J Viral Hepat 2019 01 11;26(1):4-15. Epub 2018 Dec 11.

Barts Liver Centre, Blizard Institute, Barts and The London School of Medicine & Dentistry, Queen Mary University of London, London, UK.

Despite the availability of a preventative vaccine, chronic hepatitis B (CHB) remains a global healthcare challenge with the risk of disease progression due to cirrhosis and hepatocellular carcinoma. Although current treatment strategies, interferon and nucleos(t)ide analogues have contributed to reducing morbidity and mortality related to CHB, these therapies are limited in providing functional cure. The treatment paradigm in CHB is rapidly evolving with a number of new agents in the developmental pipeline. However, until novel agents with functional cure capability are available in the clinical setting, there is a pressing need to optimize currently licensed therapies. Here, we discuss current agents used alone and/or in combination strategies along with the impact of these therapies on viral and immune responses. Novel treatment strategies are outlined, and the potential role of current therapies in the employment of pipeline agents is discussed.
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http://dx.doi.org/10.1111/jvh.13040DOI Listing
January 2019

Molecular Recalibration of PD-1+ Antigen-Specific T Cells from Blood and Liver.

Mol Ther 2018 11 16;26(11):2553-2566. Epub 2018 Aug 16.

Division of Infection and Immunity, Institute of Immunity and Transplantation, UCL, London, UK. Electronic address:

Checkpoint inhibitors and adoptive cell therapy provide promising options for treating solid cancers such as HBV-related HCC, but they have limitations. We tested the potential to combine advantages of each approach, genetically reprogramming T cells specific for viral tumor antigens to overcome exhaustion by down-modulating the co-inhibitory receptor PD-1. We developed a novel lentiviral transduction protocol to achieve preferential targeting of endogenous or TCR-redirected, antigen-specific CD8 T cells for shRNA knockdown of PD-1 and tested functional consequences for antitumor immunity. Antigen-specific and intrahepatic CD8 T cells transduced with lentiviral (LV)-shPD-1 consistently had a marked reduction in PD-1 compared to those transduced with a control lentiviral vector. PD-1 knockdown of human T cells rescued antitumor effector function and promoted killing of hepatoma cells in a 3D microdevice recapitulating the pro-inflammatory PD-L1 liver microenvironment. However, upon repetitive stimulation, PD-1 knockdown drove T cell senescence and induction of other co-inhibitory pathways. We provide the proof of principle that T cells with endogenous or genetically engineered specificity for HBV-associated HCC viral antigens can be targeted for functional genetic editing. We show that PD-1 knockdown enhances immediate tumor killing but is limited by compensatory engagement of alternative co-inhibitory and senescence program upon repetitive stimulation.
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http://dx.doi.org/10.1016/j.ymthe.2018.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225092PMC
November 2018

Liver sampling: a vital window into HBV pathogenesis on the path to functional cure.

Gut 2018 04 13;67(4):767-775. Epub 2018 Jan 13.

Division of Infection and Immunity, UCL, London, UK.

In order to optimally refine the multiple emerging drug targets for hepatitis B virus (HBV), it is vital to evaluate virological and immunological changes at the site of infection. Traditionally liver biopsy has been the mainstay of HBV disease assessment, but with the emergence of non-invasive markers of liver fibrosis, there has been a move away from tissue sampling. Here we argue that liver biopsy remains an important tool, not only for the clinical assessment of HBV but also for research progress and evaluation of novel agents. The importance of liver sampling has been underscored by recent findings of specialised subsets of tissue-resident immune subsets capable of efficient pathogen surveillance, compartmentalised in the liver and not sampled in the blood. Importantly, the assessment of virological parameters, such as cccDNA quantitation, also requires access to liver tissue. We discuss strategies to maximise information obtained from the site of infection and disease pathology. Fine needle aspirates of the liver may allow longitudinal sampling of the local virus/host landscape. The careful utilisation of liver tissue and aspirates in conjunction with blood will provide critical information in the assessment of new therapeutics for the functional cure of HBV.
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http://dx.doi.org/10.1136/gutjnl-2017-314873DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058064PMC
April 2018

HBV infection and HCC: the 'dangerous liaisons'.

Gut 2018 05 1;67(5):787-788. Epub 2017 Dec 1.

Cancer Research Center of Lyon (CRCL), INSERM U1052, UMR-5286 CNRS, University of Lyon, Lyon, France.

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http://dx.doi.org/10.1136/gutjnl-2017-315528DOI Listing
May 2018

TRAIL regulatory receptors constrain human hepatic stellate cell apoptosis.

Sci Rep 2017 07 17;7(1):5514. Epub 2017 Jul 17.

Division of Infection and Immunity, UCL, London, UK.

The TRAIL pathway can mediate apoptosis of hepatic stellate cells to promote the resolution of liver fibrosis. However, TRAIL has the capacity to bind to regulatory receptors in addition to death-inducing receptors; their differential roles in liver fibrosis have not been investigated. Here we have dissected the contribution of regulatory TRAIL receptors to apoptosis resistance in primary human hepatic stellate cells (hHSC). hHSC isolated from healthy margins of liver resections from different donors expressed variable levels of TRAIL-R2/3/4 (but negligible TRAIL-R1) ex vivo and after activation. The apoptotic potential of TRAIL-R2 on hHSC was confirmed by lentiviral-mediated knockdown. A functional inhibitory role for TRAIL-R3/4 was revealed by shRNA knockdown and mAb blockade, showing that these regulatory receptors limit apoptosis of hHSC in response to both oligomerised TRAIL and NK cells. A close inverse ex vivo correlation between hHSC TRAIL-R4 expression and susceptibility to apoptosis underscored its central regulatory role. Our data provide the first demonstration of non-redundant functional roles for the regulatory TRAIL receptors (TRAIL-R3/4) in a physiological setting. The potential for these inhibitory TRAIL receptors to protect hHSC from apoptosis opens new avenues for prognostic and therapeutic approaches to the management of liver fibrosis.
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http://dx.doi.org/10.1038/s41598-017-05845-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5514093PMC
July 2017

Current therapeutic approaches for HBV infected patients.

J Hepatol 2017 08 17;67(2):412-414. Epub 2017 Jun 17.

Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine & Dentistry, QMUL, London, UK. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2017.04.015DOI Listing
August 2017

IL-2 tissue-resident T cells in the human liver: Sentinels for hepatotropic infection.

J Exp Med 2017 06 19;214(6):1567-1580. Epub 2017 May 19.

Division of Infection and Immunity, Institute of Immunity and Transplantation, University College London, London, England, UK

The liver provides a tolerogenic immune niche exploited by several highly prevalent pathogens as well as by primary and metastatic tumors. We have sampled healthy and hepatitis B virus (HBV)-infected human livers to probe for a subset of T cells specialized to overcome local constraints and mediate immunity. We characterize a population of T-betEomesBlimp-1Hobit T cells found within the intrahepatic but not the circulating memory CD8 T cell pool expressing liver-homing/retention markers (CD69CD103 CXCR6CXCR3). These tissue-resident memory T cells (T) are preferentially expanded in patients with partial immune control of HBV infection and can remain in the liver after the resolution of infection, including compartmentalized responses against epitopes within all major HBV proteins. Sequential IL-15 or antigen exposure followed by TGFβ induces liver-adapted T, including their signature high expression of exhaustion markers PD-1 and CD39. We suggest that these inhibitory molecules, together with paradoxically robust, rapid, cell-autonomous IL-2 and IFNγ production, equip liver CD8 T to survive while exerting local noncytolytic hepatic immunosurveillance.
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http://dx.doi.org/10.1084/jem.20162115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461007PMC
June 2017

Immune Tolerant Chronic Hepatitis B: The Unrecognized Risks.

Viruses 2017 04 29;9(5). Epub 2017 Apr 29.

Fox Chase Cancer Center, Philadelphia, PA 19111, USA.

Chronic infection with hepatitis B virus (HBV) progresses through multiple phases, including immune tolerant, immune active, immune control, and, in a subset of patients who achieve immune control, reactivation. The first, the immune tolerant phase, is considered to be prolonged in duration but essentially benign in nature, lacking long-term consequences, and thus not recommended for antiviral therapy. This review challenges the notion that the immune tolerant phase is truly benign and considers the possibility that events during this phase may contribute significantly to cirrhosis, hepatocellular carcinoma (HCC), and the premature death of 25% of HBV carriers worldwide. Thus, earlier treatment than recommended by current guidelines should be considered. Low therapeutic coverage exacerbated by restrictive treatment guidelines may facilitate disease progression in many patients but also increase the risk of neonatal and horizontal transmission from untreated mothers to their children. While a prophylactic vaccine exists, there are many areas worldwide where the treatment of adults and the delivery of an effective vaccination course to newborns present difficult challenges.
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http://dx.doi.org/10.3390/v9050096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454409PMC
April 2017

Quantification of intrahepatic total HBV DNA in liver biopsies of HBV-infected patients by a modified version of COBAS Ampliprep/COBASTaqMan HBV test v2.0.

Med Microbiol Immunol 2017 Aug 11;206(4):295-299. Epub 2017 Apr 11.

Laboratory of Molecular Virology, Polyclinic Tor Vergata Foundation, Viale Oxford 81, 00133, Rome, Italy.

Intrahepatic total HBV DNA (it-HBV DNA) level might reflect the size of virus reservoir and correlate with the histological status of the liver. To quantitate it-HBV DNA in a series of 70 liver biopsies obtained from hepatitis B chronic patients, a modified version of the COBASAmpliprep/COBASTaqMan HBV test v2.0 was used for this purpose. The linearity and reproducibility of the modified protocol was tested by quantifying serial dilutions of a full-length HBV containing plasmid and it-HBV DNA from a reference patient. A good linear trend between the expected values and those generated by the assay was observed at different concentrations of both plasmid and reference patient (R  = 0.994 and 0.962, respectively). Differences between the values obtained in two independent runs were ≤0.3 log IU for the plasmid and ≤0.6 log IU/mg for the reference patient, showing a high inter-run reproducibility. In the 70 liver biopsies, it-HBV DNA level ranged from 1.4 to 5.4 log IU/mg, with a good linearity and reproducibility between the values obtained in two runs [R  = 0.981; median (IQR) difference of it-HBV DNA 0.05 (0.02-0.09) IU/mg]. The modified COBASAmpliprep/COBASTaqMan HBV test v2.0 allows an accurate quantitation of it-HBV DNA. Its determination may have prognostic value and may be a useful tool for the new therapeutic strategies aimed at eradicating the HBV infection.
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http://dx.doi.org/10.1007/s00430-017-0504-3DOI Listing
August 2017

Reply.

Gastroenterology 2017 04 6;152(5):1246-1247. Epub 2017 Mar 6.

Fox Chase Cancer Center, Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1053/j.gastro.2017.03.002DOI Listing
April 2017

Interferon Alpha Induces Sustained Changes in NK Cell Responsiveness to Hepatitis B Viral Load Suppression In Vivo.

PLoS Pathog 2016 08 3;12(8):e1005788. Epub 2016 Aug 3.

Hepatology, Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine & Dentistry, QMUL, London, United Kingdom.

NK cells are important antiviral effectors, highly enriched in the liver, with the potential to regulate immunopathogenesis in persistent viral infections. Here we examined whether changes in the NK pool are induced when patients with eAg-positive CHB are 'primed' with PegIFNα and importantly, whether these changes are sustained or further modulated long-term after switching to nucleos(t)ides (sequential NUC therapy), an approach currently tested in the clinic. Longitudinal sampling of a prospectively recruited cohort of patients with eAg+CHB showed that the cumulative expansion of CD56bright NK cells driven by 48-weeks of PegIFNα was maintained at higher than baseline levels throughout the subsequent 9 months of sequential NUCs. Unexpectedly, PegIFNα-expanded NK cells showed further augmentation in their expression of the activating NK cell receptors NKp30 and NKp46 during sequential NUCs. The expansion in proliferating, functional NK cells was more pronounced following sequential NUCs than in comparison cohorts of patients treated with de novo NUCs or PegIFNα only. Reduction in circulating HBsAg concentrations, a key goal in the path towards functional cure of CHB, was only achieved in those patients with enhancement of NK cell IFNγ and cytotoxicity but decrease in their expression of the death ligand TRAIL. In summary, we conclude that PegIFNα priming can expand a population of functional NK cells with an altered responsiveness to subsequent antiviral suppression by NUCs. Patients on sequential NUCs with a distinct NK cell profile show a decline in HBsAg, providing mechanistic insights for the further optimisation of treatment strategies to achieve sustained responses in CHB.
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http://dx.doi.org/10.1371/journal.ppat.1005788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972354PMC
August 2016

HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.

Gastroenterology 2016 Nov 22;151(5):986-998.e4. Epub 2016 Jul 22.

Hepatology, Centre for Immunobiology, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, London, United Kingdom.

Background & Aims: Chronic infection with hepatitis B virus (HBV) progresses through different phases. The first, called the immune-tolerant phase, has been associated with a lack of disease activity. We examined HBV-DNA integration, clonal hepatocyte expansion, HBV antigen expression, and HBV-specific immune responses in patients in the immune-tolerant phase to assess whether this designation is appropriate or if there is evidence of disease activity.

Methods: We studied HBV-DNA integration, clonal hepatocyte expansion, and expression of hepatitis B surface antigen and core antigen in liver tissues from 26 patients with chronic HBV infection (ages, 14-39 y); 9 patients were positive for hepatitis B e antigen (HBeAg) in the immune-tolerant phase and were matched for age with 10 HBeAg-positive patients with active disease and 7 HBeAg-negative patients with active disease. Peripheral blood samples were collected and HBV-specific T cells were quantified for each group.

Results: Detection of HBV antigens differed among groups. However, unexpectedly high numbers of HBV-DNA integrations, randomly distributed among chromosomes, were detected in all groups. Clonal hepatocyte expansion in patients considered immune tolerant also was greater than expected, potentially in response to hepatocyte turnover mediated by HBV-specific T cells, which were detected in peripheral blood cells from patients in all phases of infection.

Conclusions: We measured HBV-specific T cells, HBV-DNA integration, and clonal hepatocyte expansion in different disease phases of young patients with chronic hepatitis B, with emphasis on the so-called immune-tolerant phase. A high level of HBV-DNA integration and clonal hepatocyte expansion in patients considered immune tolerant indicated that hepatocarcinogenesis could be underway-even in patients with early stage chronic HBV infection. Our findings do not support the concepts that this phase is devoid of markers of disease progression or that an immune response has not been initiated. We propose that this early phase be called a high-replication, low-inflammation stage. The timing of therapeutic interventions to minimize further genetic damage to the hepatocyte population should be reconsidered.
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http://dx.doi.org/10.1053/j.gastro.2016.07.012DOI Listing
November 2016

Assessment of bone mineral density in tenofovir-treated patients with chronic hepatitis B: can the fracture risk assessment tool identify those at greatest risk?

J Infect Dis 2015 Feb 25;211(3):374-82. Epub 2014 Aug 25.

Hepatology Unit, Centre for Digestive Diseases, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London.

Background: Tenofovir disoproxil fumarate (TDF) is an established nucleotide analogue in the treatment of chronic hepatitis B. Bone mineral density loss has been described in TDF-treated patients with human immunodeficiency virus infection, but limited data exist for patients with chronic hepatitis B. Dual X-ray absorptiometry (DEXA) was used to determine bone mineral density changes in TDF-exposed patients. We evaluated the accuracy of the Fracture Risk Assessment Tool (FRAX) as an alternative to DEXA in clinical practice.

Methods: A total of 170 patients were studied: 122 were exposed to TDF, and 48 were controls. All patients underwent DEXA, and demographic details were recorded. FRAX scores (before and after DEXA) were calculated.

Results: TDF was associated with a lower hip T score (P = .02). On univariate and multivariate analysis, advancing age, smoking, lower body mass index, and TDF exposure were independent predictors of low bone mineral density. In addition, the pre-DEXA FRAX score was an accurate predictor of the post-DEXA FRAX treatment recommendation (100% sensitivity and 83% specificity), area under the curve 0.93 (95% CI, .87-.97, P < .001).

Conclusions: TDF-treated patients with chronic hepatitis B have reduced bone mineral density, but the reduction is limited to 1 anatomical site. Age and advanced liver disease are additional contributing factors, underlining the importance of multifactorial fracture risk assessment. FRAX can accurately identify those at greatest risk of osteoporotic fracture.
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http://dx.doi.org/10.1093/infdis/jiu471DOI Listing
February 2015

Chronic hepatitis B virus in young adults: the need for new approaches to management.

Expert Rev Anti Infect Ther 2014 Sep 23;12(9):1045-53. Epub 2014 Jul 23.

Hepatology Unit, Centre for Digestive Diseases, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK.

One in four patients infected with hepatitis B virus (HBV) at birth or in early childhood will develop cirrhosis or hepatocellular carcinoma. Historically, guidelines have overlooked treatment in young people, as the immune tolerant disease phase is considered synonymous with chronic infection in the young. Current treatment aims to suppress HBV replication through long-term nucleos(t)ide therapy with little emphasis on virus eradication. To achieve HBsAg loss, it is accepted that effective immune control of virus is required, mimicking that seen in those who resolve acute HBV infection. We have recently challenged the accuracy of a generic immune tolerant state in young people, thus raising a potential role for earlier treatment. Here we report on our immunological analysis of HBV in young people and the role of a dedicated clinic; we make the case for earlier intervention to achieve effective immune control leading to better outcomes.
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http://dx.doi.org/10.1586/14787210.2014.940899DOI Listing
September 2014

Up-regulation of a death receptor renders antiviral T cells susceptible to NK cell-mediated deletion.

J Exp Med 2013 Jan 17;210(1):99-114. Epub 2012 Dec 17.

Division of Infection and Immunity and Centre for Sexual Health and HIV Research, University College London, London NW3 2PF, UK.

Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8(+) T cell responses directed against HBV, but not against well-controlled viruses, in patients with chronic hepatitis B (CHB). We find that NK cells can rapidly eliminate HBV-specific T cells in a contact-dependent manner. CD8(+) T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 renders T cells susceptible to caspase-8-mediated apoptosis, from which they can be partially rescued by blockade of this death receptor pathway. Our findings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.
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http://dx.doi.org/10.1084/jem.20121172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3549717PMC
January 2013

Preserved T-cell function in children and young adults with immune-tolerant chronic hepatitis B.

Gastroenterology 2012 Sep 15;143(3):637-645. Epub 2012 Jun 15.

Infection & Immunity Program, Singapore Institute for Clinical Sciences, A*STAR, Singapore; Program Emerging Viral Diseases, Duke-NUS Graduate Medical School, Singapore, Singapore; Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address:

Background & Aims: Chronic hepatitis B (CHB) infection acquired perinatally or in early childhood has been associated with a prolonged phase of immune tolerance from viral exposure into early adulthood. The immune-tolerant phase of the disease is characterized by high levels of hepatitis B virus (HBV) DNA and normal liver biochemistry, with minimal or no fibrosis. We investigated whether the age of patients with CHB affects their antiviral immunity and whether children and young adults have a veritable state of immunologic tolerance.

Methods: We isolated T cells from different age groups of patients with CHB and used flow cytometric methods to measure production of effector and inflammatory cytokines (interferon, tumor necrosis factor, interleukin [IL]-17A, IL-22, and IL-8), T-helper (Th)2 cytokines (IL-10, IL-4), Th1 cytokines (IL-2 and IL-21), and the CC chemokine CCL3 (MIP-1). We also measured markers of T-cell exhaustion or inhibition (PD-1, LAG-3, TIM3, LAIR-1, and CTLA-4) and HBV-specific T cells.

Results: Young patients with CHB have a Th1-cell cytokine profile and a partial profile of T-cell exhaustion. Direct quantification of the HBV-specific T-cell response showed that young patients with CHB have more HBV-specific T cells with the ability to proliferate and produce cytokines than adult patients with CHB.

Conclusions: HBV infection in younger patients is not associated with an immune profile of T-cell tolerance. On the contrary, children and young adults with chronic HBV infection have an HBV-specific immune profile that is less compromised than that observed in older patients.
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http://dx.doi.org/10.1053/j.gastro.2012.06.009DOI Listing
September 2012

IL-2-engineered nano-APC effectively activates viral antigen-mediated T cell responses from chronic hepatitis B virus-infected patients.

J Immunol 2012 Feb 30;188(3):1534-43. Epub 2011 Dec 30.

Division of Bioscience, Brunel University, London UB8 3PH, United Kingdom.

Impaired function of virus-specific T cells resulting from virus persistence is one of the major mechanisms underlying the development of chronic hepatitis B viral infection. Previously, we found that IL-2 can restore the effector function of T cells rendered tolerant by Ag persistence. However, systemic administration of IL-2 induces organ pathology and expansion of T regulatory cells. In this study, we show that nano-APC with engineered HLA alleles and IL-2 deliver peptide-MHC complexes, costimulatory molecules, and IL-2 to Ag-responding T cells, resulting in enhanced expression of CD25 and activation of TCR signaling pathways, while suppressing PD-1 expression on viral-responding CD8 T cells from chronic hepatitis B virus patients. The enhanced activation of CD4 and CD8 T cells induced by IL-2-nano-APC was Ag dependent and IL-2-nano-APC did not affect T regulatory cells. At a size of 500 nm, the nano-APC effectively induce immune synapse formation on Ag-specific T cells and accumulate as free particles in the lymphoid organs. These attributes of IL-2-nano-APC or other bioadjuvant-engineered nano-APC have profound implications for their use as a therapeutic strategy in the treatment of chronic hepatitis B virus infection or other chronic viral diseases.
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http://dx.doi.org/10.4049/jimmunol.1102709DOI Listing
February 2012

Role of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 on apoptosis-Prone CD8 T cells in persistent hepatitis B virus infection.

Hepatology 2011 May;53(5):1494-503

Division of Infection and Immunity, UCL, London, UK.

Unlabelled: An excess of coinhibitory signals has been proposed to drive the T-cell exhaustion characteristic of persistent viral infections. In this study we examined the contribution of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) to CD8 T cell tolerance in chronic hepatitis B virus (HBV) infection (CHB). CD8 T cells in patients with CHB have an increased propensity to express the coinhibitory receptor CTLA-4 and this correlates with viral load. CTLA-4 is up-regulated on those HBV-specific CD8 T cells with the highest levels of the proapoptotic protein Bim, which we have previously shown mediates their premature attrition; abrogation of CTLA-4-mediated coinhibition can reduce Bim expression. Longitudinal study of CHB patients beginning antiviral therapy reveals that HBV DNA suppression induces transient reconstitution of HBV-specific CD8 T cells but does not reprogram their CTLA-4(hi) Bim(hi) tolerogenic phenotype. Blocking CTLA-4 is able to increase the expansion of interferon gamma (IFN-γ)-producing HBV-specific CD8 T cells in both the peripheral and intrahepatic compartments. The rescue of anti-HBV responses by either CTLA-4 or PD-L1 blockade is nonredundant.

Conclusion: CTLA-4 is expressed by HBV-specific CD8 T cells with high levels of Bim and helps to drive this proapoptotic phenotype. CTLA-4 blockade could form one arm of a therapeutic approach to modulate the diverse patterns of coregulation of T-cell exhaustion in this heterogeneous disease.
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http://dx.doi.org/10.1002/hep.24249DOI Listing
May 2011

Blockade of immunosuppressive cytokines restores NK cell antiviral function in chronic hepatitis B virus infection.

PLoS Pathog 2010 Dec 16;6(12):e1001227. Epub 2010 Dec 16.

Division of Infection and Immunity, UCL, London, United Kingdom.

NK cells are enriched in the liver, constituting around a third of intrahepatic lymphocytes. We have previously demonstrated that they upregulate the death ligand TRAIL in patients with chronic hepatitis B virus infection (CHB), allowing them to kill hepatocytes bearing TRAIL receptors. In this study we investigated whether, in addition to their pathogenic role, NK cells have antiviral potential in CHB. We characterised NK cell subsets and effector function in 64 patients with CHB compared to 31 healthy controls. We found that, in contrast to their upregulated TRAIL expression and maintenance of cytolytic function, NK cells had a markedly impaired capacity to produce IFN-γ in CHB. This functional dichotomy of NK cells could be recapitulated in vitro by exposure to the immunosuppressive cytokine IL-10, which was induced in patients with active CHB. IL-10 selectively suppressed NK cell IFN-γ production without altering cytotoxicity or death ligand expression. Potent antiviral therapy reduced TRAIL-expressing CD56(bright) NK cells, consistent with the reduction in liver inflammation it induced; however, it was not able to normalise IL-10 levels or the capacity of NK cells to produce the antiviral cytokine IFN-γ. Blockade of IL-10 +/- TGF-β restored the capacity of NK cells from both the periphery and liver of patients with CHB to produce IFN-γ, thereby enhancing their non-cytolytic antiviral capacity. In conclusion, NK cells may be driven to a state of partial functional tolerance by the immunosuppressive cytokine environment in CHB. Their defective capacity to produce the antiviral cytokine IFN-γ persists in patients on antiviral therapy but can be corrected in vitro by IL-10+/- TGF-β blockade.
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http://dx.doi.org/10.1371/journal.ppat.1001227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003000PMC
December 2010