HBV-specific CD8 Research Articles

Tox expression on HBV-specific CD8+ T cells is linked to clinical stage of chronic HBV infection

Tox expression on HBV-specific CD8+ T cells is linked to clinical stage of chronic HBV infection

Hepatitis B virus kinetics after nucleos(t)ide-analogue withdrawal in chronic HBeAg(-) hepatitis correlates with HBV-specific CD8+ response and HBsAg level

Hepatitis B virus kinetics after nucleos(t)ide-analogue withdrawal in chronic HBeAg(-) hepatitis correlates with HBV-specific CD8+ response and HBsAg level

AS003 - Serum and intrahepatic HBV markers and HBV-specific CD8 T cell responses after nucleos(t)ide analog therapy discontinuation in HBeAg-negative chronic hepatitis B patients

AS003 - Serum and intrahepatic HBV markers and HBV-specific CD8 T cell responses after nucleos(t)ide analog therapy discontinuation in HBeAg-negative chronic hepatitis B patients

HBV-specific CD8 T cells present higher TNF-α expression but lower cytotoxicity in hepatocellular carcinoma.

Tumor necrosis factor (TNF)-α is largely regarded as a proinflammatory cytokine, but several recent researches have demonstrated that TNF-α could possess immunoregulatory roles with potential to suppress anti-tumor immunity. Chronic hepatitis B virus (HBV) infection is a major risk factor of hepatocellular carcinoma (HCC), and HBV-specific CD8 T cells could exert anti-tumor roles in HCC patients. Here, we found that HBV-specific CD8 T cells, both in the peripheral blood and in the tumor microenvironment, were more enriched with TNF-α-expressing cells than interferon (IFN)-γ-expressing cells. Compared to IFN-γ-expressing HBV-specific CD8 T cells, TNF-α-expressing HBV-specific CD8 T cells presented lower expression of inhibitory checkpoint molecules, including programmed cell death (PD)-1, T cell immunoglobulin mucin-3 (TIM-3) and cytotoxic T lymphocyte antigen (CTLA)-4. HBV-specific CD8 T cells could mediate the lysis of autologous primary tumor cells, and the inhibition of TNF-α could further elevate their cytotoxic capacity. Subsequently, we demonstrated that TNF-α inhibition in HBV-specific CD8 T cells could significantly increase granzyme B (GZMB) and perforin 1 (PRF1) expression while having no effect towards granzyme A (GZMA) expression. The addition of exogenous TNF-α at low levels had no consistent effect on the expression of GZMA, GZMB and PRF1, but at higher levels, exogenous TNF-α significantly reduced GZMA, GZMB and PRF1 expression. Overall, these results suggested that TNF-α-expressing cells probably presented a deleterious role in HCC but were enriched in HBV-specific CD8 T cells.

MHC-E-Restricted CD8+ T Cells Target Hepatitis B Virus-Infected Human Hepatocytes.

Currently 247 million people are living with chronic hepatitis B virus infection (CHB), and the development of novel curative treatments is urgently needed. Immunotherapy is an attractive approach to treat CHB, yet therapeutic approaches to augment the endogenous hepatitis B virus (HBV)-specific T cell response in CHB patients have demonstrated little success. In this study, we show that strain 68-1 rhesus macaque (RM) CMV vaccine vectors expressing HBV Ags engender HBV-specific CD8+ T cells unconventionally restricted by MHC class II and the nonclassical MHC-E molecule in RM. Surface staining of human donor and RM primary hepatocytes (PH) ex vivo revealed the majority of PH expressed MHC-E but not MHC class II. HBV-specific, MHC-E-restricted CD8+ T cells from RM vaccinated with RM CMV vaccine vectors expressing HBV Ags recognized HBV-infected PH from both human donor and RM. These results provide proof-of-concept that MHC-E-restricted CD8+ T cells could be harnessed for the treatment of CHB, either through therapeutic vaccination or adoptive immunotherapy.

Circulating serum HBsAg level is a biomarker for HBV-specific T and B cell responses in chronic hepatitis B patients

Chronic hepatitis B (CHB) infection functional cure is defined as sustained loss of HBsAg and several therapeutic strategies are in clinical development designed to pharmacologically reduce serum HBsAg, break immune tolerance, and increase functional cure rates. However, little is known about pre-treatment HBsAg levels as an indicator of HBV immune potential. Here, we compared the phenotypes and HBV-specific response of lymphocytes in CHB patients stratified by serum HBsAg levels <500 (HBslo) or >50,000 IU/ml (HBshi) using immunological assays (flow cytometry, ICS, ELISPOT). HBshi patients had significantly higher expression of inhibitory PD-1 on CD4+ T cells, particularly among TEMRA subset, and higher FcRL5 expression on B cells. Upon HBcAg(core) or HBsAg(env)-stimulation, 85% and 60% of HBslo patients had IFNγ+TNFα+ and IFNγ+ IL2+ CD4+ T cell responses respectively, in comparison to 33% and 13% of HBshi patients. Checkpoint blockade with αPD-1 improved HBV-specific CD4+ T cell function only in HBslo patients. HBsAg-specific antibody-secreting cells (ASCs) response was not different between these groups, yet αPD-1 treatment resulted in significantly higher fold change in ASCs among patients with HBsAg <100 IU/ml compared to patients with HBsAg >5,000 IU/ml. Thus, serum HBsAg correlates with inhibitory receptor expression, HBV-specific CD4+ T cell responses, and augmentation by checkpoint blockade.

Impact of the Interaction of Hepatitis B Virus with Mitochondria and Associated Proteins.

Around 350 million people are living with hepatitis B virus (HBV), which can lead to death due to liver cirrhosis and hepatocellular carcinoma (HCC). Various antiviral drugs/nucleot(s)ide analogues are currently used to reduce or arrest the replication of this virus. However, many studies have reported that nucleot(s)ide analogue-resistant HBV is circulating. Cellular signaling pathways could be one of the targets against the viral replication. Several studies reported that viral proteins interacted with mitochondrial proteins and localized in the mitochondria, the powerhouse of the cell. And a recent study showed that mitochondrial turnover induced by thyroid hormones protected hepatocytes from hepatocarcinogenesis mediated by HBV. Strong downregulation of numerous cellular signaling pathways has also been reported to be accompanied by profound mitochondrial alteration, as confirmed by transcriptome profiling of HBV-specific CD8 T cells from chronic and acute HBV patients. In this review, we summarize the ongoing research into mitochondrial proteins and/or signaling involved with HBV proteins, which will continue to provide insight into the relationship between mitochondria and HBV and ultimately lead to advances in viral pathobiology and mitochondria-targeted antiviral therapy.

Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-Titer Hepatitis B Virus Carrier Mice

Hepatitis B virus (HBV) infection persists because the virus-specific immune response is dysfunctional. Therapeutic vaccines might be used to end immune tolerance to the virus in patients with chronic infection, but these have not been effective in patients so far. In patients with chronic HBV infection, high levels of virus antigens might prevent induction of HBV-specific immune responses. We investigated whether knocking down expression levels of HBV antigens in liver might increase the efficacy of HBV vaccines in mice. We performed studies with male C57BL/6 mice that persistently replicate HBV (genotype D, serotype ayw)-either from a transgene or after infection with an adeno-associated virus that transferred an overlength HBV genome-and expressed HB surface antigen at levels relevant to patients. Small hairpin or small interfering (si)RNAs against the common 3'-end of all HBV transcripts were used to knock down antigen expression in mouse hepatocytes. siRNAs were chemically stabilized and conjugated to N-acetylgalactosamine to increase liver uptake. Control mice were given either entecavir or non-HBV-specific siRNAs and vaccine components. Eight to 12 weeks later, mice were immunized twice with a mixture of adjuvanted HBV S and core antigen, followed by a modified Vaccinia virus Ankara vector to induce HBV-specific B- and T-cell responses. Serum and liver samples were collected and analyzed for HBV-specific immune responses, liver damage, and viral parameters. In both models of HBV infection, mice that express hepatocyte-specific small hairpin RNAs or that were given subcutaneous injections of siRNAs had reduced levels of HBV antigens, HBV replication, and viremia (1-3 log10 reduction) compared to mice given control RNAs. Vaccination induced production of HBV-neutralizing antibodies and increased numbers and functionality of HBV-specific, CD8+ T cells in mice with low, but not in mice with high, levels of HBV antigen. Mice with initially high titers of HBV and knockdown of HBV antigen expression, but not mice with reduced viremia after administration of entecavir, developed polyfunctional, HBV-specific CD8+ T cells, and HBV was eliminated. In mice with high levels of HBV replication, knockdown of HBV antigen expression along with a therapeutic vaccination strategy, but not knockdown alone, increased numbers of effector T cells and eliminated the virus. These findings indicate that high titers of virus antigens reduce the efficacy of therapeutic vaccination. Anti-HBV siRNAs and therapeutic vaccines are each being tested in clinical trials-their combination might cure chronic HBV infection.

Human Monoclonal Antibodies as Adjuvant Treatment of Chronic Hepatitis B Virus Infection.

Despite the availability of an effective prophylactic vaccine leading to sterilizing immunity, hepatitis B virus (HBV) is responsible for chronic liver disease in more than 250 million individuals, potentially leading to cirrhosis and hepatocellular carcinoma. Antiviral drugs able to completely suppress virus replication are indeed available but they are, by and large, unable to eradicate the virus. Several alternative new treatment approaches are currently being developed but none have so far captured the interest of clinicians for possible clinical development. A constant feature of chronic HBV infection is T-cell exhaustion resulting from persistent exposure to high antigen concentrations as shown by the high expression of programmed cell death protein 1 (PD-1) by HBV-specific CD8 T cells. One way of tackling this problem is to develop HBV-specific neutralizing antibodies that would clear excess envelope proteins from the circulation, allowing for nucleos(t)ide analogs or other antiviral drugs now in preclinical and early clinical development to take advantage of a reconstituted adaptive immunity. Several fully human monoclonal antibodies (mAb) have been developed from HBV-vaccinated and subjects convalescent from acute hepatitis B that show different properties and specificities. It is envisaged that such neutralizing mAb may be used as adjuvant treatment to reduce viral protein load, thus rescuing adaptive immunity in an effort to optimize the effect of antiviral drugs.

Heterogeneity of HBV-Specific CD8+ T-Cell Failure: Implications for Immunotherapy.

Chronic hepatitis B virus (HBV) infection is a major global health burden affecting around 257 million people worldwide. The consequences of chronic HBV infection include progressive liver damage, liver cirrhosis, and hepatocellular carcinoma. Although current direct antiviral therapies successfully lead to suppression of viral replication and deceleration of liver cirrhosis progression, these treatments are rarely curative and patients often require a life-long therapy. Based on the ability of the immune system to control HBV infection in at least a subset of patients, immunotherapeutic approaches are promising treatment options to achieve HBV cure. In particular, T cell-based therapies are of special interest since CD8+ T cells are not only capable to control HBV infection but also to eliminate HBV-infected cells. However, recent data show that the molecular mechanisms underlying CD8+ T-cell failure in chronic HBV infection depend on the targeted antigen and thus different strategies to improve the HBV-specific CD8+ T-cell response are required. Here, we review the current knowledge about the heterogeneity of impaired HBV-specific T-cell populations and the potential consequences for T cell-based immunotherapeutic approaches in HBV cure.

CD8+CD28- T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection.

During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28- T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28- T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28- T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28- T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28- T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28- T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28- T-cell killing. Both CD28+ and CD28- T cells in CHB expressedCXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28- T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28- T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28- T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28- T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

TNF-α/IFN-γ profile of HBV-specific CD4 T cells is associated with liver damage and viral clearance in chronic HBV infection

The role of hepatitis B virus (HBV)-specific CD4 T cells in patients with chronic HBV infection is not clear. Thus, we aimed to elucidate this in patients with chronic infection, and those with hepatitis B flares. Through intracellular IFN-γ and TNF-α staining, HBV-specific CD4 T cells were analyzed in 68 patients with chronic HBV infection and alanine aminotransferase (ALT) <2x the upper limit of normal (ULN), and 28 patients with a hepatitis B flare. HBV-specific HLA-DRB1*0803/HLA-DRB1*1202-restricted CD4 T cell epitopes were identified. TNF-α producing cells were the dominant population in patients' HBV-specific CD4 T cells. In patients with ALT <2xULN, both the frequency and the dominance of HBV-specific IFN-γ producing CD4 T cells increased sequentially in patients with elevated levels of viral clearance: HBV e antigen (HBeAg) positive, HBeAg negative, and HBV surface antigen (HBsAg) negative. In patients with a hepatitis B flare, the frequency of HBV core-specific TNF-α producing CD4 T cells was positively correlated with patients' ALT and total bilirubin levels, and the frequency of those cells changed in parallel with the severity of liver damage. Patients with HBeAg/HBsAg loss after flare showed higher frequency and dominance of HBV-specific IFN-γ producing CD4 T cells, compared to patients without HBeAg/HBsAg loss. Both the frequency and the dominance of HBV S-specific IFN-γ producing CD4 T cells were positively correlated with the decrease of HBsAg during flare. A differentiation process from TNF-α producing cells to IFN-γ producing cells in HBV-specific CD4 T cells was observed during flare. Eight and 9 HBV-derived peptides/pairs were identified as HLA-DRB1*0803 restricted epitopes and HLA-DRB1*1202 restricted epitopes, respectively. HBV-specific TNF-α producing CD4 T cells are associated with liver damage, while HBV-specific IFN-γ producing CD4 T cells are associated with viral clearance in patients with chronic HBV infection. TNF-α producing cells are the dominant population of hepatitis B virus (HBV)-specific CD4 T cells in patients with chronic HBV infection. This population of cells might contribute to the aggravation of liver damage in patients with a hepatitis B flare. HBV-specific IFN-γ producing CD4 T cells are associated with HBV viral clearance. Differentiation from HBV-specific TNF-α producing CD4 T cells into HBV-specific IFN-γ producing CD4 T cells might favor HBV viral clearance.

HBV-Induced Immune Imbalance in the Development of HCC.

Chronic hepatitis B virus (HBV) infection is one of the high-risk factors for human HCC. Despite the integration of virus DNA and the oncoprotein HBx, chronic necroinflammation and hepatocellular regeneration account for hepatocarcinogenesis. As a non-cytopathic virus, HBV is extensively recognized to mediate chronic liver damage through abnormal immune attack. However, the mechanisms driving HBV infection to HCC are poorly understood. During chronic HBV infection in humans, the adaptive immunity changes from immune tolerance to progressive immune activation, inactivation, reactivation and exhaustion, all of which may be the immune pathogenic factors for the development of HCC. Recently, the immunopathogenic mechanisms were described in mouse HBV-induced HCC models, which is absolutely dependent on the presence of HBV-specific T cell response and NK cell-derived IFN-γ, findings which are consistent with the observations from CHB and HCC patients. In this review, we summarize recent research progression on the HBV-specific CD8+ T cells, and also CD4+ T cells, B cells and non-specific immune cells and molecules underlying chronic HBV infection and eventual HCC development to demonstrate the pathogenesis of HBV-induced immune imbalance. Based on the progression, we discussed the potential of immune-based therapies and their challenges in the treatment of HBV-related HCC, including the checkpoint inhibition, genetically modified T cell transfer, therapeutic vaccines and metabolic modulation.

Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection

Therapeutic vaccination against chronic hepatitis B must overcome high viral antigen load and local regulatory mechanisms that promote immune-tolerance in the liver and curtail hepatitis B virus (HBV)-specific CD8 T cell immunity. Here, we report that therapeutic heterologous HBcore-protein-prime/Modified-Vaccinia-Virus-Ankara (MVA-HBcore) boost vaccination followed by CpG-application augmented vaccine-induced HBcAg-specific CD8 T cell-function in the liver. In HBV-transgenic as well as AAV-HBV-transduced mice with persistent high-level HBV-replication, the combination of therapeutic vaccination with subsequent CpG-application was synergistic to generate more potent HBV-specific CD8 T cell immunity that improved control of hepatocytes replicating HBV.

HBeAg seroconversion is associated with a more effective PD-L1 blockade during chronic hepatitis B infection.

Background & AimsCurrent therapies for chronic hepatitis B virus (HBV) infection control viral replication but do not eliminate the risk of progression to hepatocellular carcinoma. HBV-specific CD8 T cells are necessary for viral control, but they are rare and exhausted during chronic infection. Preclinical studies have shown that blockade of the PD-1:PD-L1 axis can restore HBV-specific T cell functionality. The aim of this study was to analyze how the clinical and treatment status of patients impacts the ability of HBV-specific T cells to respond to PD-L1 blockade.MethodsExpression patterns of the PD-1:PD-L1/PD-L2 axis were analyzed in healthy donors and chronically infected patients in different clinical phases of disease. A functional assay was performed to quantify baseline HBV-specific T cell responses in chronically infected patients. Baseline responses were then compared to those attained in the presence of an anti-PD-L1 monoclonal antibody (MEDI2790).ResultsChronically infected patients were characterized by the upregulation of PD-1 within the T cell compartment and a concomitant upregulation of PD-L1 on myeloid dendritic cells. The upregulation was maximal in HBV e antigen (HBeAg)-positive patients but persisted after HBeAg negativization and was not restored by long-term treatment. HBV reactivity, measured as frequency of HBV-specific T cells, was significantly higher in HBeAg-negative patients with lower HBV DNA levels, independently of HBV surface antigen or alanine aminotransferase levels. Anti-PD-L1 blockade with MEDI2790 increased both the number of IFN-γ-producing T cells and the amount of IFN-γ produced per cell in 97% of patients with detectable HBV reactivity, independently of patients’ clinical or treatment status.ConclusionPatients with lower levels of HBV DNA and the absence of HBeAg have more intact HBV-specific T cell immunity and may benefit the most from PD-L1 blockade as a monotherapy.Lay summaryHepatitis B virus (HBV)-specific T cell responses during chronic infection are weak due to the upregulation of inhibitor molecules on the immune cells. In this study we show that the inhibitory PD-1:PD-L1 axis is upregulated during chronic HBV infection and successful antiretroviral therapy does not restore normal levels of PD-1 and PD-L1 expression. However, in HBV e antigen-negative patients, treatment with an anti-PD-L1 antibody can increase the functionality of HBV-specific T cell responses by an average of 2-fold and is a promising new therapy for patients with chronic HBV infection.

Ex vivo Detection and Characterization of Hepatitis B Virus-Specific CD8+ T Cells in Patients Considered Immune Tolerant.

In this study, we aimed to detect and characterize ex vivo virus-specific CD8+ T cells in patients with immune-tolerant hepatitis B virus (HBV) infection. We investigated a Korean chronic hepatitis B cohort composed of 15 patients in the immune-tolerant phase, 17 in the immune-active phase, and 13 under antiviral treatment. We performed enzyme-linked immunospot (ELISpot) assays ex vivo and intracellular cytokine staining after in vitro culture. We also performed ex vivo multimer staining assays and examined the expression of programmed death-1 (PD-1) and CD127 in pentamer-positive cells. Ex vivo ELISpot revealed that HBV-specific T cell function was weaker in immune-tolerant patients than in those under antiviral treatment. In vitro culture of peripheral blood mononuclear cells for 10 days revealed that HBV-specific CD8+ T cells produced interferon-γ in some immune-tolerant patients. We detected HBV-specific CD8+ T cells ex vivo (using the HBV core18−27 pentamer) in patients from all three groups. The PD-1+ subset of pentamer+ CD8+ T cells was smaller ex vivo in the immune-tolerant phase than in the immune-active phase or under antiviral treatment. Interestingly, the proportion of PD-1+ CD8+ T cells in HBV-specific CD8+ T cells correlated with patient age when all enrolled patients were analyzed. Overall, HBV-specific CD8+ T cells are present in patients considered as immune-tolerant, although their ex vivo functionality is significantly weaker than that in patients under antiviral treatment (P < 0.05). Despite the high viral load, the proportion of PD-1 expression in HBV-specific CD8+ T cells is lower in the immune-tolerant phase than in other phases. Our results indicate appropriate stimulation may enhance the effector function of HBV-specific CD8+ T cells in patients considered as being in the immune-tolerant phase.

MMP2/MMP9-mediated CD100 shedding is crucial for inducing intrahepatic anti-HBV CD8 T cell responses and HBV clearance

CD100 is constitutively expressed on T cells and can be cleaved from the cell surface by matrix metalloproteases (MMPs) to become soluble CD100 (sCD100). Both membrane-bound CD100 (mCD100) and sCD100 have important immune regulatory functions that promote immune cell activation and responses. This study investigated the expression and role of mCD100 and sCD100 in regulating antiviral immune responses during HBV infection. mCD100 expression on T cells, sCD100 levels in the serum, and MMP expression in the liver and serum were analysed in patients with chronic HBV (CHB) and in HBV-replicating mice. The ability of sCD100 to mediate antigen-presenting cell maturation, HBV-specific T cell activation, and HBV clearance were analysed in HBV-replicating mice and patients with CHB. Patients with CHB had higher mCD100 expression on T cells and lower serum sCD100 levels compared with healthy controls. Therapeutic sCD100 treatment resulted in the activation of DCs and liver sinusoidal endothelial cells, enhanced HBV-specific CD8 T cell responses, and accelerated HBV clearance, whereas blockade of its receptor CD72 attenuated the intrahepatic anti-HBV CD8 T cell response. Together with MMP9, MMP2 mediated mCD100 shedding from the T cell surface. Patients with CHB had significantly lower serum MMP2 levels, which positively correlated with serum sCD100 levels, compared with healthy controls. Inhibition of MMP2/9 activity resulted in an attenuated anti-HBV T cell response and delayed HBV clearance in mice. MMP2/9-mediated sCD100 release has an important role in regulating intrahepatic anti-HBV CD8 T cell responses, thus mediating subsequent viral clearance during HBV infection. Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. The clearance of HBV relies largely on an effective T cell immune response, which usually becomes dysregulated in chronic HBV infection. Our study provides a new mechanism to elucidate HBV persistence and a new target for developing immunotherapy strategies in patients chronically infected with HBV.

From the Editor’s desk…: June 2019

From the Editor’s desk…: June 2019

Characterization of MHC class II-restricted T-cell receptors for T-cell therapy of HBV infection

Background & Aim Hepatitis B virus (HBV) infection remains a severe health problem with current treatment options being unable to achieve viral clearance. Since virus eradication is known to be accompanied by a strong T-cell response in patients with resolved infection, adoptive T-cell therapy represents a promising therapeutic approach. We recently demonstrated that CD8+ T cells grafted with T-cell receptors (TCR) restricted by MHC class I have the potential to cure HBV infection in vitro and in vivo. Nevertheless, also CD4+ T cells are known to play an important role in resolving HBV infection and may therefore potentially benefit T-cell therapy. We thus identified, cloned and characterized MHC class II-restricted TCRs from HBV-specific CD4+ T cells. Methods, Results & Conclusion HBV peptides were used to stimulate PBMC from donors with cleared HBV infection. HBV-specific CD4+ T cells secreting TNF-α were sorted by flow cytometry and clonally expanded. Subsequently, variable sequences of TCR α- and β-chains were identified, codon-optimized and cloned into a retroviral vector with murine constant domains. After retroviral transduction, all TCRs were expressed on primary T cells and characterized in depth regarding their MHC restriction, specificity, binding affinity and functionality. A total of 23 TCRs specific for 7 different epitopes from HBV envelope, core or polymerase protein were generated. Through retroviral titration, TCR integration was limited to under 5 integrates per cell, as determined by qPCR. Transduction rates ranging from 65-85% were measured by flow cytometry, where strength of surface expression was found to be characteristic of the TCR rather than the number of integrates. Through co-culture with partially matched lymphoblastoid cell lines, 6 different MHC class II restrictions were identified, including HLA-DRB1*01, *07, *04 and *13 or HLA-DPB1*02 and *04. 17 TCRs recognized at least 3 HBV genotype variants and 12 TCRs had a high binding affinity with EC50 values in a low nanomolar range. Similarly, 12 TCRs were able to recognize endocytosed and intracellularly processed viral antigen. 20 TCRs were capable of activating CD8+ T cells, inducing granzyme B secretion in 75-100% of transduced T cells. Taken together, our set of 23 MHC class II-restricted TCRs recognizes epitopes derived from HBV core, surface and polymerase protein. These TCRs enabled both CD4+ and CD8+ T cells to detect antigen on MHC class II in a nanomolar range, resulting in antigen-specific T-cell activation.

The effects of nucleoside/nucleotide analogues on host immune cells: the baseline for future immune therapy for HBV?

HBV is a non-cytopathic virus and the progression of liver fibrosis is attributed to the host immune response. Complete suppression of viral replication using nucleotide or nucleoside analogues (NUCs) can prevent most complications related to chronic HBV infection. Unfortunately, antiviral treatment has to be administered lifelong to the majority of patients as HBV persists in the hepatocytes. However, although NUCs are very frequently administered in clinical practice, their effects on vital parts of the host immune response to HBV are not well established. In this review we summarize the currently available data gathered from longitudinal studies that investigated treatment-associated alterations of HBV-specific CD4+ and CD8+ T-cells, regulatory T-cells and natural killer (NK) cells. These observations are important, as they can guide the design of studies that investigate the efficacy of new immune therapeutic agents. Novel experimental compounds will likely be added to ongoing NUC treatment, which leads to a functional cure in only a small minority of patients.