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Hepatitis Delta Virus Acts as an Immunogenic Adjuvant in Hepatitis B Virus-Infected Hepatocytes

Summary Hepatitis delta virus (HDV) requires hepatitis B virus (HBV) to complete its infection cycle and causes severe hepatitis, with limited therapeutic options. To determine the prospect of T cell therapy in HBV/HDV co-infection, we study the impact of HDV on viral antigen processing and presentation. Using in vitro models of HBV/HDV co-infection, we demonstrate that HDV boosts HBV epitope presentation, both in HBV/HDV co-infected and neighboring mono-HBV-infected cells through the upregulation of the antigen processing pathway mediated by IFN-β/λ. Liver biopsies of HBV/HDV patients confirm this upregulation. We then validate in vitro and in a HBV/HDV preclinical mouse model that HDV infection increases the anti-HBV efficacy of T cells with engineered T cell receptors. Thus, by unveiling the effect of HDV on HBV antigen presentation, we provide a framework to better understand HBV/HDV immune pathology, and advocate the utilization of engineered HBV-specific T cells as a potential treatment for HBV/HDV co-infection.


In Brief
Tham et al. demonstrate that HDV activates hepatocyte antigen processing and presentation capacity, increasing their activation of virus-specific T cells. HDV, a defective RNA virus that relies on HBV envelope proteins to infect hepatocytes, exacerbates HBV-related liver pathology. This immunological adjuvant-like feature can increase the anti-HBV efficacy of immunological therapy.

INTRODUCTION
The major etiological agent of chronic viral hepatitis worldwide is hepatitis B virus (HBV), a hepatotropic non-directly cytopathic DNA virus that establishes chronic infection in $240 million people. The pathological consequences of chronic HBV infection are aggravated by co-or super-infection with the hepatitis delta virus (HDV), a defective RNA virus that relies on the envelope proteins of HBV to infect human hepatocytes. 1 HDV is the only known viroid infecting humans, and according to the World Health Organization (WHO), $20 million individuals are HBV/ HDV co-infected worldwide, while new epidemiological estimations have suggested that the number of HDV + individuals may be even substantially higher (70 million people worldwide). 2 Patients with persistent HBV/HDV co-infection develop liver cirrhosis at a younger age and are more likely to progress toward hepatocellular carcinoma. 3 Conventional antiviral therapies used in HBV mono-infection (nucleoside analogs and interferon [IFN]a) have low efficacy in HBV/HDV co-infection. 4,5 Novel treatments targeting different steps of HDV infection and replications are being tested, alone or in combination with IFN-a, with some encouraging results. [6][7][8] However, there is still ample space and urgent need to develop new therapeutic approaches for the majority of HBV/HDV-co-infected patients.
We and others have advocated the use of strategies able to boost virus-specific adaptive immunity for the treatment of chronic HBV (CHB) infection. 9,10 HBV-specific CD8 T cells are associated with the control of HBV replication in animal models 11 and in patients. 12 Furthermore, the demonstration that transplantation of bone marrow containing HBV-specific T and B cells into patients with CHB can lead to an HBV functional cure 13,14 highlights the therapeutic potential of HBV-specific immunity reconstitution. The defect of HBV-specific B and T cell responses present in CHB patients [15][16][17][18][19] renders the restoration of HBV immunity, through vaccine therapy or checkpoint inhibitors, unlikely to succeed. However, reconstitution of HBV-specific immunity through the adoptive transfer of autologous T cells engineered with chimeric antigen receptors (CARs) or T cell receptors (TCRs) specific for HBV, 20,21 may bypass such defects and could lead to specific targeting of HBV-infected hepatocytes. 22 Recent studies in chimeric mice with HBV-infected human livers have shown the efficacy of these specific strategies. 21,[23][24][25] We reasoned that such approaches possess therapeutic potential also in HBV/HDV co-infection. The demonstration that HDV can persist intracellularly in replicating human hepatocytes despite blocking re-infection with Myrcludex B, 26 suggests that immune-mediated destruction of a substantial fraction of HDV-infected cells is required to substantially lower intrahepatic HDV infection. Targeting HDV/HBV-co-infected or HDV-mono-infected cells with engineered HDV-specific T cells is hindered, however, by the high mutation rate of HDV. Two recent studies have shown that HDV generates a high frequency of mutations leading to the selection of HDV variants able to escape HDV-specific CD8 T cell recognition in HBV/HDV-co-infected patients. 27,28 Thus, targeting directly the pool of HBV/ HDV-co-infected cells by transferring engineered T cells specific to the more conserved HBV-derived CD8 T cell epitopes may develop into a promising alternative strategy. However, this approach can only be successful if HDV does not interfere with the processing and presentation of HBV epitopes.
To answer this question, we established two in vitro HBV/HDV co-infection models, based on HepG2 cells transduced with human NTCP (HepG2-hNTCP) cells 29 and with normal primary human hepatocytes (PHHs). We quantified the expression of the genes associated with antigen presentation in HBV-mono-infected cells. Subsequently, we tested whether HDV co-infection modulates the processing and presentation of two distinct HBV CD8 T cell epitopes (one immunoproteasome-dependent [human leukocyte antigen {HLA}-A0201/HBs183-91] and one immunoproteasome-independent [HLA-A0201/HBc18-27] 30 ), using two readouts: (1) direct quantification of epitope complexes with TCR-like antibodies and (2) testing the ability of HBV/ HDV-co-infected cells to activate HBV-specific CD8 T cells. Finally, we used the human liver chimeric mouse model to test directly in vivo whether HBV/HDV co-infection alters the antiviral efficiency of adoptive T cell therapy.

RESULTS
Establishing In Vitro HBV/HDV Co-infection in Primary Human Hepatocytes and in HepG2-NTCP Cell Lines We used two in vitro models of HBV/HDV co-infection established with PHHs or HepG2-hNTCP cells 29 ( Figure 1A). Briefly, 24 h after HBV infection (MOI 3,000 genome equivalents [GE]/ cell), HDV was added at an MOI of 500 GE/cell. Seven days post-co-infection, HBV and HDV infections were tested by measuring HBV and HDV mRNA levels using NanoString technology. Customized probe sets targeting 2 specific regions in the HBV genome (genotype D) and 1 region in the HDV genome (genotype 1) were used ( Figure 1B).
HBV replication was confirmed in both HBV-mono-and HBV/ HDV-co-infected HepG2-hNTCP cells and PHHs, as seen from the high levels of HBV RNA expression ( Figure 1B, left and center), while HDV infection was detected only in HBV/HDV-co-infected HepG2-hNTCP cells and PHHs ( Figure 1B, right column). Although HDV RNA levels differed dramatically between PHHs and HepG2-hNTCP cells (4,425 mRNA counts in HepG2-hNTCP versus 68,863 mRNA counts in PHHs), HBV RNAs were only slightly higher in PHHs, showing that HBV infection was similar in both cell types. To quantify HDV infection at a single-cell level and determine the frequency of infected PHH-producing HDV, PrimeFlow RNA assay, a flow cytometry-based method for detecting HDV RNA, was applied. HDV RNA was detected in $20% of HBV/HDV-co-infected PHHs ( Figure 1C), while no coinfected cells were visualized with this technology in HepG2-NTCP cells ( Figure S1). Furthermore, we analyzed the expression of HBV antigens in HBV-mono-and HBV/HDV-co-infected cultured HepG2-hNTCP cells and PHHs by staining with antibodies specific for HBV surface antigen (HBsAg) and core antigen (HBcAg). Flow cytometry analysis showed that HepG2-hNTCP cells either HBV mono-or HBV/HDV co-infected were on average 35% HBsAg + and 48% HBcAg + . HBV-mono-infected PHH cultures were 90% HBsAg + and 80% HBcAg + , which was reduced to 75% HBsAg + and 45% HBcAg + in HBV/ HDV-co-infected PHHs, indicating that HDV infection lowers HBV antigen expression, which was more evident for HBcAg ( Figure 1D).

HDV Activates the Antigen Processing and Presentation Machinery of HBV-Infected Hepatocytes
The ability of HDV to trigger the expression of innate immune genes has been shown in both in vitro 31,32 and in vivo models. [33][34][35] To further characterize the transcriptional profile of immune signaling in HBV/HDV infection, we comprehensively evaluated the impact of HDV infection on HBV-infected cells using NanoString technology. The expression level of 579 human immune-related genes was analyzed in HBV-mono-infected and HBV/HDV-infected PHHs and HepG2-hNTCP cells. Globally, uninfected and HBV-mono-infected cells showed a very similar gene expression profile, while HDV infection modified profoundly the expression of immune genes. Figure 2A shows the heatmap of immune genes expressed in cultured PHHs, while Figures 2B and 2C display a pairwise comparison of gene expression between the different infection statuses of HepG2-hNTCP cells and PHHs, respectively. HBV RNAs were detected exclusively in HBV-infected HepG2-hNTCP cells and PHHs compared to uninfected cells, and the RNA of HDV antigen (HDAg) was detected only in HDV-infected cells. Consistent with the data in Figure 1B, the level of mRNA coding for HDAg was lower in HepG2-NTCPs than in PHHs (4,425 mRNA counts in HepG2-hNTCP versus 68,863 mRNA counts in PHHs).
HBV infection alone did not induce any significant changes in the immune gene expression in both cell types. In contrast, 30 and 78 immune-related genes were upregulated (R2-fold) in HBV/HDV-co-infected HepG2-hNTCPs ( Figure 2B) and PHHs ( Figure 2C), respectively. The immune-related gene expression changes were more pronounced in PHH compared to HepG2-hNTCP cells (the range of fold change in HBV/HDV-co-infected HepG2-hNTCP cells was between 2-and 4-fold compared to 2-and 10-fold in co-infected PHHs), reflecting the different infection rates and/or the lower integrity of immune response genes in hepatoma cells.
A Gene Ontology analysis classifying all of the upregulated immune-related genes in HBV/HDV-infected PHHs revealed that the biological processes related to antiviral immunity, cytokine production, and antigen processing and presentation pathways  Figures 2D-2I. HDV activated a plethora of IFN-stimulated genes such as Mx1, IRF7, STAT1, and STAT2, as previously described 31,33,34 ( Figure 2D). The previously reported ability of HDV to induce the production of IFN-b and IFN-l but not IFN-a 34 was confirmed at the mRNA level (Figure 2E). We detected in HBV/HDV-co-infected PHHs a robust increase in CXCL-10 and CXCL-11 ($104 normalized counts) mRNA, chemokines involved in the recruitment of inflammatory cells, and activated T cells 34,36 ( Figure 2E). The expression of genes coding for co-inhibitory and co-stimulatory molecules was also analyzed. Most co-inhibitory molecules were not detected, with only a minimal increased value (<500) of mRNA levels of LAG3 and CD274 (programmed death-ligand 1 [PD-L1]) in HBV/HDV-co-infected compared to HBV-mono-infected PHH cultures ( Figure 2G). Across the co-stimulatory molecules analyzed, only CD40 mRNA expression was increased in HBV/ HDV-co-infected hepatocytes ( Figure 2H).
Notably, all of the genes analyzed related to antigen presentation and processing were clearly upregulated upon HDV infection in PHH (Figures 2F and 2I). Beta 2 microglobulin (B2M) and the proteasome subunits PSMB-8 and PSMB-9 reflected the largest mRNA count differences between HBV/HDV-co-infected and HBV-mono-infected PHHs. Differential upregulation in these 3 genes ranged between 4-and 7-fold. A similar pattern of gene expression changes was also confirmed in HepG2-hNTCP HBV/HDV-co-infected cells, although at a lower level ( Figure S2).

Increased Expression of Antigen Processing and Presentation Genes in HBV/HDV-Co-infected Human Liver Biopsies
To confirm our in vitro findings, we analyzed the liver biopsies of patients with hepatic diseases presenting different etiologies pathologies). Liver biopsy samples from 10 patients with chronic hepatitis/cirrhosis (3 without viral hepatitis, 5 HBV, and 2 HBV/ HDV) that were previously embedded in an optimal cutting temperature (OCT) compound, were cryosectioned and tissues were extracted for RNA analysis using the NanoString human immunology panel supplemented with HBV and HDV RNA-specific probes ( Figure 3A). The presence of HBV RNA in the HBV-infected livers and of HDV RNA in the two HDV/HBV-co-infected livers confirmed the ability of our method to assess the mRNA expression profile in cryopreserved liver specimens. Despite the fact that all of these hepatic tissues are derived from the liver biopsies of patients with ongoing inflammatory events (chronic active hepatitis or cirrhosis), the two HBV/HDV-infected livers displayed a pattern of gene expression showing a superior activation of type I IFN genes in comparison to non-HDV-infected livers. The level of HLA class I molecules did not differ between HDV-and non-HDV-infected livers. In contrast, the expression of genes involved in antigen processing (TAP1, TAP2, PSMB-8, and PSMB-9) were upregulated in HBV/HDV livers in comparison to mRNA levels detected in the liver biopsies of different etiologies ( Figure 3B), even though the limited number of samples did not allow us to perform any statistical analysis.

HBV/HDV Infection Increases HBV-Epitope Presentation
Next, we investigated whether the increased expression of genes associated with antigen processing and presentation had functional consequences. We quantified HBV-epitope/HLA class I complexes on HBV-mono-infected and HBV/HDV-infected PHHs by using antibodies specific for two HLA-A0201/ HBV epitope complexes, HLA-A0201/HBc18-27 (HBc18 TCR-L monoclonal antibody [mAb]) and HLA-A0201/HBs183-91 (HBs183 TCR-L mAb) that were previously used in the liver biopsies of HLA-A0201 + CHB patients 37 and in HBV-mono-infected hepatocytes in vitro. 30,37 Notably, we recently demonstrated that the processing and presentation of these two different HBV epitopes is differentially regulated. 30 While the HBc18 epitope was constitutively presented in HBV-infected hepatocytes, HBs183 epitope processing and presentation appear to require the induction of the immunoproteasome within the infected cells, since IFN-g treatment of HBV mono-infected hepatocytes was required for its visualization. 30 Here, we stained HBV-mono-infected and HBV/HDV-co-infected HLA-A0201 hepatocytes ( Figures 4B and 4C) with the two TCR-L antibodies and HDV PrimeFlow RNA probes. Single-cell analysis performed with flow cytometry allowed us to decipher whether the HBV epitopes presentation was incremented preferentially by HDV replication within the HBV/HDV-infected cells or by the effect of cytokines released by HDV-infected cells in the cell culture media. Presentation of both HBV epitopes was clearly boosted by HDV infection; however, it was detected not only on HBV/ HDV-infected hepatocytes but also on hepatocytes that were negative for HDV RNA ( Figure 4B). A more robust effect was observed for the presentation of the HBs183 epitope (p = 0.001), even though HBc18 epitope presentation was also increased (p = 0.03). Direct visualization of HDV-infected cells confirmed the boosting of HLA-A0201/HBs183 complexes on HBV/HDV-infected cells appearing as punctuated structures lining the infected cells ( Figure 4C). Thus, HDV infection boosts the presentation of HBV epitopes on HBV/HDV-co-infected hepatocytes and neighboring HBV-mono-infected cells.

HDV Infection of HBV-Infected Hepatocytes Increases the Activation of HBV-Specific CD8 T Cells
To test directly the effect of HDV infection on HBV-specific CD8 T cell function, we co-cultured previously characterized 38 HLA-A0201 restricted HBc18 and HBs183-specific CD8 T cells with the HBV-mono-and HBV/HDV-co-infected HLA-A0201 + target cells. After a 5 h incubation, T cell function was measured by intracellular cytokine staining (ICS) of T cells using anti-IFN-gand anti-TNF-a-specific antibodies ( Figure 4A, right panel). The  Article ll OPEN ACCESS frequency of IFN-g and TNF-a producing HBs183-specific CD8 T cells was significantly higher (p = 0.03) upon co-culture with HBV/HDV-infected PHHs or HepG2-hNTCP cells compared to HBV-mono-infected cells ( Figure 4D, left panel). HBc18-specific CD8 T cell activation by HBV/HDV-infected cells was instead not significantly augmented in all of the experiments in comparison with HBV-mono-infected cells ( Figure 4D, right panel). HDV infection of HBV-infected PHHs and HepG2-hNTCP cells does not suppress the activation of HBV-specific CD8 T cells, and in addition, could enhance the ability of specific CD8 T cells to recognize the immunoproteasome-dependent HBs183 epitope.

IFN-b Increases HBV Epitope Presentation
HDV infection triggers the production of IFN-b and IFN-l in hepatocytes. [31][32][33][34][35] The direct quantification of HBV epitopes on cells infected with HBV and HDV ( Figures 4B and 4C) showed an increased HBV epitope presentation not only on co-infected hepatocytes but also on mono-infected cells. This suggests that antiviral cytokines triggered by HDV infection can boost the presentation of some HBV epitopes in a bystander fashion on HBVmono-infected cells. To assess this possibility, we measured directly the ability of IFN-b and IFN-l to boost HBV-specific CD8 T cell recognition of HBV-infected HLA-A0201 + hepatocytes and HepG2-hNTCP cells ( Figure 4E). A titration assay using recombinant human IFN-a, IFN-b, and IFN-l was performed on HepG2-hNTCP cells to gauge their ability to increase HLA class I expression ( Figure S3). The doses of cytokines able to maximally increase HLA class I expression on HepG2-hNTCP cells (100 IU/mL IFN-a, 5 ng/mL IFN-b, and 100 ng/mL IFN-l) were thereafter selected for use in CD8 T cell-recognition experiments. After the respective cytokine incubation, HBV-mono-infected PHHs and HepG2-hNTCP cells were co-cultured with HBc18-and HBs183-specific T cells. Their ability to produce IFN-g or TNF-a was analyzed by ICS. Incubation of HBVmono-infected PHHs and HepG2-hNTCP cells with all 3 types of IFNs had minimal effect on HBc18-specific CD8 T cell activation ( Figure 4E, upper panel). In contrast, IFN-b-but not IFN-a-or IFN-l-treated cells increased the activation of HBs183-specific CD8 T cells ( Figure 4E, lower panel). These data confirmed previous evidence showing that HBs183 epitope processing is dependent on the activation of immunoproteasomes mediated by type I and II IFNs 30 and show that IFN-b can boost the antigen presentation of selected HBV epitopes.

T Cells of Patients Chronically Co-infected with HBV/ HDV Can Be Efficiently Used to Engineer HBV-Specific TCR-Redirected T Cells
To test whether the reconstitution of HBV-specific T cell responses through the adoptive transfer of HBV-specific TCR-redirected T cells could have therapeutic potential, we tested the ability of T cells obtained from 6 chronic HBV/HDV-co-infected patients to expand in vitro, express the HBs183 TCR, and produce cytokines after antigen-specific recognition ( Figure 5A). These were compared with the T cells of 6 healthy individuals.
Our results show that T cells of healthy controls and of chronically HBV/HDV-co-infected patients expressed the introduced TCRs at the same frequency and displayed similar levels of activation and cytokine production after antigen-specific recognition. The ability of T cells from HBV/HDV patients to be engineered into efficient HBV-specific TCR-redirected T cells was confirmed also with a different TCR specific for the HBs370-379 (HBs370) epitope ( Figure 5B).

Antiviral Efficacy of TCR-Redirected T Cells In Vivo
We then tested in an animal model of HBV infection whether HDV modulates the antiviral efficacy of adoptive T cell therapy. We generated humanized chimeric mice reconstituted with HLA-A0201 hepatocytes and infected them with HBV and HDV viruses. 39 The humanized mice were infected with HDV after having established stable HBV viremia titers (8-10 weeks post-HBV inoculation) ( Figure 6A). T cell therapy was performed using TCR-redirected T cells engineered from T cells of a healthy individual, expanded in vitro with anti-CD3 and interleukin-2 (IL-2), and electroporated with mRNA coding for the HBs183-specific TCR or with the EBV-LMP2A 426-34 (EBV)-specific TCR. Both HBs183-TCR and EBV-TCR recognize their cognate epitope (HBs183-91 and LMP2A426-34) in association with HLA-A0201 molecules. A detailed description of the production of mRNA TCR-redirected T cells is described in the STAR Methods and was previously published. 21,40 Note that due to ethical and practical restrictions, human hepatocytes and lymphocytes used to reconstitute the mice were not derived from the same donor but were matched based on presenting an HLA-A02 haplotype. HBV/HDV-infected mice received 3 doses of 0.5 million HBV-specific TCR T cells (n = 3) or EBV-specific TCR T cells (n = 3) at the indicated intervals (0, 4, and 8 days), while mice that were infected in parallel with similar viral titers were left untreated and served as controls ( Figure 6B). A progressive reduction in HBV DNA was observed after the infusion of HBs183-specific TCR T cells with a mean reduction in HBV viremia of 1.9log at day 12 ( Figure 6B). Notably, this was a 10-fold greater reduction in HBV viremia than what was observed in HBV-mono-infected mice ( Figure 6C). 21 Adoptive transfer of HBs183-specific TCR T cells in HBV/HDV-infected mice also caused a substantial reduction in intrahepatic covalently closed circular DNA (cccDNA) (mean 1.1log), HBV pregenomic RNA (pgRNA) (mean 1.3log), and total HBV RNA levels (mean 1.0log) in only 12 days of treatment ( Figure 6E). We also observed a moderate decrease in circulating HBsAg levels in 3/3 mice receiving 3 infusions of HBs183-specific TCR T cells (0.  Figure 6D). Adoptive transfer of EBV-specific TCR-T cells also exerted a degree of antiviral effect against HBV. While the reduction of cccDNA (0.15log) was not triggered by the adoptive transfer of EBV-TCR T cells ( Figure 6E), we observed a moderate decrease in HBV viremia (mean 0.5log), intrahepatic HBV RNA (0.4log), and HBsAg in 2/3 HBV/HDV-co-infected mice (mean 0.48log reduction; Figures 6B and 6E). HDV viremia was also lowered by TCR T cell transfer, but without a substantial difference between the adoptive transfer of HBV-TCR T cells and EBV-TCR T cells (mean 0.6 and 1.0log, respectively; Figure 6F). This antiviral effect of adoptively transferred T cells independent of TCR specificity was unexpected and likely due to an alloreactive T cell activation that is detected only in HBV/HDV-co-infected mice. The adoptive transfer of T cells engineered with a non-HBV-specific TCR did not cause any alteration in HBV virological parameters in comparison to non-treated mice in HBV-mono-infected mice ( Figure 6C). 21 The differential effect of activated T cells engineered with a non-HBV-specific TCR confirmed the ability of HDV to modify the liver environment and to increase, through the upregulation of inflammatory chemokines, the intrahepatic recruitment and activation of specific and also alloreactive T cells. This was confirmed using RNA in situ hybridization technology showing a more pronounced enhancement of human chemokines such as CXCL10 ( Figure 6G) in human hepatocytes of HBV/HDV-infected mice in comparison to HBV-mono-infected mice. These in vivo data corroborate the findings observed in our NanoString analysis, which showed a robust upregulation of CXCL10 transcripts in HBV/HDV-infected hepatocytes ( Figure 2E). These

DISCUSSION
Viruses that establish chronic infection usually develop strategies to dampen host immunity. HDV appears to use a different strategy. In addition to its known ability to activate IFN responses, [31][32][33][34][35] we demonstrated here that HDV increases the efficiency of the antigen processing and presentation machinery of the infected and neighboring hepatocytes. By collecting data in in vitro HDV infection systems, liver biopsies, and mice with humanized livers, we showed that HDV infection not only enhances the gene expression of HLA class I molecules, B2M, Article ll OPEN ACCESS immunoproteasome, and co-stimulatory molecules genes but it also increases the presentation of viral epitopes, and as a direct consequence, the efficiency of T cell recognition of infected hepatocytes.
The enhanced HLA class I presentation of viral epitopes could explain the high incidence of escape mutations found in HDV epitopes, 28 but it can have a different effect in HBV. The compact nature of the HBV DNA genome poses a limit on the generation of mutated HBV viruses, and this makes HBV unlikely to generate virus progeny able to escape HBV-specific CD8 T cells. This can therefore increase the chances to control HBV in HBV/ HDV-co-infected individuals. Epidemiological evidence supports this hypothesis: a longitudinal study of HBV-mono-and HBV/HDV-infected patients demonstrated that within 4 years, 10% of the dual-infected patients became HBsAg À , a much higher rate than in HBV-mono-infected patients, in whom only 3% lost serum HBsAg. 41 Furthermore, HBV clearance in HBV/HDV-co-infected patients was associated with an increased number of HDV antigen-expressing hepatocytes, 42 a historical observation that supports the concept that HDV can act as an adjuvant for the immunological control of HBV. Nevertheless, this positive outcome occurs only in a minority of HBV/ HDV-co-infected patients, 43 since most adult CHB patients have severe functional impairment of HBV-specific adaptive immunity. 9 Consequently, in the presence of a low frequency of HBV-specific CD8 T cells, the increased activation of intrahepatic T cells would not result in more chances of HBV clearance, but only in increased liver inflammation. This scenario explains the higher intrahepatic inflammatory levels and more severe liver pathology frequently present in patients with HDV/HBV co-infection in comparison to those harboring only HBV. 3 We think, however, that the ability of HDV to boost hepatic antigen presentation could, apart from the described pathogenic effects, be used to increase the therapeutic efficacy of T cell-based therapies in HBV/HDV-co-infected patients. T cell therapy with lymphocytes modified with CAR or with HLA class I-restricted TCR is starting to be considered as a therapeutic option not only for cancer but also for chronic viral diseases. 44 We have recently shown that the adoptive transfer of HBV-specific TCR-redirected T cells in a patient with HBsAg + HCC relapses drastically reduced the HBsAg values. 45 The data provided in this work, showing that HDV infection of HBV-infected humanized chimeric mice increased the anti-HBV activity of adoptively transferred HBV-TCR redirected T cells, encourage the use of HBV-TCR T cells in HBV/HDVco-infected patients. This possibility is further supported by the demonstration that the T cells of HBV/HDV chronically co-infected patients are amenable to TCR-T cell engineering and can be expanded in vitro to reach a functionality that is identical to that of healthy subjects.
Although the overall decrease in HBV and HDV viral loads observed in our experiments was objectively limited, we used a low number of human T cells (only 0.5 million HBV-TCR T cells per mouse) that only transiently expressed the virus-specific TCRs. This strategy was selected to limit inflammatory responses in the liver. 21 Nevertheless, increasing the number of infused lymphocytes or using treatment strategies that combine the use of TCR T cells with a blocker of re-infection, such as neutralizing antibodies or entry inhibitors (i.e., Myrcludex B), could increase the antiviral efficacy of such therapies. 25 A caveat of using a T cell therapy-targeting HBV to address the problem of HDV co-infection is the fact that HDV can persist in non-HBV-infected hepatocytes 46 and endure cell division. 26 Therefore, HDV-mono-infected cells will not be targeted by HBV-specific TCR T cells, unless HBV envelope proteins are produced from HBV DNA integrations into the host genome. However, the pathological impact of the presence of clusters of HDV-only-infected cells in the natural history of HBV/HDV chronic infection is still unknown.
In summary, by demonstrating that HDV acts as an immunogenic adjuvant in HBV-infected hepatocytes, we unveil a previously unknown feature of HDV that can explain its ability to increase liver inflammation in HBV/HDV-co-infected patients. In addition, we show that its ability to boost HBV epitope presentation increases the efficacy of T cell-based therapy in a in vivo mouse model of HBV/HDV infection. We believe that our findings broaden the therapeutic strategies that can be applied to HBV/ HDV-co-infected patients, who are still the only patients with hepatotropic infections without any US Food and Drug Administration (FDA)-approved therapy. 5

Limitations of Study
This work describes experiments done in vitro and in humanized chimeric mouse models that demonstrate the ability of HDV to increase the antigen presentation of HBV epitopes and HBVspecific T cell activation. Whether HDV could enhance the efficacy of HBV-specific TCR T cells in the treatment of chronic HBV/HDV infection needs to be directly tested in patients. The humanized chimeric mouse model has limitations in the measurement of the efficacy of TCR T cell therapy: there is a partial HLA match between human hepatocytes and human T cells, a situation that is different from the proposed therapy in which patients will be treated with autologous lymphocytes.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

DECLARATION OF INTERESTS
A.B. is a co-founder of LION TCR, a biotech company developing T cell receptors for the treatment of virus-related cancers and chronic viral diseases. P.L. advises and is on the speakers' bureaus for Janssen, MYR Pharmaceuticals, GlaxoSmithKline, Gilead Sciences, AbbVie, Roche, Eiger, Alnylam, Bristol-Myers Squibb, and Merck Sharp & Dohme. A.L. is on the speakers' bureaus of MYR Pharmaceuticals and Gilead. The other authors declare no competing interests.