The immune landscape in hepatitis delta virus infection—Still an open field

Hepatitis delta virus (HDV) is known to cause the most aggressive and severe form of viral hepatitis, yet it remained under‐diagnosed but does require early diagnosis for accurate disease staging. Antibody to HDV (anti‐HDV) is the primary screening tool and should be assessed in patients with hepatitis B surface antigen (HBsAg) positivity, as HDV is a satellite RNA virus of hepatitis B. Additionally, the viral load (HDV RNA) should be assessed in those with positive anti‐HDV, to differentiate between active infection and resolved hepatitis delta. Data regarding immune responses in HDV are limited but show dysfunctional adaptive and innate immunity. Many studies however fail to distinguish between active and resolved infection. Limited treatments are available for HDV, but promise has been shown with the newly approved Bulevirtide, a first‐in‐class HBV entry inhibitor. Thus immune response during therapy requires further investigation, along with additional targets for HDV cure.

HDV, and although limited research has been undertaken into clinical outcomes on relation to HDV genotypes, some data have suggested that genotype 1 is associated with poorer clinical outcomes including HCC development. 4 It is, however, important to differentiate between active HDV infection and resolved HDV infection. In many instances, the latter being referred to when subjects have cleared infection, where HDV RNA is undetectable (or below the lower limit of detection), but anti-HDV antibody positive, thus having previously been infected but cleared infection. Subjects with evidence of HDV RNA are those with active HDV infection. Anti-HDV positive, HDV RNA negative subjects may have little chance of reactivation, but despite this are currently excluded from clinical trials for HBV cure. Those with active infection (and circulating HDV RNA) and those whom are only anti-HDV (resolved hepatitis delta), however, are often studied and grouped together, yet their virological and immune profiles may be disparate, but there are few studies that differentiate between these clinical profiles and thus a lack of data which separates these distinct clinical phases/phenotypes. It would be critical to differentiate between peripheral and intrahepatic viral and immune profiles of these different subjects.

| AdaptiveimmuneresponsesinHDV
HDV is thought to utilize different mechanisms from HBV and HCV mono-infection to modulate the immune system. Similar to HBV, HDV, being a stealth-like virus, also evades IFNα-mediated immune responses, which promotes viral persistence and cell survival. 5 Antigen-specific CD8 + T cells can recognize the HDV antigen on infected cells and mediate their killing, but evidently this also leads to the death of uninfected healthy hepatocytes. Along with this, the clonal expansion of CD4 + T cells leads to cytokine release (IL-2, IL-10, IFNγ), stimulating the immune-mediated killing of HDV-infected cells but this may occur in an unregulated manner, resulting in liver necrosis and progressive liver disease. 6 In line with this, studies have shown that IL-2 and IFNγ production from HDV-specific T cells, as well as elevated IP-10 responses exerted by activated monocytes, also contribute to the inflammatory milieu in chronic active HDV infection, thus where there is ongoing inflammation and/or liver damage. 7 In several human studies, low-level HBV-specific CD4 + and CD8 + T-cell responses have been detected, following in vitro expansion in patients with chronic active HDV infection, and in those patients with acute HDV infection, expectedly strong CD8 + T cells responses have been detected, leading to the control of infection, and therefore indicating the importance of their role in the control of HDV as with other hepatotropic infections. Data from a recent study analysing blood and liver samples from active HDV-infected patients, with evidence of HDV RNA, demonstrated the high expression of the innate-like NK cell receptor NKG2D on HDV-specific and global intrahepatic CD8 + T cells, associated with TCR-independent activation. The expression of NKG2D on CD8 + T cells directly correlated with liver inflammation, suggesting a role for non-antigenspecific bystander T cell-related liver inflammation in chronic active HDV infection, indicating the lack of antigen-specific immunity leading to disease progression. 8 Overall, the adaptive immune response appears weak in HDV, similar to that observed in HBV and HCV.
What remains unclear in these studies is the full clinical HDV profile of these patients, including the level of circulating HDV RNA, and whether subjects have active or resolved HDV infection, which may impact the immune response.
Current knowledge regarding immune responses against HDV is largely restricted to the analysis of peripheral blood mononuclear cells (PBMCs) in patients with HDV infection. Interestingly, comparative to patients with HBV and HCV infections, a high frequency of cytotoxic perforin-positive CD4 + T cells have been found in the blood of patients with HDV chronic infection, but differences in those with active hepatitis delta compared to those with resolved HDV was not evident. 9 The HDV-specific T cell response generated in patients with chronic active HDV infection, however, remains weak and insufficient to contain the infection. 10 Low-level HDV-specific CD4 + and CD8 + T-cell responses have been detected following in vitro expansion in a number of subjects, but no correlation was observed between the magnitude of HDVspecific CD4 + or CD8 + T-cell responses and the level of viremia or clinical status of patients, thus not differentiating between active and resolved HDV infection. In one study, a HBV patient super-infected with HDV was longitudinally followed during the acute phase of HDV infection, where a strong CD8 + T cell response was detected initially, followed by a decrease in HDV viral load, suggesting a role for T cell responses in the control of HDV viremia, analogous to HBV mono-infection. 11 Another study identified six CD8 + T cell HDAg epitopes stimulating PBMCs from a cohort of patients with HDV infection treated with lonafarnib/ritonavir, but even in this instance some patients (4/28) demonstrated undetectable HDV RNA. The ex vivo activation state here, correlated with ALT levels and IFNγ production following after peptide stimulation and correlated inversely with the HDV RNA. The majority of the HDV-specific CD8 + T cells demonstrated a a memory-like phenotype (PD-1 + CD127 + ) and also expressed the activation marker CD38, along with the transcription factor TCF1, critical for CD8 + T cell memory generation and were capable of low-level IFNγ responses. 12 More circumspect immunological analyses differentiating between active and resolved HDV infection are however required. It is likely that adaptive immune responses in resolved HDV infection may be similar to those patients with HBV (likely with HBeAg negative chronic infection) disparate to those with active HDV infection, whom, as previously shown may demonstrate more aggressive immunopathology with an exhausted T-cell profile.

| InnateimmuneresponsesinHDV
The observed specific adaptive immune response in HDV remains weak and thus consideration of innate immunity has also been undertaken. HDV patients display a higher frequency of natural killer (NK) cells, but with a profile of less activated NK cells, low expression of the activating receptors CD244 and CD48 resulting in impaired cytolytic activity and reduced cytokine production. The proportion of CD56 bright NK cells subset (less mature and immunoregulatory) is also increased in relation to the more mature and cytolytic CD56 dim subset and in line with this, these HDV patients also produce high levels of IFNγ and TNFα, which in this regard may result in more aggressive liver disease. 13,14 The inhibition of NK cell activity appears a common escape mechanism of hepatitis viruses, which evidently leads to ongoing inflammatory activity as opposed to controlled cytolytic killing of infected hepatocytes. Thus, as noted even with adaptive immune responses, non-specific immune activation leading to inflammation ensues over virus-specific factors. 14  It may be that the balance of IFNγ and TNFα is reversed so that more TNFα is produced as observed with CD4 + T cells in HBV, which leads to a more aggressive inflammatory response. 15,16 Regardless of the inflammatory response seen, no correlation between NK cell phenotype and viral load was noted arguing against a direct effect specific to any single hepatitis virus on NK cells. 14 Determining the effects of treatment on HDV immunity, it was noted that pegylated interferonα (PEG-IFNα-2a) led to a significant change in NK cell differentiation status, with an increase in immature circulating NK cells. Peripheral blood NK cells of untreated HDVinfected patients were, however, phenotypically similar to healthy controls, suggesting that the HDV effect on NK cells may be limited to the liver, but this was not investigated in this study. 13 In previous studies increased NK cell activity in HBV was linked to liver injury, here Lunemann et al. reported no correlation between NK cell phenotype and disease severity in untreated HDV patients. Instead, a higher frequency of CD56 dim NK cells before treatment was associated with responsiveness to PEG-IFNα-2a, 13

InfectionType Adaptiveimmuneresponse Innateimmuneresponse
Active HDV infection (HDV Ab+, HDV RNA+) tive HDV infection a more 'pathogenic' innate immune profile may be present, with a greater number of NK cells, but these may be poorly functional and recover once viremia. MAIT cell function may also recover in resolved HDV and be similar to HBV mono-infection, but these findings need to be confirmed. Table 1

| IntrahepaticimmuneresponsesinHDV
Histopathological features from LB's in HDV are similar to those seen in other types of chronic viral hepatitis, with piecemeal necrosis, portal inflammation, cytoplasmic dissociation, polished nuclei loaded with viral antigens and the presence of apoptotic bodies. The degree of inflammation, however, is more marked and necrosis is enhanced in co-infection or super-infection, and the presence of delta antigen can be determined immunohistochemically. 22 It is well known that the liver is enriched in innate immune cells like NK, natural killer T cells (NKT), ILC and γδT cells and presents with a reversal of the CD4:CD8 ratio when compared to that of the peripheral compartment. 23 The tissue microenvironment can have an effect on local lymphocytes, which may be key determinants in liver-related diseases and infections. 24 Thus, what is known regarding peripheral blood lymphocytes may not be representative of the intrahepatic compartment; however, the analysis of the cells in the blood of HDV-infected patients remains the most accessible surrogate for the study of the intrahepatic immune response, since LB is not widely used in clinical practice. Nevertheless, direct liver sampling is still required for the study of the immune response, virological markers and gene expression/transcriptome profiling. 25 Some data exist delineating tissue-specific immune responses in HDV, but these still require further evaluation to fully understand innate and adaptive immune interactions in the liver, mandating liver sampling and differentiating between those subjects with active and resolved HDV infection. 8 Fine-needle aspiration (FNA) has been shown to be a safe and well-tolerated technique allowing broad analysis of the intrahepatic compartment, with significant correlation noted in comparison with data from LB. 26 Its reduced invasiveness can allow for repeated sampling and monitoring of the kinetics of the immune response during different disease stages on treatment. 27 FNAs were demonstrated to be able to sample tissue-resident subsets of T 28 and NK cells 29 in the context of HBV infection, as well as to provide viable hepatocytes and myeloid cells. 26 It would also be of interest to explore the role of other non-parenchymal cell types, and their response to the cytokines present in the active HDV-infected liver compared with that in resolved infection.

| CON CLUS IONS
Due to the complex cellular composition of the liver, only a comprehensive analysis of the site of infection can lead to a better understanding of all the players involved in chronic HDV and their specific roles in the observed pathology. Unlike HBV and HCV, determinants of the immune response in HDV infection are limited, and this remains an area of unmet need for research to find improved treatment strategies and limit HCC development, and a more circumspect approach to analysing peripheral and intrahepatic immune responses in HDV. Dissecting the immune responses further in these distinct compartments and correlating them will viral and clinical markers is work that is urgently required to better understand the natural history of HDV infection.

CO N FLI C TO FI NTE R E S T
USG has no conflicts of interest to declare.

DATAAVA I L A B I L I T YS TAT E M E N T
The data that support the findings will be available in N/A at https:// wiley.atypo nrex.com/submi ssion Board/ 0a910 9a8-491c-42f8-a4b7-e649b 06bf9 d3/addit ional Infor mation following an embargo from the date of publication to allow for commercialization of research findings.