Taking into account that CHB outcome relies on equilibrium between the virus and the host, in the next paragraphs it will be explained how different virological and immunological parameters could be considered or not as predictors to safely discontinue NA treatment.
Noncytopathic viruses, such as HBV, have developed evolutionary mechanisms to remain hidden from the immune system, which is an advantage for their persistence. HBV virus is not highly infectious but produces long-lasting disease that allows it to spread the infection over time. The host/HBV relationship is a dynamic process in which the virus tries to decrease its visibility, whereas the host attempts to prevent and eradicate infection with minimal collateral damage to itself.
Several viral markers have been proposed as potential biomarkers for a safe NA discontinuation, and they are discussed below.
Serum HBV RNA reflects the transcriptional activity of liver cccDNA, and its decline seems to be a good predictor of HBeAg seroconversion. Nevertheless, it is commonly undetectable in HBeAg(-) cases, making it useless as a biomarker for stoppage of NA treatment in this increasing population. Moreover, improvement of the HBV RNA assay to make it more sensitive and reproducible, as well as studies in bigger cohorts, are essential before considering it as a potential biomarker for monitoring safe discontinuation of NA therapy in HBeAg(+) patients.
Hepatitis B core (HBc) is an inner nucleocapsid surrounding the viral DNA and is the target of specific T cell response against the virus. AntiHBc is the first antibody to appear after HBV exposure and it represents a classical serological marker for HBV infection. The role of antiHBc as a predictor of NA discontinuation, however, has not been fully examined, but it was recently reported that baseline antiHBc level is a strong predictor for HBeAg seroconversion during PEG-IFN-α or NA therapy. Moreover, there was a trend for an inverse association between antiHBc and clinical relapse after long-term ETV treatment cessation in an Asiatic CHB cohort. AntiHBc, as a predictor, needs to be further assessed and validated in non-Asiatic cohorts, to verify if it could be useful.
HBV core-related antigen (HBcrAg) includes HBcAg, HBeAg and a pre-core protein (p22cr), and its quantification closely correlates with intrahepatic cccDNA level[58,59]. In HBeAg(+) CHB patients, the dynamics of HBcrAg accurately predict spontaneous HBeAg seroconversion and the combination of HBsAg together with HBcrAg quantification help to predict safe discontinuation after NA treatment cessation. However, most of this research has been performed in Japan with first-generation NAs, so further validation with the currently available NAs and different areas of study is lacking.
In summary, some virological markers could be useful predictors of response in HBeAg(+) patients, but improvement of the assays together with further cohort validation is still needed for HBeAg(-) cases. The other side of the balance is the host’s immune defence against the virus, presented in the next section.
To achieve control of the HBV infection, a functional adaptive immune response, in particular the cellular immune response, is essential; meanwhile, whether and how HBV triggers the components of the innate immune system remain controversial topics. Even though the humoral response is an effective line of defence against reinfection, in the setting of CHB, the virus persists despite high levels of HBV-specific antibodies due to antigen overload, and only hepatitis B surface antibody is associated with disease resolution.
Primed HBV-specific CD4 T cells are crucial to allow the adequate activation of HBV-specific CD8 T cells by secretion of proinflammatory cytokines, including IFN-α. Afterwards, HBV-specific CD8 T cells play a major role in the resolution of spontaneous infection because they can specifically recognise the infected hepatocytes. Moreover, they can clear the virus by inducing apoptosis of the infected cell as well as by proinflammatory cytokine production to eliminate the virus without causing cell death.
CD4- and CD8-specific HBV responses are vigorous, polyclonal and multispecific in acute-resolving cases, whereas are profoundly impaired in chronically infected patients[6,65-68]. During CHB, HBV-specific T cell responses gradually lose their functionality and are finally deleted due to the high and persistent antigen exposure, in order to avoid host-induced tissue damage, in a process called T cell exhaustion. T cell exhaustion is characterised by high and sustained expression of several negative pathways (i.e., PD-1, immunoregulatory cytokines and so on)[70-75].
The role of HBV-specific CD4 T cell features as a predictor for NA cessation has not been intensely studied. It could be explained mainly by two reasons. First, the frequency of these cells in the chronic setting of the disease is very low. Second, due to the nature of CD4 responses, in vitro stimulation assays are difficult because these cells are only successfully stimulated by professional antigen-presenting cells. Even though a robust HBV-specific CD4 T cell response is observed in acute resolving cases, and they are essential to support HBV-specific CD8 T cells, the difficulty of assessment makes them less useful than HBV-specific CD8 T cells or other surrogates when trying to find an easy and reproducible immunological marker to stop NA therapy safely.
CHB is one of the best models to study CD8 T cell exhaustion. In the different stages of the natural history of HBV infection, there are different virus-host interactions, reflected by different immune features of HBV-specific CD8 T cells. Bearing in mind that several studies have shown that after long-term NA treatment interruption the majority of patients remain with a viral response after long follow-up[29,32], we could infer that the host’s immunity is controlling HBV replication.
After a long-term NA treatment cessation, HBV-specific CD8 T cells could be given a second chance to fight the virus. If these cells have been restored by the reduced viremia that had been induced by the antiviral therapy at that point, these cases would be able to control the infection in a similar way to chronic infection cases. Therefore, patients with viral control are likely to have a good immune response against the virus, whereas cases with virologic rebound may have a dysfunctional response.
Thus, changes in HBV-specific CD8 T cell phenotype may predict acquisition of antiviral control before HBsAg loss. Taking into account the vital role of HBV-specific CD8 T cells during the natural history of the disease, and its in-depth characterisation achieved over the last two decades, it is presumable that those different features according to viral control could give hints to answer one of the most critical questions regarding CHB management: What kind of patients could benefit from NA therapy interruption?
Boni et al have extensively studied several immune subsets in different groups of chronically infected patients, including those under NA therapy. In the LMV treated patients, they found an initial improvement of HBV-specific T cell effector capacities against different HBV epitopes (HBcAg, HBeAg) after DNA fall, followed by a decline at 6 mo after the treatment has been stopped; this biphasic behaviour is irrespective of clinical outcome. It appears that the first-generation NAs lack the potency needed for HBV-specific T cell restoration.
Succeeding experiments in larger cohorts under the first- and second-generation NA therapies demonstrated that HBV-specific CD8 T cell effector abilities were similar between patients after several years of antiviral treatment and acute resolving cases featured by a PD-1+ phenotype (Figure 1). PD-1 up-regulation arises on HBV-specific T cells following acute and chronic infection. In the setting of acute infection, PD-1 up-regulation is transient, returning to low levels after viral clearance. However, in chronic infection, PD-1 up-regulation is sustained, and the blockade of PD-1/PD-L1 interaction has shown promising results in restoring virus-specific T cell functionality[80-83]. Therefore, a PD-1+ phenotype could mean both activation before clearance or exhaustion after persistent and high antigenemia.
Figure 1 Potential immunological biomarkers for safe nucleos(t)ide analogue discontinuation.
A possible strategy to shorten NA therapy would be to check if HBV-specific CD8 T cells have reacquired their antiviral function after long-term therapy. Effective HBV-specific CD8 T cells control the virus: PD-1+ HBV-specific CD8 T cells against different epitopes, able to mount a robust response (IFN-γ, IL-2 production) after antigen encounter, may be a good predictive tool of response. Low inflammatory profile of NK cells may likewise reflect a good point to end therapy. High levels of IP-10 also could point to anti-viral control. Exhausted HBV-specific CD8 T cells lose control of the virus: High and sustained PD-1 expression on HBV-specific CD8 T cells reflects their dysfunctionality. Lower IFN-γ and IL-2 production, along with an immunosuppressive cytokine environment (IL-10, TGF-β), renders these cells to exhaustion. An inflammatory phenotype of NK cells may reflect NK cell-mediated T cell deletion through death receptors. Low levels of IP-10 could dissuade us to stop therapy. HBV: Hepatitis B virus; IR-10: CXCL10; NA: Nucleos(t)ide analogue; NK: Natural killer; PD-1: Programmed cell death protein 1.
The most recent work studying HBV-specific T cell response as a biomarker for HBV therapy discontinuation demonstrated that the patients who did not relapse to NA stoppage featured, during NA treatment, an increased frequency of functional PD-1+ HBV-specific T cells directed against nucleocapsid and polymerase HBV proteins (Figure 1). The PD-1+ expression on functional HBV-specific T cells may reflect an activated, nonexhausted phenotype. Along these lines, patients with functional HBV-specific CD8 T cells, positive for PD-1, may no longer need NA treatment and should be considered for treatment cessation.
However, the current method is complicated to move from bench to bedside because it involves the study of rare populations by multicolour flow cytometry. Hence, the development of an assay to directly quantify PD-1+ HBV-specific CD8 T cells would be of great interest. Even though the final effectors to clear HBV are the HBV-specific CD8 T cells, it is essential to consider the interplay between them and other components of the immune system to fully understand immunity against HBV and their potential as surrogate biomarkers.
The natural enrichment of natural killer (NK) lymphocytes in the human liver underscores their potential importance in the control of hepatotropic viruses, such as HBV. During CHB, NK cells express an inhibitory phenotype with altered functionality[85,86] and have predilection for apoptosis of HBV-specific T cells, resulting in HBV-specific T cell deletion after death ligand-death receptor interaction. Boni et al showed a low inflammatory profile of NKs after successful NA therapy, similar to healthy controls. In line with the previously commented work, this lower inflammatory status of NKs correlated with a better HBV-specific T cell response (Figure 1). Moreover, a partial restoration of blood NK cells was shown following long-term ETV, in terms of antiviral cytokine production compared to naïve CHB.
So, why should study of NKs - instead of HBV-specific CD8 T cells - be useful? The study of NK cell inflammation does not involve multimers nor intracellular cytokine staining, as used to assess HBV-specific CD8 T cell responses, resulting in more easily reproducible experiments. A low inflammatory profile of NK cells can be evaluated by surface staining and may reflect an HBV-specific T cell restoration and subsequent control without the need of therapy. Studies in bigger cohorts after stoppage of NA treatment are needed to address if successful NA discontinuation correlates with a lower inflammation phenotype of NK cells.
The third signal of T cell activation requires an adequate cytokine profile, and long-term NA therapy has been shown to modulate it. Successful viral repression leads to antiviral response stimulation by promoting proinflammatory cytokines such as IFN-γ[90,91] and IL-2[92,93], as well as by decreasing regulatory effectors such as IL-10[91,94] and TGF-β. At least theoretically, the measurement of these cytokines together with HBV-specific T cells or NK cells could also give us clues to establish a good cessation point for therapy (Figure 1).
Not only are the phenotype and functionality of the different immune subsets important components of an adequate milieu during CHB but also the trafficking of HBV-specific T cells to the infected liver. The migration of lymphocytes to the liver is a complicated process involving adhesion, rolling, triggering and transendothelial migration. Chemokines and their receptors play an essential role in this multistep pathway[96,97].
After the analysis of several plasma chemokines, the one that appears to be a promising surrogate of HBsAg loss under NA therapy is CXCL10 (IP-10). IP-10 is a small protein, secreted by hepatocytes in response to viruses and the subsequent recruitment of proinflammatory CD4 and CD8 T cells to the infected liver (Figure 1). It was previously reported that baseline serum IP-10 levels were higher in patients with HBsAg loss during NA therapy and, in line with those findings, another work examined the serum IP-10 kinetics during ETV therapy. Interestingly, they found that IP-10 levels started to significantly increase after the 3rd year of treatment with ETV, which is in line with the timing observed to be necessary to achieve a sustained virological response in the different stopping-treatment studies[29,32].
It is likely that after a prolonged and effective viral replication suppression under NA treatment, the migration process to the liver is restored and HBV-specific T cells are functional and able to clear the remaining infected hepatocytes, thus reflecting the HBsAg decline.