L. Jansen

An intrahepatic transcriptional signature of enhanced immune activity predicts response to peginterferon in chronic hepatitis B

Differences in intrahepatic gene expression patterns may be associated with therapy response in peginterferon‐treated chronic hepatitis B (CHB) patients.

HBsAg loss in patients treated with peginterferon alfa-2a and adefovir is associated with SLC16A9 gene variation and lower plasma carnitine levels

BACKGROUND & AIMS Achievement of HBsAg loss remains the hallmark of chronic hepatitis B treatment. In order to identify host factors contributing to treatment-induced HBsAg loss, we performed a genome-wide screen of single nucleotide polymorphisms (SNPs) and studied its immunological consequence. METHODS Chronic hepatitis B patients (40 HBeAg-positive and 44 HBeAg-negative) treated with peginterferon alfa-2a and adefovir were genotyped for 999,091 SNPs, which were associated with HBsAg loss at week 96 (n = 9). Plasma carnitine levels were measured by tandem-mass spectrometry, and the effect of carnitine on the proliferative capacity of hepatitis B virus (HBV)-specific and non-specific CD8 T cells was studied in vitro. RESULTS One polymorphism, rs12356193 located in the SLC16A9 gene, was genome-wide significantly associated with HBsAg loss at week 96 (p = 1.84 × 10(-8)). The previously reported association of rs12356193 with lower carnitine levels was confirmed in our cohort, and baseline carnitine levels were lower in patients with HBsAg loss compared to patients with HBsAg persistence (p = 0.02). Furthermore, we demonstrated that carnitine suppressed HBV-specific CD8 T cell proliferation. CONCLUSIONS In chronic hepatitis B patients treated with peginterferon and adefovir, we identified strong associations of SLC16A9 gene variation and carnitine levels with HBsAg loss. Our results further suggest that a lower baseline plasma carnitine level increases the proliferative capacity of CD8 T cells, making patients more susceptible to the immunological effect of this treatment. These novel findings may provide new insight into factors involved in treatment-induced HBsAg loss, and play a role in the prediction of treatment outcome.

Viral minority variants in the core promoter and precore region identified by deep sequencing are associated with response to peginterferon and adefovir in HBeAg negative chronic hepatitis B patients

Background and aim: Precore (PC) and basal core promoter (BCP) mutations are associated with responses to interferon‐based treatment in HBeAg‐positive chronic hepatitis B (CHB) patients. Here, we identify viral minority variants in these regions and assess association with response to peginterferon‐alfa (Peg‐IFN) and adefovir combination therapy. Patients and methods: Ultra‐deep pyrosequencing analysis of the BCP and PC region was performed for 89 CHB patients (42 HBeAg‐positive; 47 HBeAg‐negative), at baseline and during treatment. Specifically, associations of individual positions with the HBeAg‐negative phenotype were studied, as well as the association of the most prevalent mutations with combined response in HBeAg‐positive and –negative patients at week 72 (HBeAg negativity, HBV‐DNA <2000 IU/mL and ALT normalization at 24 weeks of treatment‐free follow‐up). Results: The mutations most strongly correlated with the HBeAg‐negative phenotype were at positions 1762/1764 and 1896/1899 in the BCP and PC region, respectively. No major changes in nucleotide composition of these positions were observed during treatment. In HBeAg‐negative patients, a combined presence of 1764A and 1896A was correlated with lower ALT levels (p = 0.004), whereas the presence of 1899A was correlated with higher age (p = 0.030), lower HBV‐DNA level (p = 0.036), and previous IFN therapy (p = 0.032). The presence of 1764A/1896A or the absence of 1899A at baseline, was associated with lower response rates, after adjustment for HBV genotype (p = 0.031 and p = 0.017) and HBsAg level (p = 0.035 and p = 0.022). Conclusion: We identified novel correlations between common BCP and PC variants with response to Peg‐IFN and adefovir in HBeAg‐negative patients. Ultimately, this may guide the selection of those patients most likely to benefit from Peg‐IFN‐based treatment.

HLA-C and KIR combined genotype as new response marker for HBeAg-positive chronic hepatitis B patients treated with interferon-based combination therapy

Current treatment for chronic hepatitis B infection (CHB) consists of interferon‐based therapy. However, for unknown reasons, a large proportion of patients with CHB do not respond to this treatment. Hence, there is a pressing need to establish response markers to select patients who will benefit from therapy and to spare potential nonresponders from unnecessary side effects of antiviral therapy. Here, we assessed whether HLA‐C and KIR genotypes were associated with treatment outcome for CHB. Twelve SNPs in or near the HLA‐C gene were genotyped in 86 CHB patients (41 HBeAg positive; 45 HBeAg negative) treated with peginterferon alfa‐2a + adefovir. Genotyping of killer immunoglobin‐like receptors (KIRs) was performed by SSP‐PCR. One SNP in HLA‐C (rs2308557) was significantly associated with combined response in HBeAg‐positive CHB patients (P = 0.003). This SNP is linked to the HLA‐C group C1 or C2 classification, which controls KIR binding. The combination of KIR2DL1 with its ligand HLA‐C2 was observed significantly more often in HBeAg‐positive patients with a combined response (13/14) than in nonresponders (11/27, P = 0.001). Patients with the KIR2DL1/C2 genotype had significantly higher baseline ALT levels (136 vs 50 U/L, P = 0.002) than patients without this combination. Furthermore, KIR2DL1‐C2 predicted response independent of HBV genotype and ALT at baseline. HLA‐C and KIR genotype is strongly associated with response in HBeAg‐positive CHB patients treated with interferon‐based therapy. In combination with other known response markers, HLA‐C/KIR genotype could enable the selection of patients more likely to respond to interferon‐based therapy.

Deep sequencing identifies hepatitis B virus core protein signatures in chronic hepatitis B patients

Background We aimed to identify HBc amino acid differences between subgroups of chronic hepatitis B (CHB) patients. Methods Deep sequencing of HBc was performed in samples of 89 CHB patients (42 HBeAg positive, 47 HBeAg negative). Amino acid types were compared using Sequence Harmony to identify subgroup specific sites between HBeAg‐positive and ‐negative patients, and between patients with combined response and non‐response to peginterferon/adefovir combination therapy. Results We identified 54 positions in HBc where the frequency of appearing amino acids was significantly different between HBeAg‐positive and ‐negative patients. In HBeAg negative patients, 22 positions in HBc were identified which differed between patients with treatment response and those with non‐response. The fraction non‐consensus sequence on selected positions was significantly higher in HBeAg‐negative patients, and was negatively correlated with HBV DNA and HBsAg levels. Conclusions Sequence Harmony identified a number of amino acid changes associated with HBeAg‐status and response to peginterferon/adefovir combination therapy. HighlightsThe HBV core protein is a promising target for the design of new antiviral treatments.We identified amino acid changes in HBV core protein associated with HBeAg status and response to antiviral treatment.HBc Amino acid variation was significantly higher in HBeAg negative patients, and negatively correlated with HBV DNA levels.

Peg-interferon plus nucleotide analogue in patients with chronic hepatitis B with low viral load – Authors' reply

www.thelancet.com/gastrohep Vol 2 September 2017 629 Authors’ reply We thank Ankur Jindal and colleagues for their comments and would like to clarify the issues they raised regarding our study. First, Jindal and colleagues raise concerns about the inclusion of patients with chronic hepatitis B with a low viral load and normal concentrations of alanine aminotransferase. Previous prediction models were based on patients with hepatitis B e antigen (HBeAg)-negative chronic hepatitis B who achieved sustained immune control after peg-interferon monotherapy. The rationale of this study was formed on the basis of our finding that, in HBeAg-negative patients with high viral load, low baseline concentrations of hepatitis B surface antigen (HBsAg) were associated with functional cure after combination therapy. Because HBeAg-negative patients with low viral load generally have low concentrations of HBsAg, we hypothesised that they might benefit from combination treatment. Furthermore, we hypothesised that immunomodulation might be effective in patients with a low viral load because they have residual hepatitis B virus-specific T-cell activity that is possibly sensitive to boost with immunomodulatory agents. The second issue raised was about the study design. We agree with Jindal and colleagues that adefovir is no longer an option for monotherapy. However, the high rate of functional cure in our previous study might be due to the additional immunomodulatory effect of adefovir, which formed our rationale to include a group with adefovir and tenofovir with peginterferon. We calculated the sample size on the basis of the proportion of functional cure observed in our previous study using combination treatment. Given the low proportion of HBsAg loss (4·4%) in our recent study, we do not expect that increasing the sample size would substantially affect our conclusions. Finally, we did not design our study to differentiate the effect of combination therapy and peg-interferon monotherapy on HBsAg loss. However, a clinically relevant difference is not expected since combination therapy did not show a clear benefit in our study compared with controls. Our findings do not support the treatment of patients with low viral load with combination therapy. The side-effects of interferon-based therapy, which occurred as expected in our study, further limit its use. As described in our randomised trial, we agree that these limitations highlight the need for the development of new antiviral compounds for patients with chronic hepatitis B. LJ, FS, and AdN declare no competing interests. HWR serves as a consultant for AbbVie, BMS, Boehringer Ingelheim, ENYO, Gilead Sciences, Janssen-Cilag, Merck, PRA Health Sciences, Regulus, Roche, and R-Pharm and received a grant and research support from AbbVie, Bristol Myers Squibb, Boehringer Ingelheim, ENYO, Gilead Sciences, Janssen-Cilag, Merck, PRA Health Sciences, Regulus, Replicor, and Roche.

HBsAg loss after peginterferon‐nucleotide combination treatment in chronic hepatitis B patients: 5 years of follow‐up

Combining peginterferon‐alfa‐2a (pegIFN) with a nucleotide analogue can result in higher rates of HBsAg loss than either therapy given alone. Here, we investigated the durability of the response to combination therapy in chronic hepatitis B (CHB) patients after 5 years of follow‐up. In the initial study, 92 CHB patients (44 HBeAg‐positive, 48 HBeAg‐negative) with HBV DNA >100 000 c/mL (~20 000 IU/mL) and active hepatitis were treated for 48 weeks with pegIFN 180 μg/week and 10 mg adefovir dipivoxil daily. For the long‐term follow‐up (LTFU) study, patients were followed up for 5 years after the end of treatment. At year 5, 70 (32 HBeAg‐positive, 38 HBeAg‐negative) patients remained in the study. At year 5, 19% (6/32) of HBeAg‐positive patients and 16% (6/38) of HBeAg‐negative patients lost HBsAg, and no HBsAg seroreversion was observed. The 5‐year cumulative Kaplan‐Meier estimate for HBsAg loss was 17.2% for HBeAg‐positive patients and 19.3% for HBeAg‐negative patients. Fourteen of sixteen patients who lost HBsAg at any time point during follow‐up developed anti‐HBs antibodies (>10 IU/L). At year 5, in total 63% (20/32) of HBeAg‐positive and 71% (27/38) of HBeAg‐negative patients were retreated with nucleos(t)ide analogues during follow‐up. The cumulative Kaplan‐Meier estimate for retreatment was 60% of patients at year 5. At year 5 of follow‐up, 18% of CHB patients treated with pegIFN/nucleotide analogue combination therapy had durable HBsAg loss and 88% of these had developed anti‐HBs antibodies.