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Background and Aims: In patients with chronic hepatitis B (CHB) infection, activation of toll-like receptor 8 may induce antiviral immunity and drive functional cure. Selgantolimod, a toll-like receptor 8 agonist, was evaluated in patients with CHB who were virally suppressed on oral antiviral treatment or viremic and not on oral antiviral treatment., Approach and Results: In this phase 1b study, patients were randomized 4:1 to receive either selgantolimod or placebo once weekly. Virally suppressed patients received either 1.5 mg (for 2 weeks) or 3 mg (for 2 weeks or 4 weeks). Viremic patients received 3 mg for 2 weeks. The primary endpoint was safety, as assessed by adverse events (AEs), laboratory abnormalities, and vital sign examination. Pharmacokinetic and pharmacodynamic parameters were assessed by plasma analysis. A total of 38 patients (28 virally suppressed, 10 viremic) were enrolled from six sites in Australia, New Zealand, and South Korea. Twenty patients (53%) experienced an AE and 32 (84%) had laboratory abnormalities, all of which were mild or moderate in severity. The most common AEs were headache (32%), nausea (24%), and dizziness (13%). With a half-life of 5 hours, no accumulation of selgantolimod was observed with multiple dosing. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40 and IL-1RA, which are important for the expansion and activity of multiple T- cell subsets and innate immunity., Conclusion: Selgantolimod was safe and well-tolerated in virally suppressed and viremic patients with CHB and elicited cytokine responses consistent with target engagement. Further studies with longer durations of selgantolimod treatment are required to evaluate efficacy., (© 2021 by the American Association for the Study of Liver Diseases.)

Unlabelled: On-treatment anemia is associated with higher sustained virological response (SVR) rates during peginterferon plus ribavirin (RBV) therapy. Inosine triphosphatase (ITPA) variants causing ITPase deficiency have been shown to protect against RBV-induced anemia. However, ITPase activity has not been associated with SVR. To study this discrepancy, we examined the relationships between ITPase activity, on-treatment anemia, SVR, and RBV levels in hepatitis C virus genotype 1 (HCV-1) patients from the CHARIOT study. ITPA genotype (rs7270101, rs1127354) was used to define ITPase activity in 546 patients. Plasma RBV levels were measured using high-performance liquid chromatography (HPLC). Relationships between ITPase activity, on-treatment hemoglobin (Hb) levels, RBV levels, and SVR were tested using regression modeling, survival analysis, and locally weighted scatterplot smoothing (LOWESS) plot analysis. Hb decline was independently associated with SVR (P<0.0001). ITPase deficiency was present in 35%. ITPase deficiency strongly protected against Hb decline (P<0.0001), but was not associated with SVR (P=0.28). The probability of SVR increased with lower nadir Hb for both wild-type and deficient ITPase activity, but the association curve shifted to describe a parallel relationship at higher Hb levels in patients with ITPase deficiency. In a subset (n=203), we tested the hypothesis that the association between Hb decline and SVR reflected RBV levels rather than actual Hb level. RBV levels were associated with on-treatment Hb decline and SVR, but not ITPase activity. In regression models, adjustment for RBV levels attenuated the association between Hb decline and SVR., Conclusion: ITPase deficiency protects against RBV-induced anemia, but is not associated with SVR. Our data suggest that the relationship between Hb decline and SVR is not mechanistic, but is linked to RBV levels., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Although threshold levels for hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) titers have recently been proposed to guide therapy for chronic hepatitis B (CHB), their relationship to circulating hepatitis B virus (HBV) DNA and intrahepatic HBV replicative intermediates, and the significance of emerging viral variants, remains unclear. We therefore tested the hypothesis that HBsAg and HBeAg titers may vary independently of viral replication in vivo. In all, 149 treatment-naïve CHB patients were recruited (HBeAg-positive, n = 71; HBeAg-negative, n = 78). Quantification of HBeAg and HBsAg was performed by enzyme immunoassay. Virological characterization included serum HBV DNA load, HBV genotype, basal core promoter (BCP)/precore (PC) sequence, and, in a subset (n = 44), measurement of intrahepatic covalently closed circular DNA (cccDNA) and total HBV DNA, as well as quantitative immunohistochemical (IHC) staining for HBsAg. In HBeAg-positive CHB, HBsAg was positively correlated with serum HBV DNA and intrahepatic cccDNA and total HBV DNA (r = 0.69, 0.71, 0.76, P < 0.01). HBeAg correlated with serum HBV DNA (r = 0.60, P < 0.0001), although emerging BCP/PC variants reduced HBeAg titer independent of viral replication. In HBeAg-negative CHB, HBsAg correlated poorly with serum HBV DNA (r = 0.28, P = 0.01) and did not correlate with intrahepatic cccDNA nor total HBV DNA. Quantitative IHC for hepatocyte HBsAg confirmed a relationship with viral replication only in HBeAg-positive patients., Conclusion: The correlation between quantitative HBsAg titer and serum and intrahepatic markers of HBV replication differs between patients with HBeAg-positive and HBeAg-negative CHB. HBeAg titers may fall independent of viral replication as HBeAg-defective variants emerge prior to HBeAg seroconversion. These findings provide new insights into viral pathogenesis and have practical implications for the use of quantitative serology as a clinical biomarker.

Unlabelled: Hepatitis B virus (HBV)-specific T cells play a key role in clearance of the virus and in the pathogenesis of liver disease. Peripheral blood (n = 25) and liver biopsies (n = 19) were collected from individuals with chronic untreated HBV infection. Whole blood, cultured peripheral blood mononuclear cells (PBMCs), and cultured liver-infiltrating lymphocytes (LILs) were each stimulated with an overlapping peptide library to the whole HBV genome. The expression of T helper 1 (Th1) cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin 2 (IL-2)] and interleukin 10 (IL-10) was analyzed by intracellular cytokine staining and flow cytometry. In ex vivo whole blood, more lymphocytes produced Th1 cytokines than IL-10. When comparing cultured LILs with cultured PBMCs, we found a significantly higher magnitude of CD8(+) T cells from the liver producing IL-10 (P = 0.044), primarily in hepatitis B e antigen positive (HBeAg(+)) individuals. A positive correlation resulted between the magnitude of HBV-specific TNF-alpha(+) CD4(+) T cells in the liver and the degree of liver inflammation and fibrosis (P = 0.002 and P = 0.006, respectively)., Conclusion: The differences in cytokine production from HBV-specific T cells in blood and liver may explain the capacity for HBV to persist in the absence of significant hepatic destruction and highlights the balance between cytokine-mediated viral control and liver damage.

Unlabelled: Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-alpha) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF-alpha production., Conclusion: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response.

Activation of macrophages by endotoxin is assumed responsible for increased circulating tumor necrosis factor alpha (TNF-alpha) and soluble TNF receptor (sTNFR) levels in cirrhosis. Relevant to this is expression of Toll-like receptor (TLR) 4 and TLR2, which is critically involved in production of TNF-alpha in response to endotoxin and Gram-positive microbial stimuli, respectively. The first studies on this in cirrhosis are reported here. In 36 cirrhotic patients and 32 controls, we measured (1) circulating endotoxin, TNF-alpha, and sTNFR levels; (2) peripheral blood mononuclear cell (PBMC) expression of TLR4 and TLR2, and (3) in vitro TNF-alpha production by PBMCs stimulated with endotoxin or Staphylococcus aureus enterotoxin B (SEB). PBMC expression of TLR2, circulating TNF-alpha levels, and in vitro TNF-alpha production were reassessed after supplementation with a synbiotic regimen known to increase intestinal levels of Gram-positive bacteria. Endotoxin, TNF-alpha, and sTNFR levels were significantly increased in cirrhosis. Endotoxin levels did not correlate significantly with other parameters. PBMC expression of TLR2 but not TLR4 was significantly up-regulated in cirrhosis and correlated significantly with serum TNF-alpha and sTNFR levels. In vitro TNF-alpha production by PBMCs stimulated by SEB was significantly blunted. Supplementation with the synbiotic regimen resulted in significant up-regulation of PBMC expression of TLR2. Serum TNF-alpha levels were further increased and in vitro TNF-alpha production further reduced in most patients. In conclusion, up-regulation of PBMC expression of TLR2 but not TLR4 occurs in cirrhosis, which implies, contrary to previous assumptions, an important stimulatory role for Gram-positive microbial components but not endotoxin. TLR2 likely contributes to increased circulating TNF-alpha and sTNFR levels in cirrhosis.