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Background and Aims: Natural killer (NK) cells play a crucial role in the clearance of human viruses but their activity is significantly impaired in patients infected with chronic hepatitis B (CHB). Cooperation with dendritic cells (DCs) is pivotal for obtaining optimal NK cell antiviral function; thus, we investigated whether HBV might impact the ability of DCs to sustain NK cell functions., Approach and Results: Human DCs were poor stimulators of interferon-gamma (IFN-γ) production by NK cells when exposed to HBV, while maintaining the capability to trigger NK cell cytotoxicity. HBV prevented DC maturation but did not affect their expression of human leukocyte antigen class I, thus allowing DCs to evade NK cell lysis. Tolerogenic features of DCs exposed to HBV were further supported by their increased expression of IL-10 and the immunosuppressive enzyme indoleamine 2,3-dioxygenase, which contributed to the impairment of DC-mediated NK cell IFN-γ production and proliferation, respectively. HBV could also inhibit the expression of inducible immunoproteasome (iP) subunits on DCs. In fact, NK cells could induce iP subunit expression on DCs, but they failed in the presence of HBV. Remarkably, circulating blood DC antigen1 (BDCA1) + DCs isolated from patients with CHB were functionally compromised, hence altering, in turn, NK cell responses., Conclusions: The abnormal NK-DC interplay caused by HBV may significantly impair the efficacy of antiviral immune response in patients with CHB., (© 2021 by the American Association for the Study of Liver Diseases.)

Unlabelled: To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n = 11; HBeAg-negative, n = 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or "a" determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r = -0.431; P = 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r = 0.607; P = 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the above-mentioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells., Conclusion: In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Unlabelled: Occult HBV infection is characterized by the persistence of HBV DNA in the liver of individuals negative for HBV surface antigen (HBsAg). Occult HBV may exist in the hepatocytes as a free genome, although the factors responsible for the very low viral replication and gene expression usually observed in this peculiar kind of infection are mostly unknown. Aims of this study were to investigate whether the viral genomic variability might account for the HBsAg negativity and the inhibition of the viral replication in occult HBV carriers, and to verify in vitro the replication capability of occult HBV strains. We studied liver viral isolates from 17 HBV patients, 13 with occult infection and 4 HBsAg-positive. Full-length HBV genomes from each case were amplified and directly sequenced. Additionally, full-length HBV DNA from eight occult-HBV and two HBsAg-positive cases were cloned and sequenced. Finally, three entire, linear HBV genomes from occult cases were transiently transfected in HuH7 cells. Direct sequencing showed the absence of mutations capable of interfering with viral replication and gene expression in the major viral population of each case. Cloning experiments showed highly divergent HBV strains both in HBsAg-positive and HBsAg-negative individual cases (range of divergence 1.4%-7.1%). All of the 3 transfected full-length HBV isolates showed normal patterns of replication in vitro., Conclusion: Multiple viral variants accumulate in the liver of occult HBV-infected patients. Occult HBV strains are replication-competent in vitro, suggesting that host, rather than viral factors are responsible for cryptic HBV infection.

No data are available about the amount of hepatitis B virus (HBV) genomes in liver of patients with chronic HBV infection. The aim of this study was to quantify the intrahepatic HBV DNA in hepatitis B surface antigen (HBsAg)-positive patients with either active or suppressed viral replication and in HBsAg-negative subjects with occult HBV infection. We optimized the Roche "Amplicor HBV Monitor" kit for quantifying liver HBV DNA and analyzed hepatic DNA extracts and serum samples from 19 HBs-Ag-positive and 43 HBsAg-negative individuals. Eight of the HBsAg carriers had active HBV replication, and for 3 of them we analyzed samples obtained before and at the end of 1 year of lamivudine treatment. Five hepatitis Delta virus (HDV) coinfected patients and 6 healthy HBsAg carriers had inhibited HBV activity. Among the HBsAg-negative subjects 21 had occult HBV infection and 22 had no evidence of HBV infection. The median of HBV genomes per microgram of liver DNA milliliter of serum was 34,500 to 2,620,000 in patients with active viral replication, 20,000 to 3,900, 000 before and 10,000 to 2,800 at the end of therapy in lamivudine-treated individuals, 9,800 to 600 in HDV-infected individuals, and 7,450 to 17,400 in healthy HBsAg carriers. These data indicate that cases with suppressed HBV activity, despite the very low levels of viremia, maintain a relatively high amount of intrahepatic viral genomes. This virus reservoir is likely involved in HBV reactivation, which is usually observed after stopping lamivudine treatment. Finally, the analysis of cases with occult HBV infection showed that the assay we used was able to specifically detect and quantify as few as 100 copies of viral genomes per microgram of liver DNA.

Controversial data were recently published concerning the association of hepatitis B virus (HBV) variants with fulminant hepatitis (FH). In this study, we first analyzed the complete nucleotide sequences of HBV genomes isolated from serum samples from a surgeon and his mother, who was accidentally infected by the son; both died of FH. The infecting viruses were genetically almost identical in both patients; all the clones examined carried a double nucleotide mutation in the start codon of the pre-S2 region that prevented the synthesis of the corresponding protein. Analyses of different serum samples from the son revealed only wild-type precore sequences in a high viremic serum, whereas hepatitis B e antigen (HBeAg)-defective strains were prevalent when the viremia had decreased. Subsequently, we extended the analysis to the viral genomes isolated from 18 additional patients with acute HBV infection and different clinical behaviors: 3 of 5 patients with FH and without previous liver disease had pre-S2 start codon mutations preventing pre-S2 protein synthesis, whereas none of the 13 control cases had similar genomic rearrangements. Analysis of the precore region showed that viral populations normally producing HBeAg were the only or the prevalent viral strains in all of these cases. In summary, our results support the hypothesis that the pre-S2 protein is not essential for HBV infectivity. They also show that infection by pre-S2-defective virus is frequently associated with FH, indicating that this variant might play a pathogenetic role in cases of acute liver failure. Finally, they suggest that the emergence of HBeAg-defective viruses might be a late event in the course of FH, occurring when HBeAg-producing viruses have been mostly cleared.