Phenotypic CD8 T cell profiling in chronic hepatitis B to predict HBV-specific CD8 T cell susceptibility to functional restoration in vitro.

OBJECTIVE: Exhausted hepatitis B virus (HBV)-specific CD8 T cells in chronic HBV infection are broadly heterogeneous. Characterisation of their functional impairment may allow to distinguish patients with different capacity to control infection and reconstitute antiviral function. DESIGN: HBV dextramer+CD8 T cells were analysed ex vivo for coexpression of checkpoint/differentiation markers, transcription factors and cytokines in 35 patients with HLA-A2+chronic hepatitis B (CHB) and in 29 control HBsAg negative CHB patients who seroconverted after NUC treatment or spontaneously. Cytokine production was also evaluated in HBV peptide-stimulated T cell cultures, in the presence or absence of antioxidant, polyphenolic, PD-1/PD-L1 inhibitor and TLR-8 agonist compounds and the effect on HBV-specific responses was further validated on additional 24 HLA-A2 negative CHB patients. RESULTS: Severely exhausted HBV-specific CD8 T cell subsets with high expression of inhibitory receptors, such as PD-1, TOX and CD39, were detected only in a subgroup of chronic viraemic patients. Conversely, a large predominance of functionally more efficient HBV-specific CD8 T cell subsets with lower expression of coinhibitory molecules and better response to in vitro immune modulation, typically detected after resolution of infection, was also observed in a proportion of chronic viraemic HBV patients. Importantly, the same subset of patients who responded more efficiently to in vitro immune modulation identified by HBV-specific CD8 T cell analysis were also identified by staining total CD8 T cells with PD-1, TOX, CD127 and Bcl-2. CONCLUSIONS: The possibility to distinguish patient cohorts with different capacity to respond to immune modulatory compounds in vitro by a simple analysis of the phenotypic CD8 T cell exhaustion profile deserves evaluation of its clinical applicability.

Human Hepatitis B Virus Negatively Impacts the Protective Immune Crosstalk Between Natural Killer and Dendritic Cells.

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.

Antibiotic prophylaxis based on individual infective risk stratification in cardiac implantable electronic device: the PRACTICE study.

AIMS: In patients undergoing cardiac implantable electronic device (CIED) intervention, routine pre-procedure antibiotic prophylaxis is recommended. A more powerful antibiotic protocol has been suggested in patients at high risk of infection. Stratification of individual infective risk could guide the prophylaxis before CIED procedure. METHODS AND RESULTS: Patients undergoing CIED surgery were stratified according to the Shariff score in low and high infective risk. Patients in the 'low-risk' group were treated with only two antibiotic administrations while patients in the 'high-risk' group were treated with a prolonged 9-day protocol, according to renal function and allergies. We followed-up patients for 250 days with clinical outpatient visit and electronic control of the CIED. As primary endpoint, we evaluated CIED-related infections. A total of 937 consecutive patients were enrolled, of whom 735 were stratified in the 'low-risk' group and 202 in the 'high-risk' group. Despite different risk profiles, CIED-related infection rate at 250 days was similar in the two groups (8/735 in 'low risk' vs. 4/202 in 'high risk', P = 0.32). At multivariate analysis, active neoplasia, haematoma, and reintervention were independently associated with CIED-related infection (HR 5.54, 10.77, and 12.15, respectively). CONCLUSION: In a large cohort of patients undergoing CIED procedure, an antibiotic prophylaxis based on individual stratification of infective risk resulted in similar rate of infection between groups at high and low risk of CIED-related infection.

Flavocoxid exerts a potent antiviral effect against hepatitis B virus.

INTRODUCTION: Flavocoxid is a proprietary blend of two flavonoids, baicalin and catechin, and recent evidence has shown that bioflavonoids may exert antiviral activities. The potential antiviral activity of Flavocoxid against hepatitis B virus (HBV) was evaluated. Additionally, it was investigated if Flavocoxid used in combination with Entecavir could potentiate its anti-HBV activity. MATERIALS AND METHODS: Hepatoma cells replicating HBV were treated with Flavocoxid, or Entecavir alone or in combination for up to 5 days. Viral replicative intermediates, transcripts, and cccDNA levels were evaluated in HBV-replicating cells by real-time PCR, Southern and Northern blotting. Expression profiling was performed using TaqMan low-density arrays. RESULTS: Flavocoxid treatment induced a reduction of HBV replicative intermediates, the amount of transcripts, and HBsAg levels. Flavocoxid and Entecavir combination therapy further decreased the amount of HBV replicative intermediates, compared to Flavocoxid alone. Importantly, Flavocoxid alone or in combination with Entecavir also induced a reduction of cccDNA. Gene-expression analysis showed that Flavocoxid activates type I IFNs-signaling and dampens the HBV-induced inflammatory response. CONCLUSIONS: Flavocoxid inhibits HBV replication by targeting multiple steps of viral life cycle. These results indicate that the antiviral activity of Entecavir is potentiated by Flavocoxid, suggesting that this medical food might be considered as an adjuvant for anti-HBV therapy.

NS3 Variability in Hepatitis C Virus Genotype 1A Isolates from Liver Tissue and Serum Samples of Treatment-Naïve Patients with Chronic Hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) NS3 resistance-associated substitutions (RASs) reduce HCV susceptibility to protease inhibitors. Little is known about NS3 RASs in viral isolates from the liver of chronic hepatitis C (CHC) patients infected with HCV genotype-1a (G1a). AIM: The objective of this work was to study NS3 variability in isolates from the serum and liver of HCV-G1a-infected patients naïve to direct-acting antivirals (DAAs). METHODS: NS3 variability of HCV-G1a isolates from the serum and liver of 11 naïve CHC patients, and from sera of an additional 20 naïve CHC patients, was investigated by next-generation sequencing. RESULTS: At a cutoff of 1%, NS3 RASs were detected in all the samples examined. At a cutoff of 15%, they were found in 54.5% (6/11) and 27.3% (3/11) of the paired liver and serum samples, respectively, and in 22.5% (7/31) of the overall serum samples examined. Twenty-six out of thirty-one (84%) patients showed NS3 variants with multiple RASs. Phylogenetic analysis showed that NS3 sequences clustered within 2 clades, with 10/31 (32.2%) patients infected by clade I, 15/31 (48.8%) by clade II, and 6/31 (19.3%) by both clades. CONCLUSIONS: Though the number of patients examined was limited, NS3 variants with RASs appear to be major components of both intrahepatic and circulating viral quasispecies populations in DAA-naïve patients.

Assessing Genomic Mutations in SARS-CoV-2: Potential Resistance to Antiviral Drugs in Viral Populations from Untreated COVID-19 Patients.

Naturally occurring SARS-CoV-2 variants mutated in genomic regions targeted by antiviral drugs have not been extensively studied. This study investigated the potential of the RNA-dependent RNA polymerase (RdRp) complex subunits and non-structural protein (Nsp)5 of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to accumulate natural mutations that could affect the efficacy of antiviral drugs. To this aim, SARS-CoV-2 genomic sequences isolated from 4155 drug-naive individuals from southern Italy were analyzed using the Illumina MiSeq platform. Sequencing of the 4155 samples showed the following viral variant distribution: 71.2% Delta, 22.2% Omicron, and 6.4% Alpha. In the Nsp12 sequences, we found 84 amino acid substitutions. The most common one was P323L, detected in 3777/4155 (91%) samples, with 2906/3777 (69.9%) also showing the G671S substitution in combination. Additionally, we identified 28, 14, and 24 different amino acid substitutions in the Nsp5, Nsp7, and Nsp8 genomic regions, respectively. Of note, the V186F and A191V substitutions, affecting residues adjacent to the active site of Nsp5 (the target of the antiviral drug Paxlovid), were found in 157/4155 (3.8%) and 3/4155 (0.07%) samples, respectively. In conclusion, the RdRp complex subunits and the Nsp5 genomic region exhibit susceptibility to accumulating natural mutations. This susceptibility poses a potential risk to the efficacy of antiviral drugs, as these mutations may compromise the drug ability to inhibit viral replication.

Mitochondrial DNA is a target of HBV integration.

Hepatitis B virus (HBV) may integrate into the genome of infected cells and contribute to hepatocarcinogenesis. However, the role of HBV integration in hepatocellular carcinoma (HCC) development remains unclear. In this study, we apply a high-throughput HBV integration sequencing approach that allows sensitive identification of HBV integration sites and enumeration of integration clones. We identify 3339 HBV integration sites in paired tumour and non-tumour tissue samples from 7 patients with HCC. We detect 2107 clonally expanded integrations (1817 in tumour and 290 in non-tumour tissues), and a significant enrichment of clonal HBV integrations in mitochondrial DNA (mtDNA) preferentially occurring in the oxidative phosphorylation genes (OXPHOS) and D-loop region. We also find that HBV RNA sequences are imported into the mitochondria of hepatoma cells with the involvement of polynucleotide phosphorylase (PNPASE), and that HBV RNA might have a role in the process of HBV integration into mtDNA. Our results suggest a potential mechanism by which HBV integration may contribute to HCC development.

Frequency of somatic mutations in TERT promoter, TP53 and CTNNB1 genes in patients with hepatocellular carcinoma from Southern Italy.

Somatic mutations in the TERT promoter and in the TP53 and CTNNB1 genes are considered drivers for hepatocellular carcinoma (HCC) development. They show variable frequencies in different geographic areas, possibly depending on liver disease etiology and environmental factors. TP53, CTNNB1 and TERT genetic mutations were investigated in tumor and non-tumor liver tissues from 67 patients with HCC and liver tissue specimens from 41 control obese subjects from Southern Italy. Furthermore, TERT expression was assessed by reverse transcription-quantitative PCR. Neither CTNNB1 mutations or TP53 R249S substitution were detected in any case. The TP53 R72P polymorphism was found in 10/67 (14.9%) tumors, but was not found in either non-tumor tissues (P=0.001) or controls (P=0.009). TERT gene promoter mutations were found in 29/67 (43.3%) tumor tissues but were not found in either non-tumor (P<0.0001) or control liver specimens (P<0.0001). The most frequent mutation in the tumors was the known hot spot at -124 bp from the TERT ATG start site (-124G>A, 28 cases, 41.8%; P<0.0001). A new previously never reported TERT promoter mutation (at -297 bp from the ATG, -297C>T) was found in 5/67 (7.5%) tumors, in 0/67 (0%) non-tumor (P<0.0001), and in 0/41 (0%) controls (P=0.07). This mutation creates an AP2 consensus sequence, and was found alone (1 case) or in combination (4 cases) with the -124 bp mutation. The mutation at -124 and -297 bp induced a 33-fold (P<0.0001) and 40-fold increase of TERT expression levels, respectively. When both mutations were present, TERT expression levels were increased >300-fold (P=0.001). A new TERT promoter mutation was identified, which generates a de novo binding motif for AP2 transcription factors, and which significantly increases TERT promoter transcriptional activity.

Behaviour of occult HBV infection in HCV-infected patients under treatment with direct-acting antivirals.

BACKGROUND: There is controversial data on possible occult HBV reactivation in HCV patients successfully treated with direct-acting antivirals (DAA). However, diagnosis of occult HBV infection (OBI) was not performed by gold standard procedures in any study. METHODS: By using several highly sensitive assays, we examined serially collected serum samples from 40 HBV-surface-negative DAA-treated HCV patients with OBI identified by testing liver biopsy specimens through nested-PCR technique. Serum samples were obtained at four time points from each patient (at baseline, at 4 weeks after starting, at the end and 12 weeks after stopping therapy) and tested for HBV DNA by nested-PCR and real-time PCR techniques. RESULTS: All tested serum samples were negative by both quantitative HBV surface antigen (HBsAg) and HBV core-related antigen assays. 26/40 patients were anti-HBs-positive and in all of them, the amount of this antibody was stable at the four time points evaluated. Serum HBV DNA was detected in 10 samples at baseline, in 6 samples 4 weeks after starting therapy, in 11 samples at the end of therapy and in 21 samples 12 weeks after stopping treatment (P=0.001). Aminotransferase values dropped within the normal levels at week 4 of therapy and persisted normal over time in all cases. CONCLUSIONS: A slight increase in the amount of HBV DNA 3 months after stopping DAA therapy was the only parameter showing a possible reappearance of HBV activity in OBI patients cured for a concomitant HCV infection, but it was insufficient to lead toward a virological reactivation capable of inducing liver injury.

Free episomal and integrated HBV DNA in HBsAg-negative patients with intrahepatic cholangiocarcinoma.

There is evidence that chronic hepatitis B virus (HBV) infection is associated with an increased risk of intrahepatic cholangiocarcinoma (ICC) development, and it has been hypothesized an etiological role of HBV in the development of this tumor. Very little is known about occult HBV infection (OBI) in ICC. Aims of the study were to investigate the OBI prevalence and to characterize the HBV molecular status at intrahepatic level in OBI-positive cases with ICC. Frozen liver tumor specimens from 47 HBV surface-antigen-negative patients with ICC and 41 paired non-tumor liver tissues were tested for OBI by 4 different HBV-specific nested PCR. Covalently closed circular HBV DNA (HBV cccDNA) and viral integrations were investigated in OBI-positive cases. HBV DNA was detected in tumor and/or non-tumor specimens from 29/47 (61.7%) ICC patients. HBV cccDNA was found in tissues from 5/17 (34.5%) cases examined. HBV integration was detected in 4/10 (40%) tumor tissues tested and involved HBx and HBV-core gene sequences in 3 and 1 cases, respectively. Viral integration occurred: (a) 9,367 nucleotides upstream of the cat-eye-syndrome critical region protein-5-isoform coding sequence; (b) within the cystinosin isoform-1-precursor gene; (c) within the thromboxane-A-synthase-1 gene; (d) within the ATPase phospholipid transporting 9B gene. Occult HBV infection is highly prevalent in patients with ICC. Both free viral genomes and integrated HBV DNA can be present in these cases. These results suggest an involvement of HBV in the carcinogenic process leading to ICC development even in cases with occult infection.

Lack of the NS5B S282T mutation in HCV isolates from liver tissue of treatment-naive patients with HCV genotype-1b infection.

BACKGROUND: Despite the availability of several direct-acting antivirals (DAAs) specifically inhibiting different HCV proteins, treatment of chronic HCV infection is still a challenge also because of the possible selection of resistant viral variants under DAA therapy. Indeed, only the emergence of viruses resistant to the nucleoside inhibitors of the HCV NS5B polymerase (Pol) has not yet been reported, in spite of the fact that in vitro studies have clearly shown that an S282T amino acid change in the Pol protein may confer resistance to these drugs. On the basis of a previous study showing that viral variants resistant to HCV protease inhibitors are largely present in the liver - but not in the serum - of untreated patients, we investigated the possible natural occurrence of viral populations with the S282T change in the Pol protein, analysing viral isolates from liver and serum of HCV genotype-1b treatment-naive patients. METHODS: HCV-1b isolates from liver specimens and serum samples of 10 chronic hepatitis C patients were analysed by cloning and sequencing. RESULTS: The S282T mutation was not found in any of the viral isolates from either liver or serum samples of all the cases, although an S282G mutation of unknown virological/clinical relevance was detected in 2/19 liver isolates from one patient. CONCLUSIONS: Our study confirms that the natural selection of the S282T mutation is a rare event, thus explaining the lack of emergence and takeover of these variants under drug pressure.

NS3 genetic variability in HCV genotype-1b isolates from liver specimens and blood samples of treatment-naive patients with chronic hepatitis C.

BACKGROUND: Two distinct inhibitors of the HCV protease have been approved for the treatment of patients infected with HCV genotype-1. These drugs are highly efficient in suppressing HCV replication; however, their use is limited by the emergence of viral mutants resistant to them after a very short time of treatment. By analysing blood samples, it was shown that viral strains resistant to protease inhibitors (PIs) may exist prior to treatment. The aim of this study was to investigate the presence of viral variants resistant to PIs in isolates from liver and blood of HCV patients naive to any antiviral therapy. METHODS: Liver and blood HCV genotype-1b isolates from 10 patients with chronic hepatitis were analysed by cloning and sequencing procedures. RESULTS: The analyses of 10­15 clones from liver isolates of each patient showed that 7/10 cases had single or multiple mutations potentially conferring resistance to PIs. However, the analysis of the corresponding blood samples excluded the presence of these mutations in all cases but one, which had the Q80R mutation in all clones from both liver and plasma samples. No PI-resistant variants were detected in isolates from either liver or plasma samples of three patients. CONCLUSIONS: Naturally occurring HCV variants resistant to PIs are commonly present at the intrahepatic level and this clearly explains their usual, very early emergence under treatment; however, the identification of these variants as circulating viral populations is not unusual in untreated patients.

Risk of occult hepatitis B virus infection reactivation in patients with solid tumours undergoing chemotherapy.

BACKGROUND: Hepatitis B virus reactivation may occur in occult-infected carriers with haematological malignancies, whereas little data are available in patients undergoing chemotherapy for solid tumours. AIMS: Evaluation of cancer patients undergoing chemotherapy to investigate occult hepatitis B virus infection and its clinical-virological outcome. METHODS: Forty-four patients with solid tumours and without liver disease were prospectively enrolled and sampled before starting chemotherapy and between the second and third chemotherapy cycles (time points 1 and 2, respectively); 24 were also sampled 6 months after the end of chemotherapy (time point 3). At each time point, subjects were tested for liver biochemistry, hepatitis B serology and occult infection. RESULTS: No sample tested positive for virus surface antigen. Twelve subjects (27.3%) were antibody positive to hepatitis B virus. Overall, occult infection was detected in 4 cases (9%), with positive HBV DNA at time points 1 and 2 (one case), at time point 1 only (one case), only at time points 2 and 3 (two cases), respectively. No occult-infected carrier experienced liver biochemistry flares and/or viral surface antigen positivity. CONCLUSIONS: Occult hepatitis B virus infection may occur in subjects with solid tumours, although the risk of its reactivation under chemotherapy appears to be very low.

Hepatitis B virus (HBV) induces the expression of interleukin-8 that in turn reduces HBV sensitivity to interferon-alpha.

High levels of serum interleukin-8 (IL-8) have been detected in chronic hepatitis B (CHB) patients during episodes of hepatitis flares. We investigated whether hepatitis B virus (HBV) may directly induce IL-8 production and whether IL-8 may antagonize interferon-alpha (IFN-α) antiviral activity against HBV. We showed that CHB patients had significantly higher IL-8 levels both in serum and in liver tissue than controls. In HBV-replicating HepG2 cells, IL-8 transcription was significantly activated. AP-1, C/EBP and NF-kB transcription factors were concurrently necessary for maximum IL-8 induction. Moreover, HBx viral protein was recruited onto the IL-8 promoter and this was paralleled by IL8-bound histone hyperacetylation and by active recruitment of transcriptional coactivators. Inhibition of IL-8 increases the antiviral activity of IFN-α against HBV. Our results indicate that HBV activates IL-8 gene expression by targeting the epigenetic regulation of the IL-8 promoter and that IL-8 may contribute to reduce HBV sensitivity to IFN-α.

Hepatitis E virus infection as a cause of acute hepatitis in Southern Italy.

BACKGROUND: Hepatitis E virus (HEV) is a major cause of acute hepatitis in developing countries, whereas it is not considered a major health problem in Western World. AIMS: To investigate the spread of HEV and its possible role in causing acute hepatitis in Southern Italy. METHODS: Four hundred and thirty patients observed from April to December 2009 were studied and grouped as follows: 55 individuals with acute hepatitis (AH), 33 of whom cryptogenic; 321 individuals with chronic liver diseases (CLD), (278 Italians and 43 immigrants); 54 individuals without liver disease (control-group). Serum samples from all cases were tested for IgG anti-HEV antibodies and those positive to this test as well as all AH cases were also tested both for IgM anti-HEV and HEV RNA. RESULTS: Two of 33 (6%) cryptogenic AH cases were associated with HEV infection as shown by positive IgM anti-HEV test. Both these patients had not travelled to areas at high HEV endemicity. HEV RNA was not found in any sample tested. IgG anti-HEV antibodies were detected in 5.7% of Italians with CLD and 3.7% of the control-group. No immigrant was found positive for any HEV marker. CONCLUSION: Autochthonous HEV infection is present in Southern Italy where it may cause AH.