Session 5: Management of viral hepatitis I
Tracks
Thursday, 23 May
| Thursday, May 23, 2019 |
| 13:15 - 14:45 |
Speaker
Prof. Pietro Lampertico
Director of Division of Gastroenterology and Hepatology
Fondazione Policlinico - University of Milan
Novel compounds for HBV therapy
13:15 - 13:45
Prof. Valentina Svicher
Rome
University Of Rome Tor Vergata
Novel markers on HBV / HDV co-infection
13:45 - 14:15
Dr. Romina Salpini
Phd Student
University of Rome Tor Vergata
The integration of Hepatitis B virus in relevant regions of human genome is a common event in the setting of HBeAg negative chronic infection despite limited liver disease: implications for an altered cell metabolism
Abstract
Background
HBV integration into human genome has been primarily described in HCC and cirrhotic patients with long-term chronic hepatitis B, shedding light on the contribution of integrated HBV DNA in tumorigenesis. Differently, limited data are still available on the occurrence of HBV integration in HBeAg-negative patients with a limited evolution of HBV-related liver disease.
In this light, we aimed to analyze intrahepatic reservoir and the occurrence of HBV integration events in the setting of HBeAg-negative chronic infection with no/mild liver fibrosis, including patients with low levels of viraemia.
Material and Methods
Liver tissues from 40 HBeAg-negative patients were studied. Patients were classified according to viraemia: group-1 (HBV-DNA<2,000IU/ml; n=8), group-2 (HBV-DNA 2,000-20,000IU/ml; n=14), group-3 (HBV-DNA>20,000IU/ml; n=18). cccDNA, intrahepatic total (it)-HBV-DNA were evaluated by Real-Time PCR and pgRNA by digital-droplet PCR. Whole Exome Sequencing [Illumina, median(IQR) coverage: 115x (90x-140x)] was performed in all 40 patients. HBV integration into human exons and into the flanking intronic regions was identified by recognition of chimeric HBV-human sequences applying a bioinformatic pipeline based on Virus-Clip software. The role of genes involved in HBV integration was analysed by GeneCards and Protein Atlas Databases. The threshold of parameters predicting HBV integration was defined by AUROC.
Results
Overall, median(IQR) serum HBV-DNA and HBsAg are 3.9(3.4-5.3) logIU/ml and 3,980(1,260-10,000)IU/ml and 80% has no/mild liver fibrosis (Ishak score< 2).
Group-1 and -2 show a comparable intrahepatic reservoir in terms of cccDNA, it-HBV-DNA and pgRNA. Conversely, compared to group-2, group-3 is characterized by higher cccDNA (median[IQR]:2.6[2.3-2.7] vs 2.0[0.9-2.3]log copies/1000cells, P=0.01), it HBV-DNA (median[IQR]:3.9[3.5-4.4] vs 3.1[2.2-3.9]log copies/1000cells, p=0.005) as well as by more elevated levels of pgRNA (190[7-770] vs 3.3 (1.5-12) copies/1000cells, p=0.02).
HBV integration is detected in 35.4% of pts, more frequently in group-3 than in group-1 and -2 (55.6% vs 18.2%, p=0.03). Among the 17 overall recognized integration events, 11 involves HBx-encoding region, followed by preCore-Core- (N=3) and Pol/S-regions (N=3). In most cases (64.7%), HBV integration events are observed within intronic regions, mainly close to RNA splicing-site, at a distance <100 nucleotides from the proximal exones (47.1%). These regions are known to be critical for a proper splicing and, in turn, for mRNAs production. Notably, HBV integration often localizes in human genes, regulating cell proliferation and, thus, directly involved in hepatocarcinogenesis (NUP85, COL18A1, AGBL5, ANKRD52 and ELAC-2). Furthermore, HBV is also found integrated in genes regulating lipid or drug metabolism (CYP2UI, LMF-1) or in those regulating antiviral or inflammatory response (IFITM-1, NR3C1).
Finally, a higher amount of serum HBsAg is the only factor positively correlated with the occurrence of HBV integration (p<0.001). By AUROC, HBsAg>5,000IU/ml identifies HBV integration with the best diagnostic-accuracy (83.5%), 92% sensitivity and 73% specificity.
Conclusions
In HBeAg-negative infection, HBV integration occurs frequently in highly-viremic patients and in a not negligible percentage of low-viremic patients. Localization of HBV integrations suggests that this event can be involved not only in carcinogenesis but also in mechanisms regulating hepatocyte metabolism, antiviral immunity and inflammatory response.
HBV integration into human genome has been primarily described in HCC and cirrhotic patients with long-term chronic hepatitis B, shedding light on the contribution of integrated HBV DNA in tumorigenesis. Differently, limited data are still available on the occurrence of HBV integration in HBeAg-negative patients with a limited evolution of HBV-related liver disease.
In this light, we aimed to analyze intrahepatic reservoir and the occurrence of HBV integration events in the setting of HBeAg-negative chronic infection with no/mild liver fibrosis, including patients with low levels of viraemia.
Material and Methods
Liver tissues from 40 HBeAg-negative patients were studied. Patients were classified according to viraemia: group-1 (HBV-DNA<2,000IU/ml; n=8), group-2 (HBV-DNA 2,000-20,000IU/ml; n=14), group-3 (HBV-DNA>20,000IU/ml; n=18). cccDNA, intrahepatic total (it)-HBV-DNA were evaluated by Real-Time PCR and pgRNA by digital-droplet PCR. Whole Exome Sequencing [Illumina, median(IQR) coverage: 115x (90x-140x)] was performed in all 40 patients. HBV integration into human exons and into the flanking intronic regions was identified by recognition of chimeric HBV-human sequences applying a bioinformatic pipeline based on Virus-Clip software. The role of genes involved in HBV integration was analysed by GeneCards and Protein Atlas Databases. The threshold of parameters predicting HBV integration was defined by AUROC.
Results
Overall, median(IQR) serum HBV-DNA and HBsAg are 3.9(3.4-5.3) logIU/ml and 3,980(1,260-10,000)IU/ml and 80% has no/mild liver fibrosis (Ishak score< 2).
Group-1 and -2 show a comparable intrahepatic reservoir in terms of cccDNA, it-HBV-DNA and pgRNA. Conversely, compared to group-2, group-3 is characterized by higher cccDNA (median[IQR]:2.6[2.3-2.7] vs 2.0[0.9-2.3]log copies/1000cells, P=0.01), it HBV-DNA (median[IQR]:3.9[3.5-4.4] vs 3.1[2.2-3.9]log copies/1000cells, p=0.005) as well as by more elevated levels of pgRNA (190[7-770] vs 3.3 (1.5-12) copies/1000cells, p=0.02).
HBV integration is detected in 35.4% of pts, more frequently in group-3 than in group-1 and -2 (55.6% vs 18.2%, p=0.03). Among the 17 overall recognized integration events, 11 involves HBx-encoding region, followed by preCore-Core- (N=3) and Pol/S-regions (N=3). In most cases (64.7%), HBV integration events are observed within intronic regions, mainly close to RNA splicing-site, at a distance <100 nucleotides from the proximal exones (47.1%). These regions are known to be critical for a proper splicing and, in turn, for mRNAs production. Notably, HBV integration often localizes in human genes, regulating cell proliferation and, thus, directly involved in hepatocarcinogenesis (NUP85, COL18A1, AGBL5, ANKRD52 and ELAC-2). Furthermore, HBV is also found integrated in genes regulating lipid or drug metabolism (CYP2UI, LMF-1) or in those regulating antiviral or inflammatory response (IFITM-1, NR3C1).
Finally, a higher amount of serum HBsAg is the only factor positively correlated with the occurrence of HBV integration (p<0.001). By AUROC, HBsAg>5,000IU/ml identifies HBV integration with the best diagnostic-accuracy (83.5%), 92% sensitivity and 73% specificity.
Conclusions
In HBeAg-negative infection, HBV integration occurs frequently in highly-viremic patients and in a not negligible percentage of low-viremic patients. Localization of HBV integrations suggests that this event can be involved not only in carcinogenesis but also in mechanisms regulating hepatocyte metabolism, antiviral immunity and inflammatory response.
Ms. Stephanie Popping
Phd Student Virology
Erasmus Medical Center
The transmission dynamics of acute HCV infections in HIV-positive men who have sex with men in the Netherlands is suitable for targeted risk reduction strategies
Abstract
Background
Recently, several outbreaks of acute hepatitis C virus (HCV) infections have been observed among HIV infected men who have sex with men(MSM) in Europe. In the Netherlands, prior to the unrestricted availability of direct-acting antivirals(DAA) a stable and high incidence rate of 10/1000 person-years was reported. In addition, reinfection occurred in 20-25% within two years. Fortunately, the overall HCV incidence among HIV-positive MSM declined with 51% after the widespread use of DAAs in 2015. Yet, reinfection rates remained soaring in this population. HIV-positive MSM whom acquire HCV are often engaged in high-risk behaviour as for example chemsex and unprotected anal intercourse with multiple partners. In order to target risk reduction strategies or to establish a search and destroy strategy as with HIV, more in-depth knowledge is required regarding the transmission dynamics of acute HCV. Therefore, we aim to identify how the acute HCV infections among HIV-positive MSM are linked to specific transmission networks.
Methods
We included a total of 45 HIV-positive MSM, whom were diagnosed with an acute HCV genotype 1 infection in one of the ten HIV treatment centres across the Netherlands and were enrolled in the Dutch Acute HCV in HIV study(DAHHS 1) between 2013 and 2014. All patients received a DAA regimen containing boceprevir. Target enrichment for viral nucleic acid separation and deep sequencing with the Illumina MiSeq platform with 500bp v2 reagent sets were run multiple times to recover full-length HCV genomes. All generated sequences were aligned and for each query sequence the 100 highly similar sequences were selected through BLAST. After preforming a model test with IQtree and removal of insufficient and duplicate sequences, a maximum likelihood tree was constructed in MEGA X64 using the general time reversible nucleotide substitution model allowing for among-site variation. The robustness of phylogenetic clustering was tested by a bootstrap analysis with 1,000 replicates. Transmission networks were evaluated using ClusterPicker, with a cluster being defined as a group sequences with a genetic distance of at most 1.5% and a bootstrap support value of 100%. The most recent common ancestor was estimated with a coalescent-based HKY model with a Bayesian statistical framework.
Results
We are the first study in which all new HCV infections among HIV-positive MSM in the Netherlands were included. Four major genotype 1a transmission clusters were identified including 37(84%) patients. The largest cluster accounted for 13(30%) of the new genotype 1a HCV infections among HIV-positive MSM. Interestingly, all clusters were indicative of recent outbreaks, highlighted by small genetic distances and a most recent common ancestor after the year 2000, when the first cases of HCV infection in HIV-positive MSM were reported.
Conclusion
We used well-defined data from the Netherlands to identify the acute HCV distribution among HIV-positive MSM. Our data showed indeed that the HCV epidemic is a young epidemic and that mostly all of the acute infections are linked within major transmission networks. This is crucial information that highlights the possibility of targeted risk reduction strategies. In addition, this information can contribute into curbing the epidemic.
Recently, several outbreaks of acute hepatitis C virus (HCV) infections have been observed among HIV infected men who have sex with men(MSM) in Europe. In the Netherlands, prior to the unrestricted availability of direct-acting antivirals(DAA) a stable and high incidence rate of 10/1000 person-years was reported. In addition, reinfection occurred in 20-25% within two years. Fortunately, the overall HCV incidence among HIV-positive MSM declined with 51% after the widespread use of DAAs in 2015. Yet, reinfection rates remained soaring in this population. HIV-positive MSM whom acquire HCV are often engaged in high-risk behaviour as for example chemsex and unprotected anal intercourse with multiple partners. In order to target risk reduction strategies or to establish a search and destroy strategy as with HIV, more in-depth knowledge is required regarding the transmission dynamics of acute HCV. Therefore, we aim to identify how the acute HCV infections among HIV-positive MSM are linked to specific transmission networks.
Methods
We included a total of 45 HIV-positive MSM, whom were diagnosed with an acute HCV genotype 1 infection in one of the ten HIV treatment centres across the Netherlands and were enrolled in the Dutch Acute HCV in HIV study(DAHHS 1) between 2013 and 2014. All patients received a DAA regimen containing boceprevir. Target enrichment for viral nucleic acid separation and deep sequencing with the Illumina MiSeq platform with 500bp v2 reagent sets were run multiple times to recover full-length HCV genomes. All generated sequences were aligned and for each query sequence the 100 highly similar sequences were selected through BLAST. After preforming a model test with IQtree and removal of insufficient and duplicate sequences, a maximum likelihood tree was constructed in MEGA X64 using the general time reversible nucleotide substitution model allowing for among-site variation. The robustness of phylogenetic clustering was tested by a bootstrap analysis with 1,000 replicates. Transmission networks were evaluated using ClusterPicker, with a cluster being defined as a group sequences with a genetic distance of at most 1.5% and a bootstrap support value of 100%. The most recent common ancestor was estimated with a coalescent-based HKY model with a Bayesian statistical framework.
Results
We are the first study in which all new HCV infections among HIV-positive MSM in the Netherlands were included. Four major genotype 1a transmission clusters were identified including 37(84%) patients. The largest cluster accounted for 13(30%) of the new genotype 1a HCV infections among HIV-positive MSM. Interestingly, all clusters were indicative of recent outbreaks, highlighted by small genetic distances and a most recent common ancestor after the year 2000, when the first cases of HCV infection in HIV-positive MSM were reported.
Conclusion
We used well-defined data from the Netherlands to identify the acute HCV distribution among HIV-positive MSM. Our data showed indeed that the HCV epidemic is a young epidemic and that mostly all of the acute infections are linked within major transmission networks. This is crucial information that highlights the possibility of targeted risk reduction strategies. In addition, this information can contribute into curbing the epidemic.
