CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

viral parameters were quantified by qPCR and droplet digital (dd)PCR (LOD 3 x 10-6 copies/cell for cccDNA). cirB-RNA was quantified with the automated cobas(R) HBV RNA assay that preferentially detects viral RNAs derived from cccDNA vs. integrated sequences (LOD 5 copies/mL). Serum levels of HBV DNA, hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) were determined, as well as hepatitis B e antigen (HBeAg) status. Results: cccDNA and 3.5-kb RNA were quantifiable in all but one CLB/FNA pair, showing the highest levels in untreated HBeAg+ patients. When comparing cccDNA and 3.5-kb RNA levels in CLB/FNA samples, no statistically significant differences were identified. The analysis of CLB/serum samples showed that all HBeAg+ chronic hepatitis (CH) patients had quantifiable cirB-RNA, compared to only 57% of HBeAg- CH and 14% of HBeAg- chronic infection (CI) untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity and serum HBcrAg in both untreated and treated patients. Combined undetectability of cirB-RNA and HBcrAg in HBeAg- patients identified a subgroup with the lowest levels of transcriptionally active cccDNA. Conclusion: In the frame of HBV cure programs, we provide a proof of concept that the less invasive FNA can be used to assess intrahepatic cccDNA using a ddPCR assay. Moreover, we show the performance and relevance of quantifying cirB-RNA as an indicator of cccDNA transcriptional activity in both untreated and NUC-treated patients. These results support the use of both approaches in clinical trials to evaluate the HBV reservoir during the development of new antivirals and immunomodulatory agents. Intrahepatic cccDNA and Circulating HBV Markers in HBeAg-Negative Chronic Infection in Senegal Adrià Ramírez Mena 1 , Aleksei Suslov 1 , Hubert Akotia 2 , Pascal Bittel 3 , Andreas Limacher 3 , Judicaël Tine 2 , Christoph Niederhauser 3 , Bruce Wembulua Shinga 2 , Melissa S. Pandi 2 , Ndeye Fatou Ngom 2 , Stefan Wieland 1 , Markus Heim 1 , Moussa Seydi 2 , Gilles Wandeler 1 1 University Hospital Basel, Basel, Switzerland, 2 Centre Hospitalier Universitaire de Fann, Dakar, Senegal, 3 University of Bern, Bern, Switzerland Background: A better understanding of the relationship between the intrahepatic hepatitis B virus (HBV) activity and peripheral biomarkers of HBV infection is urgently needed in order to improve treatment monitoring and outcomes in high prevalence settings. We aimed to determine the relationship between intrahepatic HBV cccDNA and plasma HBV DNA, quantitative HBsAg (qHBsAg) and total HBV RNA levels in HBeAg-negative individuals from the SEN-B cohort in Senegal. Methods: We collected paired core liver biopsies and serum/plasma samples from untreated HBeAg-negative participants enrolled in SEN-B. We measured qHBsAg and HBV DNA on site using e411 cobas® and cobas®/TaqMan® (Roche Diagnostic Systems). HBV RNA levels were measured from cryopreserved plasma samples using cobas® 8800 investigational assay (Roche Molecular Systems) with a lower limit of quantification (LLOQ) of 10 copies/mL. cccDNA was extracted from snap-frozen liver samples using modified Hirt protocol, followed by ExoV nuclease treatment, and quantified by an HBV specific digital droplet PCR. The individual correlation between levels of intrahepatic cccDNA and i) HBV DNA, ii) qHBsAg and iii) HBV RNA was assessed using Spearman correlation coefficients. Results: Fifty SEN-B participants were included with a median age of 31 years (interquartile range 26-37) and 16/50 (32%) were female. One individual (3%) had ALT >40 IU and 13/50 (26%) had liver fibrosis defined as a Metavir stage ≥F2. HBV DNA >2,000 IU/mL was found in 19/50 (38%) participants and qHBsAg >1,000 IU/mL in 28/50 (56%). HBV RNA was detected in 26/50 (52%) participants, of whom 12/50 (24%) had >10 copies/mL. Median cccDNA (copies/ cells) was similar between HBV RNA-negative and positive individuals (p=0.74). We observed no significant correlation between the intrahepatic cccDNA and plasma HBV DNA levels (r=0.21, p=0.15), as well as between the intrahepatic cccDNA and serum qHBsAg levels (r=0.02, p=0.87). There was a moderate positive correlation between cccDNA and total plasma HBV RNA among persons with detectable levels (r=0.52, p=0.006). Conclusion: HBV RNA was the only circulating marker which correlated with intrahepatic cccDNA in our well-characterized group of persons with HBeAg-negative HBV infection in Senegal. Further research is needed to better understand the underlying mechanisms and implications of this correlation. The figure, table, or graphic for this abstract has been removed.

therapies for treatment of chronic viral infections, an ever increasing number of potent medicines will come available. One, developed in our own laboratories, is a lipophilic ProTide of TFV called M1TFV that exhibited sustained suppression of HBV DNA in mouse models of human disease [Sci. Adv.8,eade9582(2022)]. However, like other ProTide prodrugs, the reported chemical synthesis of M1TFV necessitates costly chiral catalysts and time-consuming separation of the Sp and Rp diastereomers generated at the phosphorous atom. To overcome these limitations we developed alternative amino acid free hydrophobic and lipophilic double ester crystalline TFV prodrugs. Two nanoformulated diester candidates elicited enhanced and sustained HBV DNA suppression in transgenic C57BL/6 Tg05 mice compared to limited efficacy for TAF ProTide. Methods: Three amino acid free lipophilic double ester prodrugs of TFV were synthesized through a non-catalytic esterification of TFV monophenyl ester with either an aryl substituted lipid (M4TFV), octadecanol (M5TFV) or docosanol (M6TFV). The synthesized prodrugs were characterized and nano-formulated using biocompatible surfactants to produce stable aqueous solid drug nanosuspenisons (NM4TFV, NM5TFV and NM6TFV). Control treatment consisted of TFV alafenamide solid drug nanosuspensions (NTAF). Additional controls were infected, untreated animals. Antiviral efficacy was tested in HBV Tg05 mice. Drug formulations were administered as single intramuscular injections at 168 mg/kg TFV equivalents. HBV DNA viral load in blood was measured every other week. Results: Three diester prodrugs of TFV were synthesized in high chemical yields and formed predominantly one of the two stereoisomers at the phosphorous chiral center. These prodrugs were crystalline and compatible with scalable formulation approaches for producing surfactant stabilized aqueos nanosuspenisons. Notably, a single IM injection of NM5TFV or NM6TFV diester prodrug nanosuspensions in Tg05 mice demonstrated enhanced and sustained efficacy compared to limited anti-HBV activity for TAF ProTide or arylated NM4TFV diester prodrug (Figure 1). Conclusion: NM5TFV and NM6TFV diester prodrug formulations produced sustained monthslong suppression of HBV DNA in Tg05 mice without recorded adverse events.

Poster Abstracts

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Evaluation of the Hepatitis B Virus Reservoir With Fine Needle Aspiration and Serum Biomarkers Barbara Testoni 1 , Armando Andres Roca Suarez 1 , Marie-Laure Plissonnier 1 , Caroline Scholtes 1 , Floriana Facchetti 2 , Marinta Heil 3 , François Villeret 1 , Yasmina Chouik 1 , Massimo Levrero 1 , Upkar Gill 4 , Patrick Kennedy 4 , Pietro Lampertico 2 , Fabien Zoulim 1 1 L’Université Claude Bernard Lyon 1 , Lyon, France, 2 Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy, 3 Roche Molecular Systems, Inc, Pleasanton, CA, USA, 4 Barts Liver Centre, London, United Kingdom Background: Novel treatment strategies aimed at hepatitis B virus (HBV) cure must eliminate or silence the covalently closed circular (ccc)DNA reservoir, which measurement is challenging as it requires invasive sampling methods. To address this unmet medical need, we evaluated the HBV reservoir by: i) analyzing viral markers in fine needle aspirates (FNAs) as a less invasive alternative to core liver biopsy (CLB), and ii) quantifying circulating HBV RNA (cirB-RNA) as an indicator of cccDNA transcriptional activity. Methods: We collected matched CLB/FNA/serum samples from chronic hepatitis B patients (n=9), as well as paired CLB/serum samples from untreated (n=92) and nucleos(t)ide analog (NUC)-treated (n=30) individuals. Intrahepatic

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