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THU-216
Rapid turnover of HBV cccDNA in nucleoside-treated chronic
Hepatitis B patients during drug resistance emergence and
Breakthrough
Qi Huang1, Bin Zhou2, Dawei Cai1, Yuhua Zong1, YaoboWu2, Shi Liu2,
Haitao Guo3, Jian Sun2, Jinlin Hou2, Richard Colonno1. 1Assembly
Biosciences, South San Francisco, United States; 2Nanfang Hospital,
Hepatology Unit and Department of Infectious Diseases, Guangzhou,
China; 3Indiana University School of Medicine, Department of
Microbiology and Immunology, Indianapolis, United States
Email: [email protected]
Background and aims: Current low cure rates in patients chronically-
Infected with Hepatitis B Virus (HBV) using standard of care
Therapies are believed to be due to an inability to eliminate cccDNA
Reservoirs in patients. It remains unclear whether persistence is due
To the longevity of cccDNA or its efficient replenishment by either de
Novo infection or intracellular amplification.
Method: Methodologies were optimized to enable analysis of distinct
Populations of cccDNA, pgRNA and viral DNA in both CHB patient sera
And liver biopsies. Pan-genotypic primers were designed to amplify
The HBV reverse transcriptase (RT) gene. HBV cccDNA from snap frozen
Liver biopsies were extracted by a modified Hirt method,
Digested with T5 exonuclease, amplified by PCR. Intrahepatic rcDNA
And HBV RNA were purified with QIAamp MinElute Virus kit from
Flow-through of Hirt extraction. HBV DNA and pgRNAwere extracted
From patient sera using a QIAamp MinElute Virus kit. Aliquots were
Digested by DNase I and used as a template for RT-PCR. Sanger
Sequencing results of PCR and RT-PCR fragments were analyzed using
SequencherTM software (Gene Codes) and the percentage of signature
Lamivudine/telbivudine resistance (NucR) mutations was calculated
By population sequencing or clone-based sequencing following TA cloning.
Results: The emergence and disappearance of NucR mutations was
Retrospectively monitored as a sensitive genetic marker of cccDNA
Turnover/evolution in a panel of patients with paired patient liver and
Blood samples from two clinical studies (EFFORT and ML18376).
Critical to the success of this genetic approach, we found a strong
Relationship between the population sequences found in serum
pgRNA, intrahepatic HBV RNA and cccDNA samples, validating serum
RNA as a surrogate marker of cccDNA sequences at time points when
Liver biopsy samples were unavailable. The observed changes in NucR
Viral variants (DNA) suggest the near complete turnover of wild-type
pgRNA and cccDNA populations, with serum HBV DNA, pgRNA
And cccDNA populations observed to switch between WT and
NucR populations in as little as 3-4 months in some patients.
Compensatory mutations that affect fitness likely account for the
Range of timelines observed for complete viral genetic turnover in
Throughput patients. In all cases, turnover rates were measured in
Several months, not years, as previously predicted.
Conclusion: Serum pgRNA was validated as a surrogate genetic
Biomarker for cccDNA populations. Overall, cccDNA pools appear to be dynamic, with rapid decay of existing populations and complete
Turnover in as little as 3-4 months. These findings support the
Potential for a finite treatment period for regimens that can fully
Suppress viral replication and inhibit regeneration of new cccDNA
Molecular. |
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发表于 2019-3-29 16:10