Genetic accessibility, not superior fitness, determines which anti-HBs antibody escape variants are selected in patients
- 1Weill Cornell Medicine
Abstract
Background and Aims: Genetic diversity within the hepatitis B virus (HBV) surface antigen (HBsAg) drives the emergence of immune escape variants, but studying this diversity in vivo has been limited by the difficulty of establishing infection from patient isolates. We aimed to perform unbiased in vivo screening of variants in the HBs antigenic loop for antibody escape and fitness, using synthetic DNA launch of HBV in human hepatocyte chimeric mice. Approach and Results: Direct intrahepatic injection (IHI) of barcoded, recombinant cccDNA established infection from up to 32,000 independent launch events of a genotype A strain and successfully launched clinical consensus sequences across the major genotypes. Using IHI, we performed deep mutational scanning of 4,608 codon variants across the HBs antigenic loop. Under control antibody treatment, diverse variants expanded, revealing differences in variant fitness in the absence of adaptive immune pressure. Anti-HBs monoclonal antibody treatment suppressed wild-type viremia while permitting breakthrough of resistant variants from the library. Sequencing identified strong enrichment for substitutions at G145 (G145Q, G145H, G145R). Notably, G145Q, rather than the clinically dominant G145R, showed the greatest fitness advantage, indicating that G145R's clinical prevalence does not reflect superior intrinsic fitness. Conclusions: Unbiased in vivo screening reveals that clinically observed HBV escape mutations reflect which substitutions are genetically accessible by single nucleotide change and compatible with the overlapping polymerase reading frame, rather than which confer optimal fitness. This approach establishes a platform for high-resolution mapping of viral variant fitness under antiviral selection pressure.
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BibTeX
@misc{yu2026genetic,
title = {{Genetic accessibility, not superior fitness, determines which anti-HBs antibody escape variants are selected in patients}},
author = {Y. Yu and E. Michailidis and L. L. Seifert and M. Zhang and C. Zou and C. Freije and G. Dangas and A. Athanasiadis and A. Puschnik and A. Hanson and E. Rocha and Y.-P. Chen and X. Hong and C. Quirk and Y. Zhou and M. A. Schmid and F. A. Lempp and C. M. Rice and W. Schneider and Y. P. De Jong},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.06.756817},
url = {https://www.biorxiv.org/content/10.64898/2026.10.06.756817v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Genetic accessibility, not superior fitness, determines which anti-HBs antibody escape variants are selected in patients
AU - Y. Yu
AU - E. Michailidis
AU - L. L. Seifert
AU - M. Zhang
AU - C. Zou
AU - C. Freije
AU - G. Dangas
AU - A. Athanasiadis
AU - A. Puschnik
AU - A. Hanson
AU - E. Rocha
AU - Y.-P. Chen
AU - X. Hong
AU - C. Quirk
AU - Y. Zhou
AU - M. A. Schmid
AU - F. A. Lempp
AU - C. M. Rice
AU - W. Schneider
AU - Y. P. De Jong
PY - 2026
DA - 2026/10/06
PB - bioRxiv
DO - 10.64898/2026.10.06.756817
UR - https://www.biorxiv.org/content/10.64898/2026.10.06.756817v1
AB - Background and Aims: Genetic diversity within the hepatitis B virus (HBV) surface antigen (HBsAg) drives the emergence of immune escape variants, but studying this diversity in vivo has been limited by the difficulty of establishing infection from patient isolates. We aimed to perform unbiased in vivo screening of variants in the HBs antigenic loop for antibody escape and fitness, using synthetic DNA launch of HBV in human hepatocyte chimeric mice. Approach and Results: Direct intrahepatic injection (IHI) of barcoded, recombinant cccDNA established infection from up to 32,000 independent launch events of a genotype A strain and successfully launched clinical consensus sequences across the major genotypes. Using IHI, we performed deep mutational scanning of 4,608 codon variants across the HBs antigenic loop. Under control antibody treatment, diverse variants expanded, revealing differences in variant fitness in the absence of adaptive immune pressure. Anti-HBs monoclonal antibody treatment suppressed wild-type viremia while permitting breakthrough of resistant variants from the library. Sequencing identified strong enrichment for substitutions at G145 (G145Q, G145H, G145R). Notably, G145Q, rather than the clinically dominant G145R, showed the greatest fitness advantage, indicating that G145R's clinical prevalence does not reflect superior intrinsic fitness. Conclusions: Unbiased in vivo screening reveals that clinically observed HBV escape mutations reflect which substitutions are genetically accessible by single nucleotide change and compatible with the overlapping polymerase reading frame, rather than which confer optimal fitness. This approach establishes a platform for high-resolution mapping of viral variant fitness under antiviral selection pressure.
ER -