Hepatitis B virus resistance to tenofovir: fact or fiction? A systematic literature review and structural analysis of drug resistance mechanisms

Abstract

Background: Tenofovir (TFV) is a widely used treatment for chronic hepatitis B virus (HBV) infection. There is a high genetic barrier to the selection of TFV resistance-associated mutations (RAMs), but the distribution and clinical significance of TFV RAMs are not well understood. We here present assimilated evidence for putative TFV RAMs with the aims of cataloguing and characterising mutations that have been reported, and starting to develop insights into mechanisms of resistance. Methods: We carried out a systematic literature search in PubMed and Scopus to identify clinical, in vitro and in silico evidence of TFV resistance. We included peer-reviewed studies presenting original data regarding virological TFV breakthrough, using published methods to assess the quality of each study. We generated a list of RAMs that have been reported in association with TFV resistance, developing a 'long-list' (all reported RAMs) and a 'short-list' (a refined list supported by the most robust evidence). We assessed the potential functional and structural consequences by mapping onto the crystal structure for HIV reverse transcriptase (RT), as the structure of HBV RT has not been solved. Results: We identified a 'long-list' of 37 putative TFV RAMs in HBV RT, occurring within and outside sites of enzyme activity, some of which can be mapped onto a homologous HIV RT structure. A 'short-list' of nine sites are supported by the most robust evidence. If clinically significant resistance arises, it is most likely to be in the context of suites of multiple RAMs. Other factors including adherence, viral load, HBeAg status, HIV coinfection and NA dosage may also influence viraemic suppression. Conclusion: There is emerging evidence for polymorphisms that may reduce susceptibility to TVF. However, good correlation between viral sequence and treatment outcomes is currently lacking; further studies are essential to optimise individual treatment and public health approaches.

Keywords: HBV; Hepatitis B virus; RAMs; TAF; TDF; TFV; Tenofovir; resistance.

Figure 1.. Mutations associated with TFV resistance located within and outside the active sites of the HBV RT enzyme.

Polymerase numbering shown in the grey bars is based on genotype A sequence (accession number X02763). Yellow bar represents RT; green bars represent subdomains which are designated finger, palm and thumb; orange rectangles represent active sites of the enzyme referred to as regions A-G. Mutations associated with TFV resistance (n=37 sites) are listed according to their location within active sites of the enzyme (orange table) or outside active sites (green table). The sites shown in bold represent the nine mutations in our short-list with best literature support (evidence summarised in Table 1). Note that in most cases, individual mutations are unlikely to be sufficient to mediate resistance, and a resistant phenotype arises only as a result of combinations of ≥2 polymorphisms. TFV, tenofovir; HBV, hepatitis B virus; RT, reverse transcriptase; RAM, resistance-associated mutation; TDF, tenofovir disoproxil fumarate; TAF, tenofovir alafenamide.

Figure 2.. Heatmap showing identity comparison matrix of reference sequence alignment of HBV RT and HIV RT.

Chart shows a comparison based on sequences downloaded from HIV sequence database and Hepatitis B Virus Database and aligned using MAFFT version 7. HIV reference sequence is HIV HXB2 ( K03455). HBV reference sequences are Geno A – FJ692557, Geno B - GU815637, Geno C – GQ377617, Geno D - KC875277, Geno E - GQ161817. HBV, hepatitis B virus; HIV, human immunodeficiency virus; RT, reverse transcriptase.

Figure 3.. A section of the reference sequence alignment of HBV RT and HIV RT.

Sequences downloaded from HIV sequence database and Hepatitis B Virus Database. Sequences were aligned using MAFFT version 7. HIV subtype B reference sequence is shown in light green (accession number K03455). HBV reference sequences are shown in yellow (Geno-A: FJ692557; Geno-B: GU815637; Geno-C: GQ377617; Geno-D: KC875277; Geno-E: GQ161817). Sites of TFV resistance are highlighted in red, based on the data assimilated in this study. HIV tenofovir RAMs were obtained from the online Stanford University HIV drug resistance database. Sites marked * have the same amino acid in HIV and HBV RT after alignment, and those coloured blue also share TFV resistance mutations. This section is shown as it contains the only two homologous TFV RAMs that we have identified using this approach. Sequence alignments and RAMs throughout the whole RT protein is shown in Suppl Table 5 (see Extended data). Note that in most cases, individual mutations are unlikely to be sufficient to mediate resistance, and a resistant phenotype arises only as a result of combinations of ≥2 polymorphisms. HBV, hepatitis B virus; HIV, human immunodeficiency virus; RAM, resistance-associated mutation; RT, reverse transcriptase; TFV, tenofovir.

Figure 4.. Cartoon to show the sites of TFV drug resistance polymorphisms, using the homologous crystal structure of HIV RT as a model.

The sequence alignment of HBV was extended with HIV RT’s p66 domain and then projected onto a high-resolution HIV RT structure (PDB code 3dlk). Sub-domains of the HIV RT are coloured and annotated. Positions associated with resistance are scattered primarily throughout the finger and palm subdomains of the p66 domain (purple space-filled representations, left whole-molecule view, purple stick representation on the zoomed in view on the right). Three aspartate residues, D83, D205 and D206 (indicated by grey space-filled representation) form the catalytic triad of the enzyme and are shown as a point of reference. Of the 37 sites identified as potential TFV RAMs, 24 residues which are visible in the structure are labelled (using HBV numbering). This excludes seven putative HBV mutations at sites which do not have a homologous site in the HIV structure (sites 78, 80, 130, 134, 153, 163 and 256), and six sites which are beyond the end of the sequence of the solved crystal HIV structure (267, 269, 278, 317, 333 and 337). Figure produced using the ICM platform. Note that in most cases, individual mutations are unlikely to be sufficient to mediate resistance, and a resistant phenotype arises only as a result of combinations of ≥2 polymorphisms. HBV, hepatitis B virus; HIV, human immunodeficiency virus; RAM, resistance-associated mutation; RT, reverse transcriptase; TFV, tenofovir.