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

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.


Introduction
Nucleot(s)ide analogues (NA) are the most widely used antiviral treatments for chronic hepatitis B virus (HBV) infection 1 , with tenofovir (TFV) being a safe, cheap, and widely available agent. NA agents inhibit the action of HBV reverse transcriptase (RT), acting as DNA chain terminators. NA therapy can be effective in suppressing HBV viraemia, thus reducing the risks of inflammation, fibrosis and hepatocellular carcinoma (HCC) as well as lowering the risk of transmission 1 . However, NAs are not curative due to the persistent intracellular hepatic reservoir of HBV covalently closed circular DNA (cccDNA). Long-term administration is therefore typically required 2 , with a potential risk of selection of resistance-associated mutations (RAMs) in the virus 3,4 . RAMs are mostly likely to arise in the context of high viral replication, arising as a result of the error prone RT enzyme 1 .
Lamivudine (3TC), telbivudine (LdT) and adefovir (ADV) have been phased out of use in HBV management, mainly due to the predictable selection of RAMs over time 1 . The best recognised 3TC RAM arises at RT-M204, representing the second position of the tyrosine-methionine-aspartate-aspartate ('YMDD') motif in viral RT 1,4 . Resistance to TFV, formulated either as tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) (Suppl Fig 1, Extended data 5 ), remains controversial. Unlike other NAs, TFV has a high genetic barrier to resistance 1 , corroborated by studies that report no resistance after many years of treatment 6 . An on-line tool, 'geno2pheno hbv', lists only one position (RT N236T) in association with reduced TFV susceptibility, while other reports also include A181T/V 7 . However, there are emerging reports of a wider range of amino acid substitutions that are associated with reduced TFV sensitivity, described in both treatment-experienced and treatment-naïve individuals with chronic HBV infection (CHB) 8,9 . There is some degree of homology between the sequence, structure and function of HIV and HBV RT enzymes, explaining why certain NAs are active against both viruses 10 . Although no crystal structure has been resolved for HBV RT, some studies have modelled this enzyme based on the HIV crystal structure 10-12 , suggesting that insights into HBV drug resistance mechanisms might be inferred from what is known about HIV.
A better understanding of the role of NA therapy in driving HBV elimination at a population level is crucial to underpin efforts to move towards international targets for elimination by the year 2030 3,13 . For populations in which HIV and HBV are both endemic, as exemplified by many settings in sub-Saharan Africa, there are particular concerns about drug resistance in HBV, given the widespread population exposure to TDF as a component of first-line antiretroviral therapy (ART) for HIV 3 . In order to progress towards these targets, many more people will need to be treated in the decade ahead.
We have therefore undertaken a systematic approach to assimilate the current evidence for the development of clinical or virological HBV breakthrough during TFV therapy. The evidence on this topic is not currently sufficiently advanced to underpin definitive conclusions regarding specific genetic signatures that underpin TVF resistance, or the extent to which these are significant in clinical practice. However, we add to the field by providing a comprehensive summary of relevant publications, together with a quality appraisal of the evidence. We used this process to assimilate a 'long-list' (all putative TFV RAMs) and a 'short-list' (a refined catalogue containing only the polymorphisms most robustly supported by existing data). We highlight gaps in the existing data and the urgent need for more research.

Search strategy and quality appraisal
We undertook a systematic search of PubMed and Scopus in February 2019, using PRISMA criteria (Suppl Fig 2, Extended data 5 ). Data extraction was performed independently using the search terms ("Hepatitis B virus" [Mesh] OR "hepatitis b" OR HBV) AND (Tenofovir OR TDF OR TAF OR "Tenofovir alafenamide" OR "Tenofovir Disoproxil Fumarate") AND (resista* OR drug muta* OR DRMs OR RAMs OR viremia OR replica*). We reviewed the titles and abstracts matching the search terms and included those reporting virological HBV breakthrough after exposure to TFV, only including studies that presented original data and had undergone peer review. All retrieved articles were in English, therefore no exclusion in relation to language was required. For each study, we extracted information on type of study, characteristics of study participants, sequencing method, genotype, HBV treatment used, mutations associated with TFV resistance and method used to define TFV resistance. We used the Joanna Briggs Institute Critical Appraisal tool checklist to assess for quality of case reports 14 . For assessment of quantitative studies, we used the BMJ adapted Quality Assessment Tool for Quantitative Studies 15 .

Appraisal of putative sites of drug resistance
Recognising that there is sparse and varied evidence to support TDF RAMs, we divided our data into two categories. First, we generated a 'long list' of all polymorphisms that have been reported in association with TFV resistance, to summarise all the available data in the most inclusive way. We then refined this into a 'short list' including just those sites supported by the most robust evidence, based on reporting in ≥2 studies and a combination of in vivo and in vitro evidence.

Sequence analysis
To assess similarity between HIV and HBV RT, we downloaded HIV (HXB2 -K03455); and HBV reference sequences (Geno A -FJ692557, Geno B -GU815637, Geno C -GQ377617, Geno D -KC875277, Geno E -GQ161817) from publicly available repositories: HIV sequence database 16 and Hepatitis B Virus Database 17 . We aligned amino acid RT sequences using MAFFT version 7 18 . The alignment illustrates regions of similarity and differences between HIV and HBV RT. We obtained RAMs associated with HIV resistance to TFV published in the Stanford University HIV drug resistance database under drug resistance summaries for nucleoside RT inhibitors. 19 .

Structural analysis
The crystal structure for HBV RT has not been solved, However, the enzyme is homologous to HIV RT. In order to further assess the relevance of sites included in a 'long list' of all polymorphisms, we therefore considered evidence for a mechanistic influence by mapping HBV RAMs onto a previously solved crystal structure of HIV RT (Protein Data Bank (PDB) code 3dlk) 20 using ICM-Pro platform, which provides a direct link to the PDB 21

Results
Nature and quality of the evidence identified We identified 15 studies that met our search criteria. Although some studies used hybrid methods, we classified them broadly as seven studies arising from clinical case reports 8,9,22-26 , (one of these also presented in vitro evidence for drug resistance 9 ), four from in vitro studies 27-29 and four from longitudinal studies of CHB (with or without HIV coinfection) 30 A detailed quality assessment of each individual reference is included in Suppl Table 2 (see Extended data 5 ). Among the case reports, three were of high quality as they clearly described patients' characteristics, clinical details, diagnosis, treatment and follow-up, and concluded with take away lessons 22-24 . Three further case reports did not describe diagnostic or assessment methods 8,25,26 , and one study did not describe post-intervention follow-up 9 . The overall quality rating for four cohort studies was strong because participants selected represented the target population, characteristics of participants were clearly described, there was a clear hypothesis for the study, and inclusion/ exclusion criteria were specified 30-33 . Two studies had a weak rating because there was no description of the characteristics of participants and it was not clear to what extent participants were representative of the target population 27,34 . Two natural experimental studies were not rated because the quality assessment questions were not applicable 28,29 .
Approach to defining resistance Resistance was studied based on exposure to TDF in 13 studies and TAF in two studies; we therefore refer to TFV throughout the results section. TFV resistance was determined using a range of strategies, which can be summarised as follows: i A sequencing approach to identifying possible RAMs in HBV sequence isolated individuals in whom viraemia was not suppressed by TFV therapy, undertaken in seven studies 8,22,24-26,30,33 ; ii In vitro assays to measure the effect of TFV on viral replication in cell lines, reported by three studies 27,28,34 ; iii Approaches (i) and (ii) in combination, applied in four studies 9,23,31,32 ; iv Approach (ii) combined with an animal model, described by one study 29 .
In 12/15 studies, HBV mutations were reported in association with TFV resistance (suggesting complete virologic escape from the impact of a drug) or reduced TFV susceptibility (evidenced by incomplete suppression of viraemia, but not complete drug resistance) 9,22,23,26-32,34 . In the remaining three studies, persistent viraemia was reported among individuals with chronic HBV infection while on TFV but either no RAMs were identified 25,33 or viral sequencing was not performed due to low viral load 24 .

Location and number of RAMs associated with TFV resistance
We generated a 'long-list' of 37 different sites of polymorphism, arising both within (n=15) and outside (n=22) enzymatically active sites in RT ( Figure 1; Suppl Table 3, Extended data 5 ). HBV mutations outside active sites of the enzyme occurred in combination with RAMs located within active sites, with the exception of A194T. Only two studies reported TFV resistance arising from the selection of a single mutation, S78T and A194T 23,28 . S78T was defined by sequencing HBV from two individuals in whom viraemia was not suppressed by TDF, combined with in vitro assays 23 , while A194T was only defined in vitro 28 . In all other studies, ≥2 RAMs were required to confer TFV resistance (2 RAMs in four studies 27,29,30,34 , 3 RAMs in one study 32 , 5 RAMs in one study 9 , and ≥8 RAMs in a further four studies 8,22,26,31 ). This pattern supports the high genetic barrier to selection of TFV resistance.
We narrowed the list of 37 sites down to compile a 'short-list' of TFV RAMs that have been identified in ≥2 studies, regarding these sites as having the strongest evidence base (nine sites; Table 1). The most frequently described RAMs were L180M 22,31,32 , A181T/V 27,30,31,34 , M204I/V 9,22,31,32 , and N236T 27,30,34 , which were all identified through sequencing and tested in in vitro assays to measure the effect of TDF on viral replication in cell lines. Among these, the M204 mutation (within the 'YMDD' motif) is well established in association with 3TC resistance, commonly arising in combination with substitutions at positions V173, L180 and A181, while N236 substitutions appear to be more specifically associated with reduced susceptibility to ADV and TFV 7 . Mutations at sites 177, 194 and 249 may also be more  ). 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.
specific to TFV resistance, having been less clearly reported in association with resistance to other agents 3,35 . Polymorphisms at positions 80, 173 and 184 have been described as compensatory changes to allow the virus to accommodate the primary drug escape substitution 7 . These mutations on their own may not be sufficient to mediate TVF resistance but may be a necessary compensatory contribution to combinations of mutations that underpin resistance.
Most of these polymorphisms are located outside the active site of the enzyme, with the exception of position 256. The barrier to selection of TFV resistance may therefore be lower in certain genotypes, (Figure 1; Suppl Table 4, Extended data 5 ). The same phenomenon has been described for HCV resistance, in which certain sub genotypes are predicted to be intrinsically resistant to certain direct acting antiviral agents due to the presence of resistance associated polymorphisms in the wild type sequence 36,37 .
One study assessed the replication competence and susceptibility to TFV of mutated HBV clones in vitro and in vivo using mice models. The introduction of P177G and F249A mutations (substitutions in active sites of the RT enzyme) in HBV clones resulted in a reduction in their susceptibility to TFV 29 .

RAMs occurring as minor quasispecies
There is limited evidence for the significance of TFV RAMs occurring as minor quasispecies. One study that performed ultra-deep pyrosequencing enrolled HIV/HBV co-infected individuals on (or about to start) TFV-containing ART, reporting minor variants present at <20% with mutations (V173, L180M, A181T/V and M204V) in 2/50 TFV naïve samples and 1/14 sample obtained from a TDF-experienced individual 33 . One other study performed deep sequencing using Illumina on HBV clones 9 , revealing that RAMs S106C, H126Y, D134E, M204I/V & L269I were predominant. Only one study reported sequencing the whole HBV genome, but this was undertaken following in vitro introduction of RAMs into a clinically isolated virus 34 , so does not provide any evidence of the association between TFV RAMs and other polymorphisms that might arise on the same viral haplotype. Comparison between HIV and HBV RT HBV RT has been classified into subdomains which are further divided into regions A -G 38 , which form the main catalytic core of the enzyme (Figure 1). Alignment of sequences of HBV and HIV RT demonstrates 25-27% homology between HBV and the HXB2 HIV reference sequence (Figure 2). Comparing sites that have been reported in association with drug resistance in HBV vs HIV (Figure 3; Suppl Table 5, Extended data 5 ), we found that among our long-list of 37 HBV RAMs, two sites had identical substitutions in HIV RT (M204 and L229 in HBV 10,12 , corresponding to M184 and L210 in HIV, respectively; Stanford University HIV drug resistance database; Figure 3). Other sites reporting an association with TVF resistance in HBV have substitutions that overlap with HIV RAMs, but not all of these are associated with TFV resistance (Suppl Table 6, Extended data 5 ).
Six established TFV RAMs in HIV RT (M41L, K65R, K70E, Y115F, Q151M and T215F/Y) 19 do not correspond to an equivalent mutation in HBV RT, although three of these HIV RAMs have an HBV RAM within three amino acids up-or down-stream in the equivalent sequence, suggesting there may be homology in the mechanism through which drug resistance is mediated.  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 We mapped HBV RAMs onto the crystal structure of the likely structurally-related HIV RT (PDB code 3dlk) in order to visualise their approximate 3D locations and infer possible functional consequences (Figure 4; Suppl Table 6, Extended data 5 ). The RAMs are primarily located within the 'fingers', 'palm', 'thumb' and 'connection' subdomains of the p66 polymerase domain of HIV RT, with the majority within the 'palm'. A number of RAMs (e.g. V207, M204, F249) are spatially adjacent to the catalytically critical (and highly conserved) residues D110, D185 and D186 in HIV RT (D83, D204 and D205 in HBV), suggesting that these RAMs are highly likely to affect catalytic competency. The 22 HBV RAMs that map to HIV RT residue positions are likely to cause polymerase structure destabilisation if mutated, suggesting that many of these RAMs are likely to impact upon resistance.

Discussion
Summary of key findings TFV is a safe and effective treatment choice for CHB in the majority of cases, and large case series have not raised significant concerns about clinically significant drug resistance. However, it is important to consider the potential for the emergence of resistance, demonstrated by persistent viraemia on therapy and/or reduced virologic suppression in vitro. Based on existing evidence, TFV resistance seems likely to depend on the selection of suites of mutations (most commonly including L180M, A181V/T, M204I/V and/or N236T), overlapping with RAMs that allow escape from other NA drugs. There is also a suggestion that, rarely, single mutations can confer TFV resistance, best demonstrated for S78T.
Notably, the literature to date is limited and heterogenous, and there remains a lack of evidence about the frequency and likely impact of proposed TFV RAMs, either within individual patients or at population level. At present, we have tackled this uncertainty by dividing our catalogue of polymorphisms into a 'long-list' (all reported RAMs) and a 'short-list' (RAMs with the best evidence-base of support).
Tools that have been designed to identify drug resistance may bias against detection of relevant mutations if they do not scrutinise all relevant sites that contribute to reducing TFV susceptibility. For example, 'TRUGENE' a commercially available HBV drug resistance interpretation system, captures Overlap of TFV RAMs with RAMs to other NA agents RAMs L180M, M204I/V and A181T/V have been associated with resistance to 3TC, telbivudine (LdT) and entecavir (ETV) 3,40-43 ; their reported association with TFV resistance is of concern in suggesting that prior NA exposure can increase the likelihood of cross-resistance to TFV. A study of HIV/HBV co-infected individuals demonstrated a decreased likelihood of HBV DNA suppression with TDF among individuals exposed to prolonged 3TC treatment, possibly due to accumulation of such mutations 44 . A large study in China reported A181 and/ or N236 substitutions in 11% of the population 42 , which may underpin reduced susceptibility to TFV. The structural similarities between ADV and TFV, and similar interaction with HBV polymerase 1,2 explain why the ADV RAMs A181T/V and N236T are also reported to confer resistance to TFV 1,45 .
Although TFV has been considered effective in the context of resistance to other NAs 46 , the current evidence suggests that there may be common pathways to resistance 47 . There is some evidence showing co-location of RAMs conferring resistance to different antiviral agents on the same viral haplotype 48 . These findings suggesting cross-resistance are of concern, especially for settings in which there has been widespread use of NA therapy as a component of ART for HIV 3 .

Sites of TFV RAMs in HBV RT
Resistance to TFV can be explained by RAMs both within and outside the active site of the RT enzyme, some of which may have similar mechanisms to those described in HIV 10,38 . The mechanism of resistance in most of these polymorphisms remains unknown, but may interfere with drug access to sites of activity through steric hindrance. Mutations within active sites of the enzyme may be associated with a higher fitness cost to the virus than mutations at other locations in the RT sequence, as they are more likely to interfere with the RT function. Some polymorphisms listed as RAMs may in fact represent compensatory mutations, which are co-selected in the presence of primary RAMs. For example, substitution at position 269 has been previously described as a compensatory mutation that restores impairments to RT function 49 .
Currently, HBV genotyping is not routinely undertaken in clinical practice, so it is difficult to amass data for any potential relationship between resistance and viral genotype. However, there are some clues that genotype may be relevant. For example, C256S has been linked to TFV resistance, but S256 is wild type in genotype C (Suppl Table 4, Extended data 5 ), suggesting that the genetic barrier to TFV resistance in genotype C might be lower than in other genotypes. However, a study of >1000 individuals in China found no differences in drug resistance rates between genotype B vs genotype C infection 42 . The identification of Y9H as a TFV RAM should be viewed with caution as H9 is frequently the wildtype residue, irrespective of genotype.

Other factors associated with persistent vireamia
In addition to RAMs, there are other explanations for incomplete suppression of HBV viraemia on therapy 25,33 , including a higher baseline HBV DNA level, positive baseline HBeAg status, history of 3TC exposure, a lower nadir CD4+ T cell count in the context of HIV coinfection, and high serum HBV RNA levels 44,50,51 . Given that HBV DNA is inhibited in a dosedependent manner 2 , it is also possible that insufficient drug delivery to the infected hepatocyte could be the cause of persistent viraemia even in the absence of specific RAMs.
Incomplete adherence to therapy can also contribute to virological breakthrough 52 . Two studies included in our review assessed treatment compliance by measuring drug concentration in plasma 9,31 . Assessment of adherence in chronic HBV has been through the use of questionnaires 53 , but these are subject to self-reporting bias. Evidence of potential TFV resistance may emerge when individuals with HIV/HBV coinfection are treated with a TFV-containing regimen leading to suppression of HIV but with sustained HBV vireamia 54 .
It has been reported to take three years for 90% of HBV infected individuals to reach viraemic suppression on therapy 55 , in contrast to HIV, in which 88% of patients suppress the virus within the first year of TDF-based treatment 56 . In the studies we have reported in this review, persistent HBV viraemia on therapy could be due to the prolonged timeline for viraemic suppression; however, in most studies there was a reduction in viral load when TDF was initiated, with subsequent virological breakthrough that is more in keeping with the selection of resistance.

Implications for patient management
There are not currently sufficient data about TFV RAMs to underpin robust universal guidelines for clinical practice. However, the evidence that we have gathered here can underpin some practical recommendations: i There is an urgent need for more HBV sequencing data, together with contemporaneous viral load measurements and clinical metadata to advance understanding of the relationship between viral sequence and treatment outcomes. Sequence repositories, databases and tools for sequence analysis should regularly review the evidence for TDF RAMs in order to highlight all sites that may be significant in mediating resistance.
ii In the context of failure of viraemic suppression in a patient prescribed therapy, assessing and supporting drug compliance is crucial, ideally together with viral sequencing.
iii Therapeutic failure of TFV -whether in the presence or absence of known RAMs -should lead to an expert clinical decision about switching therapy or combining agents, as long as adherence has been optimised and supported. The presence of recognised RAMs, especially when in combination, may support a change of therapy, considering ETV or combination therapy, although guidance and options are currently limited.
iv If there is an ongoing emergence of data to suggest TFV resistance, there will be a need for expert guidelines to include practical recommendations in order to unify clinical approaches. HIV guidelines should take an active stance on incorporating recommendations for those with HBV co-infection, particularly if dual therapy regimens are adopted.
v Evidence of TFV resistance highlights the need for development of robust novel direct acting antivirals and immune therapies for HBV.

Caveats and limitations
There is sparse literature on HBV resistance to TFV, and studies are of varying quality. While there is a high genetic barrier to selection of TFV resistance, it is likely that there is under-reporting of cases of resistance, particularly in low/middle income settings in which routine monitoring of HBV viral load on treatment is not undertaken. It can be difficult to infer the impact of common polymorphisms on drug resistance phenotype; for example, it is plausible that M204I/V may be enriched among TFV resistant strains simply as a 'footprint' of prior exposure to 3TC.
Most studies to date have used Sanger sequencing, and it is possible that significant minority variants may be underrepresented, as suggested by one report in which phenotypic TFV resistance was associated with RAMs in <20% of minor variants 33 . Low HBV DNA viral loads are a further barrier to sequencing, and bias existing data towards samples from individuals with high viral loads, in which the full spectrum of relevant RAMs may not occur. It is therefore important to invest in deep sequencing platforms that offer the opportunity to explore the full landscape of HBV variants isolated from an infected individual, and to improve sensitivity of sequencing methods including both Sanger and 'next-generation' approaches. Some sequencing methods, such as Oxford Nanopore Technologies, can generate long reads that allow reconstruction of complete viral haplotypes, providing improved certainty about linkages between sites 57 . To be able to undertake an appropriate haplotypes analysis, datasets with robustly phenotyped patients (displaying clinical evidence of drug resistance), together with full length viral sequence data, would be required; such datasets have not been generated to date but are an important long-term aim.
We recognise the limitations of drawing direct comparisons between HIV and HBV RT, given the limited (<30%) sequence homology between the two enzymes, and the finding that only 2/37 sites associated with TVF resistance in HBV are homologous RAMs in HIV. This highlights a need for future work to solve the crystal structure of HBV RT.

Conclusions
We have assimilated emerging evidence for HBV polymorphisms that reduce susceptibility to TFV, also acknowledging the potential influences of other viral and host factors in cases of persistent viraemia on therapy. While the genetic barrier to resistance is high, evidenced by the large number of mutations that typically have to be selected to produce resistance, of concern is the overlap with other NA resistance mutations, and the instances in which individual amino acid polymorphisms may be sufficient to produce phenotypic resistance. Enhanced studies representing larger numbers of patients, tracking longitudinal viral sequence changes, and monitoring viral suppression over time are needed. In addition, the evolution of better in vitro models will support experiments to investigate the effect of individual and combined RAMs. In order to optimise the use of NA therapy as a tool in driving advancements towards elimination at a population level, improved insights into drug resistance are essential. If resistance emerges as a substantial clinical problem, there will be a need for consideration of synergistic drug regimens, new agents that inhibit a target other than viral RT, and for the development of new therapeutic strategies that can bring about cure.

Suzane K Ono
Departamento de Gastroenterologia, Universidade de Sao Paulo, Sao Paulo, Brazil This is an excellent systematic review on clinical, in vitro and in silico TNF resistance. The authors reviewed and listed polymorphisms associated to TNF resistance and analyzed the existence of polymorphisms according to HBV genotypes. In addition, compared the polymorphisms in the HBV and HIV RT gene sequence alignments.
Comments: Figure 1. The green box is named within active site? Is it correct? Please verify. Figure 1. As mentioned by the authors in the text, polymorphisms at position 80, 173 and 184 are described as compensatory changes, therefore the title "RAMs associated with TDF/TAF …" this could mislead the readers to believe they are all confirmed resistant mutations. The urgent need for more HBV sequencing data is important for research and better understanding of the topic; however, it is important to first check patient adherence to treatment (feasible and lower cost) and then look for RAMs. Therefore, I would suggest to change position of item "i" There is urgent need for more HBV sequencing… with item "ii" Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes

Is the statistical analysis and its interpretation appropriate? Not applicable
Are the conclusions drawn adequately supported by the results presented in the review? Yes Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
Reviewer Report 08 April 2021 https://doi.org/10.21956/wellcomeopenres.17540.r43247 © 2021 Lukhwareni A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Azwidowi Lukhwareni
Department of Medical Virology, University of Pretoria and National Health Laboratory Service, Tshwane Academic Division, Pretoria, South Africa TVF Abbreviation was not explained in full.
This is an excellent systematic review. There is still however limited data regarding HBV primary resistance against TFV or TFV. The data is even sparse in low/middle income settings such as sub Saharan Africa where routine monitoring of HBV viral load on treatment is rarely undertaken despite high usage of TFV as part of HIV/AIDS management. While some resistance mutations have been reported, phenotypic studies are lacking regarding primary TFV resistance mutation. In a recent Gilead HBV clinical studies, all the patients harbouring rtA194T or CYE/CYEI (S106C, H126Y, D134E and L269I) at baseline achieved viral suppression by week 96 after TDF or TAF treatment. No patients developed an rtA194T mutation or > 1 substitution of CYEI, and the number of patients losing any substitutions of CYEI (n = 17) was similar to the number who developed a single substitution of CYEI (n = 32) during treatment. Phenotypic evaluation of the site-directed mutant panel containing these mutations with or without other resistance mutations did not demonstrate a significant shift in TFV and TAF potency in vitro. No evidence of rtA194T and CYEI conferring resistance to TDF or TAF was observed based on the treatment responses to TDF or TAF in patients with mutations at baseline, the lack of selection of mutations after starting TDF or TAF treatment and no change in susceptibility to TFV or TAF in vitro. Overall, rtA194T and CYEI did not appear to confer resistance in any patient on a TDF or TAF regimen.

Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes
Are sufficient details of the methods and analysis provided to allow replication by others? Yes

Is the statistical analysis and its interpretation appropriate? Not applicable
Are the conclusions drawn adequately supported by the results presented in the review?