Background: HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. Methods: This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV- DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. (Continued on next page) * Correspondence: firstname.lastname@example.org; email@example.com Department of Experimental Medicine and Surgery, University of Rome Tor Vergata, Via Montpellier, 1, 00133 Rome, Italy Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 2 of 12 (Continued from previous page) Results: At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32–3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21. 2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co- presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32–3.10],P = 0.001). Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties. Conclusions: Immune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence. Keywords: HBV, HBsAg, Immune-escape, Stop-codons, Drug-resistance Background and thus determine a false-negativity or an underestima- Worldwide, around 250 million individuals have a tion of HBsAg levels, that can pose an issue for a proper chronic hepatitis B virus (HBV) infection. Among them, diagnosis and staging of chronic HBV-infection. around 1 million dies as a consequence of end-stage To date, six nucleos(t)ide analogues (NAs) have been liver disease or hepatocellular carcinoma (HCC) . approved for the treatment of HBV-infection, namely HBV is a highly evolving pathogen characterized by a lamivudine (LAM), adefovir dipivoxil (ADV), entecavir high degree of genetic-variability (a unique property (ETV), telbivudine (LdT), tenofovir (TDF), and the re- among DNA viruses) that is driven by the lack of cently approved tenofovir-alafenamide (TAF). Among proof-reading function of HBV reverse transcriptase them ETV, TDF or TAF are characterized by high gen- (RT) and exacerbated by the high speed of the HBV etic barrier to resistance , and thus they are pre- replication cycle . ferred as first-line treatment in the majority of European This high degree of HBV genetic-variability allows the Countries [13–15]. virus to react to endogenous (i.e. immune system), and Furthermore, due to the overlapping between the genes exogenous (i.e. vaccination, hepatitis B immunoglobulin, encoding reverse transcriptase (RT) and HBsAg, some RT antiviral drugs) selective pressures by further modulating drug-resistance mutations can introduce mutations in the its genome structure. major hydrophilic region of HBsAg that are capable to Among the different HBV-proteins, HBV surface reduce the binding affinity for neutralizing antibodies, antigen (HBsAg) contains the major hydrophilic re- including those induced by HBV-vaccine . Again, gion that is a dominant epitope crucial for binding to these mutations (hereafter defined as NA-induced neutralizing-antibodies. So far, around 30 immune-escape immune-escape mutations) may pose a public health mutations in HBsAg (hereafter defined as immune-associated concern for their pathogenetic potential and possibility escape mutations), have been identified [3–5]toevade of transmission to vaccinated individuals. neutralizing-antibodies, to allow persistent HBV-infection and Another type of mutation that can be detected in to promote viral fitness [2, 6]. These mutations can HBsAg is represented by stop-codons. They are associ- have relevant pathobiological implications at the time ated with the synthesis of truncated forms of HBsAg that of immunosuppression-driven HBV-reactivation, thus remain trapped in the endoplasmic reticulum. This favoring the reuptake of viral replication during the initial intracellular HBsAg accumulation can induce an oxida- weakening of immune responses [6–9]. Immune-associated tive stress that can favour the neoplastic transformation escape mutations can also hamper HBsAg-recognition by of hepatocytes . antibodies induced by vaccine, thus posing a potential Information about the prevalence of the above-mentioned threat for the global vaccination program also in the mutations in patients with chronic HBV-infection exposed setting of mother-to-child transmission . In addition, to NA in Europe is limited. Filling this gap can provide an Immune-associated escape mutations can decrease/ab- estimate of the pool for HBV-transmissions also to vacci- rogate HBsAg-binding to antibodies used in diagnostic nated individuals and/or can have a higher risk of disease assays for HBsAg-detection and -quantification [6, 10, 11], progression. Thus, this study was designed to estimate the Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 3 of 12 prevalence and characteristics of i) immune-associated es- HBsAg sequences were analyzed using SeqScape-v2.6 cape mutations ii) NA-induced immune-escape mutations software (Thermo-Fisher Scientific), then the sequences and iii) stop-codons in HBsAg in Europe. were aligned using Bioedit 7.0 software . Sequences having a mixture of wild-type and mutant residues at single positions were considered to have the mutant(s) Methods at that position. The mixed base identification was set at Study population a percentage of 20%. A multicenter survey was performed on genotypic-resistance HBsAg sequences have been submitted to Genbank with testingresults generatedduringroutine clinical assessments the following accession number: MH218870-MH219804. of patients with chronic hepatitis B attending tertiary referral centers in European countries according to Hermans et al., 2016. Inclusion criteria were: chronic hepatitis B with Mutation prevalence detectable serum HBV-DNA, exposure to ≥ 1 NA, RT/ HBsAg-sequences were analysed to define the HBsAg-sequence availability, and age ≥ 18 years . prevalence of immune-associated escape mutations, Inclusion of patients exposed to NAs allows to define NA-induced escape mutations, and stop-codons. the prevalence of immune-associated, and also of Mutations were defined as difference from HBV NA-induced escape mutations. genotype-A reference sequence (Genbank accession 935 RT/HBsAg-sequences were collected in the number: JN182318) or HBV genotype-D reference se- time-window between January 1998 and August 2012. quence (Genbank accession number: GU456636). Only 1 sequence per patient was included in the analysis. We determined the prevalence of 29 immune-associated Patient datasets were collected in the framework of the escape mutations (sQ101K, sT114R, sP120S/T/A, European Society for translational antiviral research sT123A/N, sT126N/S, sP127L, sA128V, sQ129R/N, (ESAR) from 15 countries. Countries were grouped in sG130N/R, sT131I, sM133I/L/T, sY134L, sC138Y, sC139S, geographical regions (http://unstats.un.org/unsd/)asfol- sT140S, sP142S, sD144A/E, sG145A/R, sN146S) exten- lows: Northern Europe (Denmark/Norway), Western Eur- sively retrieved from literature and known to affect ope (Austria/France/Germany/Luxembourg/Netherlands), HBsAg-recognition by antibodies [3–5, 19]. Among them, Eastern Europe (Poland/Romania), and Southern Europe sP120S/T/A, sT126N/S, sQ129R/N, sT131I/N, sM133I/L, (Greece/Italy/Serbia/Slovenia/Spain) . Israel and sP142S, sD144A/E, sG145A/R were known to act as Turkey were grouped with Southern European coun- vaccine-escape mutations [3–5, 19]. All these mutations tries . are localized in the major hydrophilic region of HBsAg known to contain the major B-cell epitopes. Data characteristics We also analyzed the prevalence of the NA-induced The following information was collected: serum immune-escape mutations sI195M, sI196S, and sE164D HBV-DNA; HBsAg; hepatitis B e antigen (HBeAg); (resulting from drug-resistance mutation rtM204 V, anti-HBe; serum–alanine aminotransferase (serum-ALT); rtM204I, and rtV173L)  and stop-codons. exposure to ≥ 1 NA (LAM, LdT, ADV, ETV, TDF, LdT). No administrative permissions were required to review patients’ records and to use related data. Statistical analysis Statistical analysis was performed using SPSS software RT/HBsAg sequencing (v19.0; SPSS Inc., Chicago, IL) and the statistical envir- RT/HBsAg sequences obtained by well-standardized onment R (version 3.2.5). Data were expressed as me- population-based sequencing procedures during routine dian (interquartile range [IQR]) for quantitative variables clinical practise were collected. Sequence data consisted and as counts and percentages for qualitative variables. of FASTA files containing nucleic acid sequence infor- Chi-Squared Test of Independence based on a 2 × 2 con- mation of the RT/HBsAg region. The ESAR quality con- tingency table was used for qualitative data, while trol procedure was applied on all submitted sequences. Mann-Whitney test for continuous data. If amino acid substitutions at immune-escape codons Univariable and multivariable logistic regression ana- were due to ambiguities consisting of > 2 bases per nu- lysis was performed in order to assess the potential asso- cleotide position or > 1 ambiguities per codon, or if in- ciations between the presence of at least one i) sertions or deletions were present causing a shift in the immune-associated escape mutation, ii) NA-induced HBsAg open-reading frame that affected immune-escape immune-escape mutation, iii) stop-codon, with several codons, sequences were excluded from the analysis . factors, including: gender, age, serum HBV-DNA at the Furthermore, there was no specific pattern of mutations time of genotypic testing, LAM, ADV, ETV, TDF, geo- linked to a specific center. graphical origin, year of collection, and HBV-genotype. Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 4 of 12 Results 1998–2002 and in 2003–2005 (15/101), showed an in- Study population crease to 27.2% (89/327) in 2006–2008 (P =0.012, using The study population included 935 patients with chronic 1998–2002 as reference), and then declined to 20.8% (68/ HBV infection exposed to ≥ 1 NA. Phylogenetic analysis 327) in 2009–2012. showed that most patients were infected with HBV Furthermore, the circulation of HBV strains with ≥ 1 genotype-D (573, 61.3%) and genotype-A (255, 27.3%). immune-associated escape mutation was significantly In the remaining patients, the following HBV-genotypes higher in genotype-D than A (25.3%[145/573] vs were detected: B (36, 3.9%), C (36, 3.9%), E (23, 2.4%), G 14.9%[38/255], P = 0.001) (Fig. 1a). This result was also (5, 0.5%), H (4, 0.4%), F (3, 0.3%). observed when the analysis was specifically focused on To provide a more robust characterization of vaccine-escape mutations (18.3%[105/573] for immune-escape mutations and stop-codons circulating genotype-D vs 7.1%[18/255] for genotype-A; P < 0.001). in Europe, the analysis was focused on 828 patients HBV genotype-D was significantly associated with the infected with HBV genotype-D and A. Table 1 shows selection of specific immune-associated escape muta- demographics, clinical, biochemical, and virological tions. This is the case of sA128V and sP120S selected characteristics of these patients. with higher prevalence in genotype-D than A (sA128V: Patients were predominantly males (70.5%) with a me- 3.3%[19/573] vs 0.8%[2/255], P = 0.032; sP120S: 5.1%[29/ dian (IQR) age of 45(38–59)years (Table 1). Median 573] vs 0.8[2/255], P = 0.003) (Fig. 2a). Conversely, the (IQR) log serum HBV-DNA was 4.4(3.2–6.4)IU/ml, and immune-associated escape mutation G130 N occurred median (IQR) ALT was 47(32–78)U/L (Table 1). more frequently in genotype-A than D (2%[5/255] vs Information on HIV-1 coinfection was known for 445 0.2%[1/573], P = 0.012) (Fig. 2a). These results were patients. Among them, 103 patients were HIV confirmed also when the analysis was focused on co-infected. LAM-treated patients, thus limiting the impact of anti-HBV drugs on the selection of these mutations Treatment history and drug resistance (sA128V: 4.4%[16/362] vs 0.5%[2/209], P = 0.008; A detailed information of anti-HBV drugs used was sP120S: 5.5%[20/362] vs 1%[2/209], P = 0.006; sG130N: available for 650 patients. Most patients were exposed to 0.3%[1/362] vs 1.9%[4/209], P = 0.063). This suggests NA mono-therapy, predominantly with LAM (62.5%, that the genetic-backbone of genotype-A and -D can 406/650) followed by ADV (4.9%, 32/650), ETV (4.8%, favour the selection of specific immune-associated es- 31/650), TDF (0.8%, 5/650) and LdT (0.5%, 3/650) cape mutations. (Table 1). Exposure to 2 NAs, either simultaneously In addition, in genotype-D, the presence of ≥ 1 or consecutively, most frequently concerned LAM + immune-associated escape mutation was significantly ADV (17.7%, 115/650), followed by LAM + TDF higher in drug-exposed patients with drug-resistance (3.2%, 21/650), LAM + ETV (2.6%, 17/650), ADV + than in patients without the drug resistance muta- ETV (0.6%, 4/650), ETV + TDF (0.5%, 3/650) and tions (29.5%[92/312] vs 20.3%[53/261], P = 0.012). In ADV + TDF (0.2%, 1/650) (Table 1). Triple exposure particular, sP120T significantly correlated with was present in 1.8% (12/650) of patients. rtM204V/I (P = 0.001): 16/20 patients with sP120T At least one drug-resistance mutation was detected in had also rtM204V/I. Moreover, patients with 54% (447/828) of patients. In particular, the primary mu- rtM204V/I + sP120T had higher serum HBV-DNA tation rtM204V (conferring full-resistance to LAM, LdT, than patients with rtM204V/I alone (5.5[3.2– and partially to ETV) was observed in 25.8% (214/828) 7.2]logIU/ml vs 4.3[3.2–6.3]logIU/ml). This associ- of patients, while rtM204I (conferring full-resistance to ation was not observed in genotype A. LAM and LdT) in 20% (166/828). Conversely, rtA181T To corroborate the correlation between and rtA181V (conferring full-resistance to ADV and as- immune-associated escape mutations and drug-resistance sociated with TDF suboptimal response) were detected mutations, the prevalence of ≥ 1 immune-associated es- in 2.3% (19/828) and 3.6% (30/828) of patients, cape mutations was also analysed in an independent data- respectively. set of drug-naïve patients (cite Additional file 1: Table S1 for demographic and virological characteristics). The per- Detection of immune-associated escape mutations centage of drug-naive patients harbouring drug-resistant At least one immune-associated escape mutation was strains is 1% (all genotype D). The only primary detected in 22.1% (183/828) of patients (min-max:1–4). drug-resistance mutations detected were rtM204I (0.4%, In 6% (50/828) of patients, ≥ 2 mutations were detected 1/245) and rtN236T (0.4%, 1/245), while the only second- (Fig. 1a). ary mutations detected were rtL180M and rtV173L, each The proportion of patients with ≥ 1 immune-associated present in 0.4% of patients. Again, the presence of ≥ 1 escape mutation was stable to around 15% (11/73) in immune-associated escape mutations in genotype D was Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 5 of 12 Table 1 Patients’ Characteristics Overall Genotype-A Genotype-D P-value (N = 828) (N = 255) (N = 573) General Median Age (IQR), years 45 (38–59) 45 (33–56) 49 (40–59) 0.001 Male, N(%) 584 (70.5) 183 (74.4) 401 (73.6) 0.810 CHB-related data Median HBV-DNA, log IU/ml (IQR) 4.4 (3.2–6.4) 4.7(3.3–6.9) 4.4 (3.2–6.3) 0.079 HBeAg positive, N(%) 183 (44.1) 71 (59.7) 112 (38) < 0.001 Median ALT, IU/L (IQR) 46.5 (32–78) 46 (30–80) 48 (32–78) 0.473 Geographical origin, N(%) Western Europe 142 (17.1) 67 (26.3) 75 (13.1) < 0.001 Northern Europe 26 (3.1) 10 (3.9) 16 (2.8) 0.519 Eastern Europe 131 (15.8) 99 (38.8) 32 (5.6) < 0.001 Southern Europe 529 (63.9) 79 (31) 450 (78.5) < 0.001 Anti-HBV drug history, N(%) Monotherapy LAM 406 (62.5) 157 (66.8) 249 (60) 0.085 ADV 32 (4.9) 10 (4.3) 22 (5.3) 0.554 ETV 31 (4.8) 7 (3) 24 (5.8) 0.107 TDF 5 (0.8) 2 (0.9) 3 (0.7) 0.857 LdT 3 (0.5) 1 (0.4) 2 (0.5) 1.000 Dual exposure LAM + ADV 115 (17.7) 27 (11.5) 88 (21.2) 0.002 LAM + TDF 21 (3.2) 14 (6) 7 (1.7) 0.003 LAM + ETV 17 (2.6) 10 (4.3) 7 (1.7) 0.045 ADV + ETV 4 (0.6) 3 (1.3) 1 (0.2) 0.137 ETV + TDF 3 (0.5) 3 (1.3) 0 (0) 0.047 ADV + ETV 1 (0.2) 0 (0) 1 (0.2) 1.000 Triple exposure LAM + ADV + ETV 5 (0.8) 1 (0.4) 4 (1) 0.450 LAM + ADV + TDF 7 (1.1) 0 (0) 7 (1.7) 0.045 Percentages are calculated on 791 patients with the datum available, 246 patients for genotype A and 545 for genotype D Percentages are calculated on 414 patients with the datum available, 119 patients for genotype A and 295 for genotype D Percentages are calculated on 650 patients with the type of anti-HBV drugs available, 235 patients for genotype A and 415 for genotype D Statistically significant difference was assessed by Chi-squared Test based on a 2 × 2 contingency table P-value in italic are statistically significant Abbreviations: ADV adefovir, ETV entecavir, IQR interquartile range, LAM lamivudine, LdT telbivudine, TDF tenofovir significantly higher in drug-exposed patients with 24.2%, P = 0.02). This increase was also observed in drug-resistant strains than in drug-naïve patients Eastern compared to Western/Northern Europe, although (29.5%[92/312] vs 21.2%[52/245], P = 0.032). No associ- not statistically significant (37.5% vs 24.2%, P = 0.17) (Fig. 3). ation was observed for genotype A (14.9%[38/255] vs By multivariable-analysis, factors independently associ- 11.3% [8/71], P =0.56). ated with higher selection of ≥ 1 immune-associated escape Our results also showed that the distribution of mutation was genotype-D (OR[95% CI]:2.20[1.32– immune-associated escape mutations differed between 3.67], P = 0.002) and age (OR[95% CI]:1.02(1.00–1.03), European regions (Fig. 3). Indeed, the percentage of HBV P = 0.013) (Table 2). A trend between the presence of ≥ 1 genotype-D infected patients with ≥ 1 immune-associated immune-associated escape mutations and higher levels escape mutation was significantly higher in Southern of serum HBV-DNA was also observed (OR[95% Europe than in Western/Northern Europe (36.7% vs CI]:1.10[0.99–1.23], P = 0.079) (Table 2). Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 6 of 12 Fig. 1 The histograms report the percentage of patients with at least one: a immune-associated escape mutation; b NA-induced immune-escape mutation; c stop-codon. The analyses included a total of 828 chronically HBV-infected patients: 573 infected with HBV genotype-D and 255 with HBV genotype-A. Statistically significant differences were assessed by Chi Square Test based on a 2 × 2 contingency table. **: 0.001; ***: P < 0.001. Immune-associated escape mutations (sQ101K, sT114R, sP120S/T/A, sT123A/N, sT126N/S, sP127L, sA128V, sQ129R/N, sG130N/R, sT131I, sM133I/L/T, sY134L, sC138Y, sC139S, sT140S, sP142S, sD144A/E, sG145A/R, sN146S) were retrieved from literature and known to affect HBsAg recognition by antibodies [2, 13, 14, 39–47]. The NA-induced immune-escape mutations I195M, I196S, and E164D result from drug-resistance mutation M204 V, M204I, and V173 L (Torresi, 2002) Detection of NA-induced immune-escape mutations Due to RT and HBsAg open reading frames overlapping, some drug-resistance mutations in RT can correspond to some NA-induced immune-escape mutations in HBsAg. The prevalence of such mutations (sI195M, sI196S, and sE164D resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) was thus investigated. At least one NA-induced immune-escape mutation was detected in 28.6% (237/828) of patients (Fig. 1b). The proportion of patients with ≥1drug-induced immune-escape mutation did not show statistically significant differences over time and ranged from 38.4% in 1998–2002 to 30.0% in 2009– Notably, HBV genotype-A was associated with a sig- nificantly higher prevalence of NA-induced immune-escape mutations (39.6% vs 23.7%, P < 0.001) (Fig. 1b). This was also confirmed by multivariable-analysis (2.03[1.32–3.10]; P = 0.001), along with LAM use (OR[95% CI]:4.60[1.87–11.31]; P = 0.001) (Table 3). In particular, the vaccine-escape mutational pattern sI195M + sE164D (resulting from rtM204V + rtV173L) was present in 7.1% (18/255) of HBV genotype-A infected patients and in 3.7% (21/573) of HBV genotype-D infected patients (P = 0.03) (Fig. 2b). Detection of stop-codons Stop-codons determine truncated HBsAg production that can be implicated in hepatocarcinogenesis. Stop-codons were observed in 8.5% of patients (9.8%[25/255] for geno- type-A vs 7.9%[45/573] for genotype-D). They occurred at 20 HBsAg-positions, including 172 (corresponding to drug-resistance mutation rtA181T) and 182, both known to increase HBV oncogenic potential (Lee et al., ). Notably, the selection of stop-codons at HBsAg-positions 182 and 199 occurred more frequently in genotype-A than D (4.7%[12/255] vs 1%[6/573], P = 0.001 and 2%[5/255] vs 0%[0/573], P = 0.001, respect- ively) (Fig. 2c). These results were confirmed also when the analysis was focused on LAM-treated patients (182: Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 7 of 12 Fig. 2 The histograms report the prevalence of a immune-associated escape mutations, b NA-induced immune-escape mutations, c stop-codons. The prevalence was calculated in the group of 255 patients infected with HBV genotype-A (yellow bars) and in the group of 573 patients infected with HBV genotype-D (green bars). Statistically significant differences were assessed by Chi Squared Test for independence based on a 2 × 2 contingency table. * P < 0.05; ** P < 0.01; *** P < 0.001. In A) a schematic representation of HBsAg functional domains is also reported: N-terminus HBsAg (encompassing amino acids [aa] 1–7), transmembrane domain 1 (TM1, aa: 8–22), loop protruding inside the virion (23-79aa), transmembrane domain 2 (TM2, aa: 80–98), major hydrophilic region (MHR, aa: 99–169) and transmembrane domain 3 and 4 (TM3/4, aa: 170–226). The MHR contains B cell-epitopes including the a-determinant (aa: 124–147) 4.3%[9/209] vs 1.1%[4/362], P = 0.013; 199: 1.9%[4/209] Discussion vs 0%[0/362], P =0.008). In this largest-to-date European survey of 828 No associations were observed between the presence NA-experienced chronically HBV-infected patients, ≥ 1 of stop-codons and the following variables: patients’ immune-associated escape and NA-induced mutation demographics, serum HBV-DNA at the time of geno- was observed in 22.1 and 28.6% of patients, respectively. typic testing, anti-HBV drugs, geographical origin, year Furthermore, in 8.5% of patients, ≥ 1 stop-codon in of collection, and HBV-genotype. HBsAg was detected. Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 8 of 12 The proportion of patients with ≥ 1 immune-associated escape mutation was stable to around 15% in 1998–2002 and in 2003–2005, and remained > 20% in 2006–2008 and in 2009–2012, suggesting a substantial circulation over time of viral strains with a reduced antigenic potential. By multivariable analysis, the selection of immune-associated escape mutations (including vaccine-escape mutations) was significantly higher in HBV genotype-D than A. HBV genotype-D is known to be more prone to the onset of HBeAg-negative chronic hepatitis characterized by an extensive accumulation of mutations in the pre-core/basal core promoter of HBV-genome in response to a potent host-based selec- tion pressure . It is conceivable that this selective pressure may also favor the generation and selection of Fig. 3 The histogram reports the percentage of patients with at immune-associated escape mutations in HBsAg, further least one immune-associated escape mutations between European exacerbating HBV-escape from immunological-pressure. regions. The prevalence was calculated in HBV genotype-D and -A Only the immune-associated escape mutation G130 N infected patients from Western/Northern (black bars), Southern (grey was detected more frequently in genotype-A than -D. bars), and Eastern Europe (light grey bars). Statistically significant This difference can be explained considering the fact differences were assessed by Chi Squared Test for independence based on a 2 × 2 contingency table. * P =0.02 that the number of nucleotide substitutions necessary to generate G130 N from the wild-type amino acid is lower in genotype-A than -D . This suggests that the Table 2 Factors associated with the presence of at least one immune-associated escape mutation by fitting a uni-multivariable logistic regression model b b Variables Univariate analysis Multivariate analysis crude OR [95% CI] p-value adjusted OR [95% CI] p-value Gender (Female vs. Male ) 1.16 (0.76–1.78) 0.483 1.20 (0.77–1.87) 0.432 Age (per 1 year increase) 1.02 (1.00–1.03) 0.010 1.02 (1.00–1.03) 0.013 HBV-DNA (per 1 log IU/ml increase) 1.03 (0.94–1.14) 0.490 1.10 (0.99–1.23) 0.079 LAM 1.16 (0.65–2.08) 0.616 1.46 (0.71–3.02) 0.307 ADV 1.44 (0.96–2.17) 0.078 1.31 (0.83–2.06) 0.250 ETV 1.28 (0.71–2.31) 0.409 2.04 (0.97–4.29) 0.060 TDF 0.76 (0.31–1.87) 0.547 1.13 (0.43–3.02) 0.803 Geographical origin South 11 West 0.71 (0.43–1.17) 0.175 1.03 (0.55–1.89) 0.937 North 0.55 (0.18–1.62) 0.276 0.72 (0.23–2.28) 0.581 East 0.75 (0.46–1.22) 0.250 1.26 (0.62–2.55) 0.519 Year of collection 1997-2002 11 2003–2005 1.07 (0.42–2.71) 0.892 0.79 (0.28–2.20) 0.651 2006–2008 2.37 (1.11–5.07) 0.026 1.65 (0.67–4.01) 0.273 2009–2012 1.79 (0.84–3.83) 0.134 1.36 (0.50–3.68) 0.547 Genotype (D vs. A ) 2.19 (1.43–3.34) < 0.0001 2.20 (1.32–3.67) 0.002 Reference group The analysis was led on 650 patients for whom type of anti-HBV drugs received was known Among 64 ETV-treated patients, 26 received LMV P-value in italic are statistically significant Abbreviations: ADV adefovir, CI Confidence interval, ETV entecavir, LAM lamivudine, OR Odd ratio, TDF tenofovir Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 9 of 12 Table 3 Factors associated with the presence of at least one drug-induced immune-associated escape mutation by fitting a uni- multivariable logistic regression model Variables Univariate analysis Multivariate analysis crude OR [95% CI] p-value adjusted OR [95% CI] p-value Gender (Female vs. Male ) 0.76 (0.51–1.14) 0.188 0.71 (0.46–1.08) 0.111 Age (per 1 year increase) 1.00 (0.99–1.01) 0.672 1.00 (0.99–1.01) 0.850 HBV-DNA (per 1 log IU/ml increase) 1.06 (0.97–1.15) 0.208 1.04 (0.94–1.15) 0.409 LAM 4.03 (1.97–8.25) < 0.0001 4.60 (1.87–11.31) 0.001 ADV 0.42 (0.27–0.65) < 0.0001 0.53 (0.33–0.86) 0.009 ETV 0.99 (0.57–1.73) 0.981 2.02 (0.95–4.29) 0.068 TDF 1.10 (0.52–2.31) 0.805 1.58 (0.67–3.73) 0.294 Geographical origin South 11 West 1.01 (0.65–1.58) 0.951 0.75 (0.43–1.32) 0.323 North 0.58 (0.21–1.58) 0.287 0.49 (0.17–1.41) 0.184 East 1.79 (1.18–2.71) 0.006 1.22 (0.64–2.33) 0.552 Year of collection 1997-2002 11 2003–2005 0.58 (0.29–1.17) 0.128 0.88 (0.41–1.92) 0.754 2006–2008 0.61 (0.34–1.08) 0.088 1.09 (0.54–2.19) 0.817 2009–2012 0.72 (0.41–1.27) 0.259 0.83 (0.36–1.88) 0.650 Genotype (A vs. D ) 2.15 (1.53–3.02) < 0.0001 2.03 (1.32–3.10) 0.001 Reference group (dummy) P-value in italic are statistically significant Abbreviations: ADV adefovir, CI Confidence interval, ETV entecavir, LAM lamivudine, OR Odd ratio, TDF tenofovir different genetic background of HBV-genotypes can correlated with rtM204V/I, and their co-presence is charac- modulate the generation of immune-associated escape terized by elevated serum HBV-DNA. This is consistent mutations, and consequently HBV-antigenicity. with an in-vitro study showing sP120T ability to rescue Recent studies highlighted the role of HBV-replication impaired by rtM204V/I . immune-associated escape mutations in The ability of immune-associated escape mutations to immunosuppression-driven HBV-reactivation [6–9, 23]. promote the fitness of HBV lamivudine-resistant strains It has been proposed that immune-associated escape can raise the issue on lamivudine-use as prophylaxis in mutations can favor the re-uptake of HBV-replication immunosuppressed-patients, and highlights the import- during the initial weakening of immune-system, particu- ance to use potent anti-HBV drugs in order to prevent larly during rituximab-treatment (known to deplete HBV-reactivation. Since the highly potent anti-HBV B-lymphocytes) . The substantial circulation of drugs will soon become generic, this will also allow to immune-associated escape mutations may thus pose an reduce the cost related to the management of issue in term of increased risk of HBV-reactivation in immunosuppressed-patients at risk of HBV-reactivation. immunosuppressed-patients. This has also implications for those European Coun- Previous in-vitro studies showed that some tries in which lamivudine is still prescribed, again sup- immune-associated escape mutations can promote the fit- porting the role of potent anti-HBV drugs for a proper ness of HBV lamivudine-resistant strains [23, 24]. We found management of patients with chronic HBV-infection. an enrichment of immune-associated escape mutations in The circulation of immune-associated escape muta- drug-exposed patients with drug-resistant strains compared tions can have important implications, since they can to drug-exposed patients with wild-type virus and to potentially affect the efficacy of the current vaccination drug-naïve patients. This highlights a strict relationship be- strategy. Indeed, several studies have highlighted the tween drug-resistance and immune-associated escape mu- presence of immune-associated escape mutations in in- tations, and suggests the ability of immune-associated dividuals who contracted HBV-infection despite com- escape mutations to stabilize drug-resistance mutations in pleted HBV-vaccination [25–27]. In a study led in viral-quasispecies. We observed that sP120T significantly Taiwan, a positive HBV-DNA was detected in 10 of 60 Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 10 of 12 individuals in which the HBsAg or anti-hepatitis B core HBsAg-positions including 172 and 182, known to pro- (HBc) was either positive or equivocal despite vaccin- mote the carcinogenic transformation of hepatocytes ation . Among them, 8 have received 3 doses of vac- [38, 39]. Notably, stop-codon at HBsAg-position 172 de- cine. Five out of 8 vaccinees harbored HBsAg mutations: rives from the drug-resistance mutation rtA181T selected 4 with immune-associated escape mutations, and 1 with under ADV- and (in some cases) LAM-treatment . a stop-codon in HBsAg . This represents an important issue probably originating Immune-escape mutations can also play a relevant role from the broad use of first-generation drugs which may in the setting of mother-to-child transmission. Currently, have fuelled the circulation of viral strains with an HBV-vaccine (in addition to immunoglobulins) is ad- increased oncogenic potential. ministered to children born to HBV-infected mothers. In a recent study, serum HBV-DNA was detected in 28% Conclusions children born from HBsAg-positive mothers, and fully “Immune-escape mutations and stop-codons develop in responded to HBV-vaccination. Among them, 62% in- a large proportion of NA-exposed patients in Europe. fected children had ≥ 1 immune-associated escape muta- These mutant isolates may potentially transmit in gen- tion, suggesting the maternal transmission of viral eral population, including vaccinated individuals, and strains with enhanced capability to evade neutralizing fuel drug-resistance emergence”. antibodies in vaccinated-children . In chronic HBV-infection, recent studies highlighted Additional file that the presence of immune-associated escape muta- tions at baseline was negatively correlated with Additional file 1: Table S1. Demographic and virological characteristics of HBV genotype-D drug-naïve patients. (DOCX 12 kb) HBsAg-loss during treatment with potent anti-HBV drugs [29, 30]. It is conceivable that the circulation of Abbreviations these mutations can hamper the full immune control of ADV: Adefovir dipivoxil; ALT: Alanine aminotransferase; CHB: HBV-infected the virus despite potent anti-HBV therapy. This issue patients; ESAR: European Society for translational antiviral research; should be considered by the recent therapeutic strategies ETV: Entecavir; HBc: Hepatitis B core; HbeAg: Hepatitis B e antigen; HbsAg: HBV surface antigen; HBV: Hepatitis B virus; HCC: Hepatocellular aimed at achieving HBV-cure. carcinoma; IQR: Interquartile range; LAM: Lamivudine; LdT: telbivudine; Finally, different studies showed that some NAs: Nucleos(t)ide analogues; OR: Odds ratio; RT: Reverse transcriptase; immune-associated escape mutations can affect TAF: Tenofovir-alafenamide; TDF: Tenofovir HBsAg-quantification by altering HBsAg-binding to Acknowledgments antibodies used in diagnostic assays [6, 31, 32]. We thank Massimiliano Bruni for data management. Antoinet van Kessel of HBsAg-amount is used to provide a more precise defin- ESAR for coordination efforts. ition of the inactive carrier status and to monitor the ef- Funding ficacy of interferon-treatment. The presence of This work was supported by the FIRB project (RBAP11YS7K_001), by the immune-associated escape mutations may cause an Italian Ministry of Instruction, University and Research (Progetto Bandiera PB05), and the Aviralia Foundation. underestimation of HBsAg-levels thus hampering the proper management of chronically HBV-infected Availability of data and materials patients. Data are available upon request. Due to the peculiar HBV-genome organization, Authors’ contributions drug-resistance mutations rtM204 V, rtM204I, and LC, VS, CFP, CAB and AMW were involved in design of the study and rtV173 L correspond to the NA-induced immune-escape supervised the overall study. LC, LEH, RS, and DDC were involved in the analyses of the study. LC and VS wrote the manuscript. LC, LE, RS, DDC,SDS, MA, mutations sI195M, sI196S, and sE164D. In our study, ZBA, GB, BB, NC, CSD, TD, FG, RK, SK, HK, IL, MML, SM, VM, OM, SP, DP, MP, EPS, HBV genotype-A was associated with a significantly FS, MS, KSJ, NT, PT, JV, AV, SZL, NW, TY, CABB, AMJW, CFP and VS provided higher prevalence of NA-induced immune-escape muta- clinical and virological data, reviewed and approved the final manuscript. tions. This is in line with previous studies showing that Ethics approval and consent to participate genotype-A is more prone to develop rtM204V than Approval was provided by: genotype-D at lamivudine failure [32–34]. The issue of NA-induced escape mutations is critical considering the – Ethic Committee of Sheba Medical Center in Israel, ongoing use of lamivudine in some European regions – Ethikkommission of the Medical University, Vienna, Austria. – Ethical Committee of the Medical School National and Kapodistrian where genotype-A is predominant [18, 35]. University of Athens, Athens, Greece. Finally, ≥ 1 stop-codon was detected in 8.5% of pa- – Ethic Committee of the Faculty of Medicine, University of Belgrade, tients. Stop-codons can determine the accumulation of Belgrade, Serbia. – Comité de Etica de Investigacion de la Provincia de Granada, truncated HBsAg in the endoplasmic-reticulum, thus in- Granada, Spain. ducing oxidative stress and in turn enhancing hepato- – Director for Research of University Clinical Center, Bosnia cytes proliferation [36, 37]. They were detected at 20 Erzegovina. Colagrossi et al. BMC Infectious Diseases (2018) 18:251 Page 11 of 12 In accordance with National Guidelines and/or legislature, approval by Ethic Statistics, Faculty of Medicine, National and Kapodistrian University of Athens, Committee was not necessary since the study was based on a retrospective Athens, Greece. Department for Virology, Medical University of Vienna, analysis of anonymized viral sequences obtained for clinical routine practice Vienna, Austria. Liver Disease Centre, Sheba Medical Centre, Ramat Gan, for the following centers: Italy (as outlined in art. 6 and art. 9 from the Israel. Department of Virology, Norwegian Institute of Public Health, Oslo, legislative decree 211/2003), The Netherlands (as outlined in 7:467 of the Norway. Institute of Microbiology, Polyclinic for Laboratory Diagnostics, Dutch Civil Code [WMO] and 7:457 [WGBO]), Denmark (as outlined in the Act University Clinical Centre Tuzla, Tuzla, Bosnia and Herzegovina. Virology on Research Ethics Review of Health Research Projects), Turkey (as outlined Laboratory, Centre Hospitalier Régional et Université “Victor Segalen”, in art. 2 from Regulations on Clinical Research, Official Gazette, Number Bordeaux, France. Institute of Virology, University-Hospital, University 28617 at Apr 13, 2013), Luxembourg (as outlined in art. 25 of 28th august Duisburg-Essen, Essen, Germany. University of Zagreb School of Medicine 1998 law on hospitals establishments), Poland (as outlined in the Polish Act and University Hospital for Infectious Diseases, Zagreb, Croatia. Department from on December 5, 1996, on “physician professions and dentists” and in of Infectious Diseases, Copenhagen University Hospital, Hvidovre, art. 37.1 Act of September 6, 2001 with changes on April 20, 2004 Copenhagen, Denmark. Refik Saydam National Public Health Agency, “Pharmaceutical Law”), Norway (as outlined in the Regional comittees of Ankara, Turkey. medical and health research ethics in Norway, reference nr 2012/896), France (as outlined in French Public Health Law CSP Art. L 1121–1.1), Rumenia (as Received: 4 August 2017 Accepted: 23 May 2018 outlined in leg. 46/2003), Slovenia (as outlined in 26 Direktive 98/44/EC). For Croatia, specific national guidelines/legislatives on this issue are currently not available, thus approval was deemed unnecessary according the Internal References Regulation of the University Hospital for Infectious Diseases, in Zagreb. For 1. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of Germany, at the time of sequences submission, there was no specific worldwide prevalence of chronic hepatitis B virus infection: a systematic requirement concerning retrospective studies, legislations covered only review of data published between 1965 and 2013. Lancet. 2015;386: clinical trials involving drugs or medical device. Part of the samples derived 1546–55. from our RESINA-cohort which is approved by the ethics “Cologne-16-460”. 2. Tong S, Revill P. Overview of hepatitis B viral replication and genetic variability. J Hepatol. 2016;64(Suppl):S4–S16. Competing interests 3. Geno2pheno [hbv]. http://hbv.bioinf.mpi-inf.mpg.de/index.php. 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