L. Carioti

Hepatitis B surface antigen genetic elements critical for immune escape correlate with hepatitis B virus reactivation upon immunosuppression

Hepatitis B virus (HBV) reactivation during immunosuppression can lead to severe acute hepatitis, fulminant liver failure, and death. Here, we investigated hepatitis B surface antigen (HBsAg) genetic features underlying this phenomenon by analyzing 93 patients: 29 developing HBV reactivation and 64 consecutive patients with chronic HBV infection (as control). HBsAg genetic diversity was analyzed by population‐based and ultradeep sequencing (UDS). Before HBV reactivation, 51.7% of patients were isolated hepatitis B core antibody (anti‐HBc) positive, 31.0% inactive carriers, 6.9% anti‐HBc/anti‐HBs (hepatitis B surface antibody) positive, 6.9% isolated anti‐HBs positive, and 3.4% had an overt HBV infection. Of HBV‐reactivated patients, 51.7% were treated with rituximab, 34.5% with different chemotherapeutics, and 13.8% with corticosteroids only for inflammatory diseases. In total, 75.9% of HBV‐reactivated patients (vs. 3.1% of control patients; P < 0.001) carried HBsAg mutations localized in immune‐active HBsAg regions. Of the 13 HBsAg mutations found in these patients, 8 of 13 (M103I‐L109I‐T118K‐P120A‐Y134H‐S143L‐D144E‐S171F) reside in a major hydrophilic loop (target of neutralizing antibodies [Abs]); some of them are already known to hamper HBsAg recognition by humoral response. The remaining five (C48G‐V96A‐L175S‐G185E‐V190A) are localized in class I/II–restricted T‐cell epitopes, suggesting a role in HBV escape from T‐cell‐mediated responses. By UDS, these mutations occurred in HBV‐reactivated patients with a median intrapatient prevalence of 73.3% (range, 27.6%‐100%) supporting their fixation in the viral population as a predominant species. In control patients carrying such mutations, their median intrapatient prevalence was 4.6% (range, 2.5%‐11.3%; P < 0.001). Finally, additional N‐linked glycosylation (NLG) sites within the major hydrophilic loop were found in 24.1% of HBV‐reactivated patients (vs. 0% of chronic patients; P < 0.001); 5 of 7 patients carrying these sites remained HBsAg negative despite HBV reactivation. NLG can mask immunogenic epitopes, abrogating HBsAg recognition by Abs. Conclusion: HBV reactivation occurs in a wide variety of clinical settings requiring immune‐suppressive therapy, and correlates with HBsAg mutations endowed with enhanced capability to evade immune response. This highlights the need for careful patient monitoring in all immunosuppressive settings at reactivation risk and of establishing a prompt therapy to prevent HBV‐related clinical complications. (Hepatology 2015;61:823–833)

Multiple Hepatitis B Virus (HBV) Quasispecies and Immune-Escape Mutations Are Present in HBV Surface Antigen and Reverse Transcriptase of Patients With Acute Hepatitis B

BACKGROUND This study characterizes and defines the clinical value of hepatitis B virus (HBV) quasispecies with reverse transcriptase and HBV surface antigen (HBsAg) heterogeneity in patients with acute HBV infection. METHODS Sixty-two patients with acute HBV infection (44 with genotype D infection and 18 with genotype A infection) were enrolled from 2000 to 2010. Plasma samples obtained at the time of the first examination were analyzed by ultradeep pyrosequencing. The extent of HBsAg amino acid variability was measured by Shannon entropy. RESULTS Median alanine aminotransferase and serum HBV DNA levels were 2544 U/L (interquartile range, 1938-3078 U/L) and 5.88 log10 IU/mL (interquartile range, 4.47-7.37 log10 IU/mL), respectively. Although most patients serologically resolved acute HBV infection, only 54.1% developed antibody to HBsAg (anti-HBs). A viral population with ≥1 immune-escape mutation was found in 53.2% of patients (intrapatient prevalence range, 0.16%-100%). Notably, by Shannon entropy, higher genetic variability at HBsAg amino acid positions 130, 133, and 157 significantly correlated with no production of anti-HBs in individuals infected with genotype D (P < .05). Stop codons were detected in 19.3% of patients (intrapatient prevalence range, 1.6%-47.5%) and occurred at 11 HBsAg amino acid positions, including 172 and 182, which are known to increase the oncogenic potential of HBV.Finally, ≥1 drug resistance mutation was detected in 8.1% of patients (intrapatient prevalence range, 0.11%-47.5% for primary mutations and 10.5%-99.9% for compensatory mutations). CONCLUSIONS Acute HBV infection is characterized by complex array of viral quasispecies with reduced antigenicity/immunogenicity and enhanced oncogenic potential. These viral variants may induce difficult-to-treat HBV forms; favor HBV reactivation upon iatrogenic immunosuppression, even years after infection; and potentially affect the efficacy of the current HBV vaccination strategy.

Ultradeep sequencing detection of the R263K integrase inhibitor drug resistance mutation

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Kinetics of hepatitis C virus RNA decay, quasispecies evolution and risk of virological failure during telaprevir-based triple therapy in clinical practice

BACKGROUND The used first generation protease inhibitors may be hampered by virological failure in partially interferon-sensitive patients. AIM To investigate early hepatitis C virus (HCV)-RNA decay and quasispecies modifications, and disclose viral dynamics underlying failure. METHODS Viraemia decay at early time-points during telaprevir treatment was modelled according to Neumann et al. (1998). NS3-sequences were obtained by population-sequencing and ultradeep-454-pyrosequencing. RESULTS 13 treatment-experienced (8 non-responders, 5 relapsers), and two cirrhotic naïve patients, received telaprevir+pegylated-interferon-α+ribavirin. Viraemia decay was biphasic. In all patients, first-phase was rapid and consistent, with a median [interquartile-range] viraemia decay of 2.8 [2.6-3.2]logIU/ml within 48h. Second-phase decay was slower, especially in failing patients: 3/3 showed <1logIU/ml decay between 48h and 2 weeks, and HCV-RNA >100IU/ml at week 2. Only one patient experiencing sustained viral response showed similar kinetics. By pyrosequencing, mutational freeze was observed in all 15 patients within the first 24h, but only in patients with sustained response afterwards. Indeed, 2/2 failing patients showed early resistance, as minor (V36A-T54A: prevalence <26% at 48h) or major (V36M/A-R155K: prevalence, 99.8% at week 2) variants. CONCLUSIONS Following telaprevir administration, first-phase HCV-RNA decay is consistent with mutational freeze and limited/no viral replication, while second-phase is significantly slower in failing patients (with appearance of resistance), suggesting the usefulness of early HCV-RNA monitoring.

Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage

ABSTRACT Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e−7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, −40.1 kcal/mol; G24E, −510 kcal/mol; E25K, −522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression.

Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro

Background An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation. Methods This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry. Results Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001). Conclusions Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.

Dynamics and phylogenetic relationships of HIV-1 transmitted drug resistance according to subtype in Italy over the years 2000–14

Background Transmitted drug-resistance (TDR) remains a critical aspect for the management of HIV-1-infected individuals. Thus, studying the dynamics of TDR is crucial to optimize HIV care. Methods In total, 4323 HIV-1 protease/reverse-transcriptase sequences from drug-naive individuals diagnosed in north and central Italy between 2000 and 2014 were analysed. TDR was evaluated over time. Maximum-likelihood and Bayesian phylogenetic trees with bootstrap and Bayesian-probability supports defined transmission clusters. Results Most individuals were males (80.2%) and Italian (72.1%), with a median (IQR) age of 37 (30-45) years. MSM accounted for 42.2% of cases, followed by heterosexuals (36.4%). Non-B subtype infections accounted for 30.8% of the overall population and increased over time (<2005-14: 19.5%-38.5%, P < 0.0001), particularly among Italians (<2005-14: 6.5%-28.8%, P < 0.0001). TDR prevalence was 8.8% and increased over time in non-B subtypes (<2005-14: 2%-7.1%, P = 0.018). Overall, 467 transmission clusters (involving 1207 individuals; 27.9%) were identified. The prevalence of individuals grouping in transmission clusters increased over time in both B (<2005-14: 12.9%-33.5%, P = 0.001) and non-B subtypes (<2005-14: 18.4%-41.9%, P = 0.006). TDR transmission clusters were 13.3% within the overall cluster observed and dramatically increased in recent years (<2005-14: 14.3%-35.5%, P = 0.005). This recent increase was mainly due to non-B subtype-infected individuals, who were also more frequently involved in large transmission clusters than those infected with a B subtype [median number of individuals in transmission clusters: 7 (IQR 6-19) versus 4 (3-4), P = 0.047]. Conclusions The epidemiology of HIV transmission changed greatly over time; the increasing number of transmission clusters (sometimes with drug resistance) shows that detection and proper treatment of the multi-transmitters is a major target for controlling HIV spread.

A recent epidemiological cluster of acute hepatitis B genotype F1b infection in a restricted geographical area of Italy

In this study, by phylogenetic analysis, we identified an epidemiological cluster involving eight individuals diagnosed with acute hepatitis B virus (HBV) infection related to unprotected sexual intercourse in a restricted area of central Italy (time period: 2011-2014). Notably, these patients (six of eight Italians) were infected by subgenotype F1b, which is not commonly found in western countries. Ultra-deep pyrosequencing confirmed a superimposable composition of HBV quasi-species in these patients. Despite the availability of effective vaccination, this study highlights the importance of not underestimating the risk of HBV infection, of continuing to set up surveillance programmes for HBV infection, and of investigating the pathogenetic potential of these atypical genotypes.

HDV Can Constrain HBV Genetic Evolution in HBsAg: Implications for the Identification of Innovative Pharmacological Targets

Chronic HBV + HDV infection is associated with greater risk of liver fibrosis, earlier hepatic decompensation, and liver cirrhosis hepatocellular carcinoma compared to HBV mono-infection. However, to-date no direct anti-HDV drugs are available in clinical practice. Here, we identified conserved and variable regions in HBsAg and HDAg domains in HBV + HDV infection, a critical finding for the design of innovative therapeutic agents. The extent of amino-acid variability was measured by Shannon-Entropy (Sn) in HBsAg genotype-d sequences from 31 HBV + HDV infected and 62 HBV mono-infected patients (comparable for demographics and virological-parameters), and in 47 HDAg genotype-1 sequences. Positions with Sn = 0 were defined as conserved. The percentage of conserved HBsAg-positions was significantly higher in HBV + HDV infection than HBV mono-infection (p = 0.001). Results were confirmed after stratification for HBeAg-status and patients’ age. A Sn = 0 at specific positions in the C-terminus HBsAg were correlated with higher HDV-RNA, suggesting that conservation of these positions can preserve HDV-fitness. Conversely, HDAg was characterized by a lower percentage of conserved-residues than HBsAg (p < 0.001), indicating higher functional plasticity. Furthermore, specific HDAg-mutations were significantly correlated with higher HDV-RNA, suggesting a role in conferring HDV replicative-advantage. Among HDAg-domains, only the virus-assembly signal exhibited a high genetic conservation (75% of conserved-residues). In conclusion, HDV can constrain HBsAg genetic evolution to preserve its fitness. The identification of conserved regions in HDAg poses the basis for designing innovative targets against HDV-infection.

Occupational HIV infection in a research laboratory with unknown mode of transmission: A case report

A laboratory worker was infected with human immunodeficiency virus (HIV) type 1 in a biosafety level 2 containment facility, without any apparent breach. Through full-genome sequencing and phylogenetic analyses, we could identify the source of infection in a replication-competent clone that unknowingly contaminated a safe experiment. Mode of transmission remains unclear. Caution is warranted when handling HIV-derived constructs.