R. Salpini

HCV Genotypes Are Differently Prone to the Development of Resistance to Linear and Macrocyclic Protease Inhibitors

Background Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed whether specific HCV-genotypes are differently prone to develop resistance to linear and macrocyclic protease-inhibitors (PIs). Methods The study includes 1568 NS3-protease sequences, isolated from PI-naive patients infected with HCV-genotypes 1a (N = 621), 1b (N = 474), 2 (N = 72), 3 (N = 268), 4 (N = 54) 5 (N = 6), and 6 (N = 73). Genetic-barrier was calculated as the sum of nucleotide-transitions (score = 1) and/or nucleotide-transversions (score = 2.5) required for drug-resistance-mutations emergence. Forty-three mutations associated with PIs-resistance were analyzed (36A/M/L/G-41R-43S/V-54A/S/V-55A-Q80K/R/L/H/G-109K-138T-155K/Q/T/I/M/S/G/L-156T/V/G/S-158I-168A/H/T/V/E/I/G/N/Y-170A/T-175L). Structural analyses on NS3-protease and on putative RNA-models have been also performed. Results Overall, NS3-protease was moderately conserved, with 85/181 (47.0%) amino-acids showing <1% variability. The catalytic-triad (H57-D81-S139) and 6/13 resistance-associated positions (Q41-F43-R109-R155-A156-V158) were fully conserved (variability <1%). Structural-analysis highlighted that most of the NS3-residues involved in drug-stabilization were highly conserved, while 7 PI-resistance residues, together with selected residues located in proximity of the PI-binding pocket, were highly variable among HCV-genotypes. Four resistance-mutations (80K/G-36L-175L) were found as natural polymorphisms in selected genotypes (80K present in 41.6% HCV-1a, 100% of HCV-5 and 20.6% HCV-6; 80G present in 94.4% HCV-2; 36L present in 100% HCV-3-5 and >94% HCV-2-4; 175L present in 100% HCV-1a-3-5 and >97% HCV-2-4). Furthermore, HCV-3 specifically showed non-conservative polymorphisms (R123T-D168Q) at two drug-interacting positions. Regardless of HCV-genotype, 13 PIs resistance-mutations were associated with low genetic-barrier, requiring only 1 nucleotide-substitution (41R-43S/V-54A-55A-80R-156V/T: score = 1; 54S-138T-156S/G-168E/H: score = 2.5). By contrast, by using HCV-1b as reference genotype, nucleotide-heterogeneity led to a lower genetic-barrier for the development of some drug-resistance-mutations in HCV-1a (36M-155G/I/K/M/S/T-170T), HCV-2 (36M-80K-155G/I/K/S/T-170T), HCV-3 (155G/I/K/M/S/T-170T), HCV-4-6 (155I/S/L), and HCV-5 (80G-155G/I/K/M/S/T). Conclusions The high degree of HCV genetic variability makes HCV-genotypes, and even subtypes, differently prone to the development of PIs resistance-mutations. Overall, this can account for different responsiveness of HCV-genotypes to PIs, with important clinical implications in tailoring individualized and appropriate regimens.

Novel HBsAg markers tightly correlate with occult HBV infection and strongly affect HBsAg detection

Occult HBV infection (OBI) is a threat for the safety of blood-supply, and has been associated with the onset of HBV-related hepatocellular carcinoma and lymphomagenesis. Nevertheless, genetic markers in HBsAg (particularly in D-genotype, the most common in Europe) significantly associated with OBI in vivo are missing. Thus, the goal of this study is to define: (i) prevalence and clinical profile of OBI among blood-donors; (ii) HBsAg-mutations associated with OBI; (iii) their impact on HBsAg-detection. OBI was searched among 422,278 blood-donors screened by Nucleic-Acid-Testing. Following Taormina-OBI-definition, 26 (0.006%) OBI-patients were identified. Despite viremia <50IU/ml, HBsAg-sequences were obtained for 25/26 patients (24/25 genotype-D). OBI-associated mutations were identified by comparing OBI-HBsAg with that of 82 chronically-infected (genotype-D) patients as control. Twenty HBsAg-mutations significantly correlated for the first time with OBI. By structural analysis, they localized in the major HBV B-cell-epitope, and in HBsAg-capsid interaction region. 14/24 OBI-patients (58.8%) carried in median 3 such mutations (IQR:2.0-6.0) against 0 in chronically-infected patients. By co-variation analysis, correlations were observed for R122P+S167L (phi=0.68, P=0.01), T116N+S143L (phi=0.53, P=0.03), and Y100S+S143L (phi=0.67, p<0.001). Mutants (obtained by site-directed mutagenesis) carrying T116N, T116N+S143L, R122P, R122P+Q101R, or R122P+S167L strongly decreased HBsAg-reactivity (54.9±22.6S/CO, 31.2±12.0S/CO, 6.1±2.4S/CO, 3.0±1.0S/CO and 3.9±1.3S/CO, respectively) compared to wild-type (306.8±64.1S/CO). Even more, Y100S and Y100S+S143L supernatants show no detectable-HBsAg (experiments in quadruplicate). In conclusions, unique HBsAg-mutations in genotype-D, different than those described in genotypes B/C (rarely found in western countries), tightly correlate with OBI, and strongly affect HBsAg-detection. By altering HBV-antigenicity and/or viral-particle maturation, they may affect full-reliability of universal diagnostic-assays for HBsAg-detection.

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)

The profile of mutational clusters associated with lamivudine resistance can be constrained by HBV genotypes

BACKGROUND/AIMS To investigate the different clusters of mutations associated with lamivudine resistance in HBV genotypes D and A. METHODS HBV reverse transcriptase sequences of 89 HBV-infected patients failing lamivudine treatment were analyzed. The association of mutations with HBV genotypes was assessed by Chi-Squared test and multivariate logistic regression analysis. Covariate analysis was based on hierarchical clustering. RESULTS In genotype A, the rtM204V (prevalence: 68.2%) was the main sign of lamivudine failure. Multivariate analysis confirmed that genotype A is the only predictor for rtM204V emergence (OR: 14.5 [95% CI: 1.3-158], P=0.02). Covariate analysis showed that rtM204V clusters with rtL180M, rtL229V (corresponding to sF220L in the HBsAg), and, interestingly, with HBsAg mutation sS207N (bootstrap=0.95). Both sF220L and sS207N co-localized in the fourth transmembrane HBsAg domain. In contrast, in genotype D the primary mutations rtM204V and rtM204I occurred with similar prevalence (39.1% versus 45.3%, P=0.47), and showed a distinct pattern of compensatory mutations. rtM204V clusters with mutations localized in the RT-B domain (rtV173L, rtL180M, and rtT184A/S) (bootstrap=0.94), while rtM204I clusters with mutations localized in the RT-A domain (rtS53N, rtT54Y, and rtL80I/V) (bootstrap=0.96) (without associations with HBsAg specific mutations). CONCLUSIONS HBV genotype plays an important role in driving RT evolution under lamivudine treatment, and thus can be relevant for therapeutic sequencing, immunological response and disease progression.

Role of hepatitis B virus genetic barrier in drug-resistance and immune-escape development

BACKGROUND Impact of hepatitis B virus genetic barrier, defined as the number and type of nucleotide substitutions required to overcome drug/immune selective pressure, on drug-resistance/immune-escape development is unknown. METHODS Genetic barrier was calculated according to Van de Vijver (2006) in 3482 hepatitis B virus-reverse transcriptase/HBV surface antigen sequences from 555 drug-naïve patients and 2927 antiviral-treated patients infected with hepatitis B virus genotypes A-G. RESULTS Despite high natural variability, genetic barrier for drug-resistance development is identical amongst hepatitis B virus genotypes, but varies according to drug-resistance mutation type. Highest genetic barrier is found for secondary/compensatory mutations (e.g. rtL80I/V-rtL180M-rtV173L), whilst most primary mutations (including rtM204V-rtA181T/V-rtI169T-rtA194T) are associated with low genetic barrier. An exception is rtM204I, which can derive from a transition or a transversion. Genotypes A and G are more prone to develop immune/diagnostic-escape mutations sT114R and sG130N. Vaccine-escape associated sT131N-mutation is a natural polymorphism in both A and G genotypes. CONCLUSION Genetic barrier and reverse transcriptase/HBV surface antigen overlapping can synergistically influence hepatitis B virus drug-resistance/immune-escape development. The different immune-escape potential of specific hepatitis B virus genotypes could have important clinical consequences in terms of disease progression, vaccine strategies and correct HBV surface antigen detection.

Drug resistance among drug-naive and first-line antiretroviral treatment-failing children in Cameroon

Background: Scale-up to antiretroviral therapy (ART) requires surveillance for HIV drug resistance. With the goal of attaining 100% pediatric ART coverage in Cameroon, strategies to limit the spread of HIV resistance among children are very important. Methods: From June 2009 through February 2011, 92 HIV-1-infected children (41 ART-naive, 51 failing first-line ART) living in Yaoundé, Cameroon, were enrolled; HIV-1 Prot-RT genotypic resistance testing (GRT) was performed using an inhouse assay. Among 40 children failing first-line ART, treatment response was evaluated at weeks 24 and 48 after treatment was changed, based on GRT results. Results: The mean age was 72 months both for children who were drug-naive and those failing ART (range: 3–144 and 12–144, respectively), with a mean viremia of 5.59 log and 4.71 log RNA copies/mL, a median CD4 of 17% (588 cells/&mgr;L) and 23% (719 cells/&mgr;L), respectively. Median time-to-treatment failure was 610 days. A prevalence of 4.9% and 90% drug resistance was observed, respectively, among children who were drug-naive and those failing first-line ART, with circulating recombinant form CRF02_AG as the most prevalent clade (58.6% and 62%, respectively). After a change to GRT-based treatment, more than 90% of children had viremia <3 log RNA copies/mL at week 24 and confirmed at week 48, with 70% achieving undetectable viremia, although without correlation to immune response; 97.5% had switched to lopinavir/ritonavir-containing regimens. Conclusion: HIV-1 drug resistance was low among ART-naive children and very high among those failing first-line ART. Treatment change based on GRT was successful for most children, with lopinavir/ritonavir regimens being very promising for second-line use.

Overlapping structure of hepatitis B virus (HBV) genome and immune selection pressure are critical forces modulating HBV evolution.

How the overlap between the hepatitis B virus (HBV) reverse transcriptase (RT) and HBV S antigen (HBsAg) genes modulates the extent of HBV genetic variability is still an open question, and was investigated here. The rate of nucleotide conservation (≤1% variability) followed an atypical pattern in the RT gene, due to an overlap between RT and HBsAg (69.9% nucleotide conservation in the overlapping region vs 41.2% in the non-overlapping region; P<0.001), with a consequently lower rate of synonymous substitution within the overlapping region [median(interquartile range)dS=3.1(1.5-7.4) vs 20.1(10.6-30.0); P=3.249×10(-22)]. The most conserved RT regions were located within the YMDD motif and the N-terminal parts of the palm and finger domains, critical for RT functionality. These regions also corresponded to highly conserved HBsAg domains that are critical for HBsAg secretion. Conversely, the genomic region encoding the HBsAg antigenic loop (where immune-escape mutations are localized) showed a sharp decrease in the extent of conservation (40.6%), which was less pronounced in the setting of human immunodeficiency virus (HIV)-driven immune suppression (48.8% in HIV-HBV co-infection vs 21.5% in mono-infected patients; P=0.020). In conclusion, the overlapping reading frame and the immune system appear to have shaped the patterns of RT and HBsAg genetic variability. Highly conserved regions in RT and HBsAg may deserve further attention as novel therapeutic targets.

Late hepatitis B virus reactivation after lamivudine prophylaxis interruption in an anti-HBs-positive and anti-HBc-negative patient treated with rituximab-containing therapy

We describe a case of an anti-HBs-positive patient who experienced hepatitis B reactivation 18 months after the discontinuation of rituximab and after 12 months of lamivudine prophylaxis. The patient carried a hepatitis B genotype D virus harbouring a single immune escape mutation, sT118K. No consensus guidelines regarding the optimal length of treatment or the best elective drug have been defined for antiviral prophylaxis for HBsAg-negative, anti-HBc- and/or anti-HBs-positive patients undergoing immunosuppressive treatment. Screening based on HBV serological markers and HBV DNA testing is a critical issue to recognise hepatitis B reactivation as early as possible. Furthermore, it is of outstanding importance to identify alternative markers (e.g. cccDNA, HBV core related antigen, etc.), that could be predictive of HBV reactivation.

Performance evaluation of an in-house human immunodeficiency virus type-1 protease-reverse transcriptase genotyping assay in Cameroon

Most commercial HIV-1 genotyping assays are hampered by high cost in resource-limited settings. Moreover, their performance might be influenced over time by HIV genetic heterogeneity and evolution. An in-house genotyping protocol was developed, and its sequencing performance and reproducibility were compared to that of ViroSeq™. One hundred ninety plasma samples from HIV-1-infected subjects in Cameroon, a resource-limited setting with a high HIV genetic variability, were processed for pol gene sequencing with an in-house protocol, ViroSeq™, or both. Only non-B subtypes were found. The in-house sequencing performance was 98.7% against 92.1% with ViroSeq™. Among 36 sequence pairs obtained using both assays, the overall rate of discordant amino acid positions was negligible (0.24%). With its high sensitivity and reproducibility, as well as its affordable cost (about half of ViroSeq™: 92 € vs. 217 €), this in-house assay is a suitable alternative for HIV-1 genotyping in resource-limited and/or in high-genetic-diversity settings.

Characterization of drug resistance mutations in naïve and ART-treated patients infected with HIV-1 in Yaounde, Cameroon.

Currently the prevalence of HIV‐1 infection in Cameroon is 5.1%, CRF02_AG subtype is responsible for about 50% of infections. Since an HIV‐1 drug resistance test is not yet available widely, accurate data on the prevalence of resistant viral strains are missing. The objective of this study was to determine HIV‐1 genetic diversity and to characterize HIV‐1 mutations conferring drug resistance among antiretroviral therapy (ART)‐naïve and ART‐treated patients. A cohort of 239 patients infected with HIV were followed‐up between January 2007 and July 2010 in Cameroon. Two hundred and sixteen plasma samples were sequenced for phylogenetic analysis and identification of drug resistance mutations in the HIV‐1 pol region. A significant genetic diversity was found: Seven pure subtypes (A1, A3, D, F1, F2, G, H), nine circulating recombinant forms (CRFs: 01_AE, 02_AG, 06cpx, 09cpx, 11cpx, 13cpx, 16cpx, 18cpx, 37cpx) and one new unique recombinant form (URF) (G/F2). The rate of transmitted drug resistance (TDR) in naïve patients was 8.2% (4/49). Around 80% of patients failing a first‐line ART harbored a virus with at least one resistance mutation to two antiretroviral (ARV) classes, and 36% of those failing a second‐line regimen carried a virus with at least one resistant mutation to three ARV classes. The high level of drug resistance observed in the cohort is alarming because this occurred as a result of only few years of treatment. Adherence to therapy, adequate education of physicians, and the appropriate use of genotypic resistance assay are critical points of intervention for the improvement of patient care. J. Med. Virol. 84:721–727, 2012.