V. Cento

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.

Study of Genotypic and Phenotypic HIV-1 Dynamics of Integrase Mutations During Raltegravir Treatment: A Refined Analysis by Ultra-Deep 454 Pyrosequencing

BACKGROUND The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.

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.

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.

Hepatitis C Virus Genetic Variability and the Presence of NS5B Resistance-Associated Mutations as Natural Polymorphisms in Selected Genotypes Could Affect the Response to NS5B Inhibitors

ABSTRACT Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant nucleoside inhibitors (NIs)/nonnucleoside inhibitors (NNIs). The study included 1,145 NS5B polymerase sequences retrieved from the Los Alamos HCV database and GenBank. The genetic barrier was calculated for drug resistance emergence. Prevalence and genetic barrier were calculated for 1 major NI and 32 NNI resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity toward the diverse HCV genotypes. Overall, NS5B polymerase was moderately conserved among all HCV genotypes, with 313/591 amino acid residues (53.0%) showing ≤1% variability and 83/591 residues (14.0%) showing high variability (≥25.1%). Nine NNI resistance variants (2 major variants, 414L and 423I; 7 minor variants, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV genotype 4 (HCV-4) (n = 14/38, 36.8%) and in all HCV-5 sequences (n = 17, 100%), respectively. Regardless of HCV genotype, the 282T major NI resistance variant and 10 major NNI resistance variants (316Y, 414L, 423I/T/V, 448H, 486V, 495L, 554D, and 559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier score development among the six HCV genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations. The genetic variability among HCV genotypes, particularly with the presence of polymorphisms at NNI resistance positions, could affect their responsiveness to NS5B inhibitors. A pretherapy HCV NS5B sequencing could help to provide patients with the full efficacy of NNI-containing regimens.

Multiclass HCV resistance to direct-acting antiviral failure in real-life patients advocates for tailored second-line therapies

Despite the excellent efficacy of direct‐acting antivirals (DAA) reported in clinical trials, virological failures can occur, often associated with the development of resistance‐associated substitutions (RASs). This study aimed to characterize the presence of clinically relevant RASs to all classes in real‐life DAA failures.

New findings in HCV genotype distribution in selected West European, Russian and Israeli regions

BACKGROUND HCV affects 185 million people worldwide and leads to death and morbidities. HCV has a high genetic diversity and is classified into seven genotypes and 67 subtypes. Novel anti-HCV drugs (Direct-Acting-Antivirals) eligibility, resistance and cure rates depend on HCV geno/subtype (GT). OBJECTIVES Analysis of epidemiological information and viral GT from patients undergoing viral genotyping in 2011-2015. STUDY DESIGN Anonymized information from 52 centers was analyzed retrospectively. RESULTS 37,839 samples were included in the study. We show that the GT distribution is similar throughout Western European countries, with some local differences. Here GTs 1 and 2 prevalences are lower and of GT4 higher than in all previous reports. Israel has a unique GT pattern and in South Russia the GT proportions are more similar to Asia. GTs 5 and 6 were detected in very low proportions. Three cases of the recombinant genotype P were reported in Munich (Germany). In addition, we observed that GT proportion was dependant on patientś gender, age and transmission route: GTs 1b and 2 were significantly more common in female, older, nosocomially-infected patients, while GTs 1a, 3 and 4 were more frequent in male, younger patients infected by tattooing, drug consume, and/or sexual practices. In infections acquired by drug consume, GTs 1a (35.0%) and 3 (28.1%) prevailed. In infections related to sexual practices lower proportion of GT3 (14.0%) and higher of GT4 (20.2%) were detected. GT4 was mostly abundant in MSM (29.6%). HIV coinfection was significantly associated with higher proportions GTs 1a and 4 (42.5% and 19.3%, respectively). CONCLUSION Genotype prevalence evolves and correlates to epidemiological factors. Continuous surveillance is necessary to better assess hepatitis C infection in Europe and to take appropriate actions.

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.

HIV-1 dual/mixed tropic isolates show different genetic and phenotypic characteristics and response to maraviroc in vitro

Dual/mixed-tropic HIV-1 strains are predominant in a significative proportion of patients, though few information is available regarding the genetic characteristics, quasispecies composition, and susceptibility against CCR5-antagonists of the primary-isolates. For this reason, we investigated in deep details, both phenotypically and genotypically, the characteristics of 54 HIV-1 primary-isolates obtained from HIV-infected patients. Tropism was assessed by multiple-cycles phenotypic-assay on U87MG-CD4(+)-CCR5(+)-/CXCR4(+)-expressing cells. In vitro selection in PBMCs of X4-tropic viral strains following maraviroc-treatment was also performed. Phenotypic-assay reported pure R5-tropic viruses in 31 (57.4%) isolates, dual/mixed-tropic viruses in 22 (40.7%), and pure X4-tropic virus in only 1 (1.8%). Among dual/mixed-tropic isolates, 12 showed a remarkably higher replication-efficacy in CCR5-expressing cells (R5(+)/X4), and 2 in CXCR4-expressing cells (R5/X4(+)). Genotypic-tropism testing showed a correlation between PSSM-scores, geno2pheno false-positive-rate, and V3-net-charge with both CCR5-usage and syncytium-inducing ability. Moreover, specific gp120- and gp41-mutations were significantly associated with tropism and/or syncytium-inducing ability. Ultra-deep V3-pyrosequencing showed the presence of a swarm of genetically distinct species with a preference for CCR5-coreceptor not only in all pure R5-isolates, but also in 6/7 R5(+)/X4-tropic isolates. In both pure-X4 and R5/X4(+)-isolates, we observed extensive prevalence of X4-using species. In vitro selection-experiments with CCR5-inhibitor maraviroc (up to 2 months) showed no-emergence of X4-tropic variants for all R5- and R5(+)/X4-isolates tested (while X4-virus remained fully-resistant). In conclusion, our study shows that dual/mixed-tropic viruses are constituted by different species, whereby those with characteristics R5(+)/X4 are genotypically and phenotypically similar to the pure-R5 isolates; thus the use of CCR5-antagonists in patients with R5(+)/X4-tropic viruses may be a therapeutic-option that deserves further investigations.

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.