Fabio Mercurio

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.

Selected amino acid mutations in HIV-1 B subtype gp41 are Associated with Specific gp120V3 signatures in the regulation of Co-Receptor usage

BackgroundThe third variable loop (V3) of the HIV-1 gp120 surface protein is a major determinant of cellular co-receptor binding. However, HIV-1 can also modulate its tropism through other regions in gp120, such as V1, V2 and C4 regions, as well as in the gp41 protein. Moreover, specific changes in gp41 are likely to be responsible for of damage in gp120-CCR5 interactions, resulting in potential resistance to CCR5 inhibitors.In order to genetically characterize the two envelope viral proteins in terms of co-receptor usage, we have analyzed 526 full-length env sequences derived from HIV-1 subtype-B infected individuals, from our and public (Los Alamos) databases. The co-receptor usage was predicted by the analysis of V3 sequences using Geno2Pheno (G2P) algorithm. The binomial correlation phi coefficient was used to assess covariation among gp120V3 and gp41 mutations; subsequently the average linkage hierarchical agglomerative clustering was performed.ResultsAccording to G2P false positive rate (FPR) values, among 526 env-sequences analyzed, we further characterized 196 sequences: 105 with FPR <5% and 91 with FPR >70%, for X4-using and R5-using viruses, respectively.Beyond the classical signatures at 11/25 V3 positions (S11S and E25D, R5-tropic viruses; S11KR and E25KRQ, X4-tropic viruses), other specific V3 and gp41 mutations were found statistically associated with the co-receptor usage. Almost all of these specific gp41 positions are exposed on the surface of the glycoprotein. By the covariation analysis, we found several statistically significant associations between V3 and gp41 mutations, especially in the context of CXCR4 viruses. The topology of the dendrogram showed the existence of a cluster associated with R5-usage involving E25DV3, S11SV3, T22AV3, S129DQgp41 and A96Ngp41 signatures (bootstrap = 0.88). Conversely, a large cluster was found associated with X4-usage involving T8IV3, S11KRV3, F20IVYV3, G24EKRV3, E25KRV3, Q32KRV3, A30Tgp41, A189Sgp41, N195Kgp41 and L210Pgp41 mutations (bootstrap = 0.84).ConclusionsOur results show that gp120V3 and several specific amino acid changes in gp41 are associated together with CXCR4 and/or CCR5 usage. These findings implement previous observations that determinants of tropism may reside outside the V3-loop, even in the gp41. Further studies will be needed to confirm the degree to which these gp41 mutations contribute directly to co-receptor use.

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.

Rapid prediction of sustained virological response in patients chronically infected with HCV by evaluation of RNA decay 48h after the start of treatment with pegylated interferon and ribavirin

The combination of pegylated interferons (PEG-IFNs) and ribavirin represents the standard of care for the treatment of chronic HCV-infected patients, yet with a success rate around 50% in genotypes 1 and 4, high costs and side effects. Therefore, early prediction of sustained virological response (SVR) is a relevant issue for HCV-patients. We evaluated the association between SVR and decline of HCV-RNA at 48h in a prospective cohort of 145 HCV-patients treated with PEG-IFNs and ribavirin (males=69.1%; genotypes 1/4=51.0%; HIV-1 coinfected=6.7%). SVR was obtained in 65.5% of patients, while 16.6% experienced relapse and 17.9% no response. The first-phase of HCV-RNA decline clearly differentiated patients with SVR from relapsers and non-responders, independently of genotype (P<0.001). In univariate and multivariate analyses, different infralogaritmic thresholds of HCV-RNA decay at 48h were tested, observing the highest predictive potential at 0.5log: decays above this threshold showed a 76.2% negative predictive value for SVR, whereas decays >0.5log indicated a 6.8 odds ratio (95% C.I.: 2.0-23.2) for SVR after controlling for genotype, baseline viremia, adherence to therapy and HIV coinfection. Decays beyond the 0.5log threshold were also strongly associated with and highly predictive of early virological response (95.0% positive predictive value, P<0.001).

Genetic and structural analysis of HIV-1 Rev responsive element related to V38A and T18A enfuvirtide resistance mutations.

Background: For the expression of late viral genes, HIV-1 efficiently exploits the nuclear export by using Rev viral protein, which specifically binds the RNA Rev Responsive Element (RRE). This region is contained within the gp120-gp41 encoding sequence. Enfuvirtide is the first approved HIV-1 fusion-inhibitor, and gp41 codons associated with primary enfuvirtide-resistance (amino-acids 36–45) are localized within the RRE structure. We previously found the co-presence of V38A+T18A resistance mutations in patients failing enfuvirtide. Methods: Collecting 476 and 135 HIV-1 B-subtype gp41 sequences from enfuvirtide-naïve and enfuvirtide-treated patients, respectively, two mutations previously found associated with enfuvirtide treatment, T18A and V38A, were analyzed. Moreover, the RNA secondary structure was displayed by CONTRAfold-software and the gp41 evolutionary pathways by a mutagenetic tree. Results: By modeling the RRE structure, we show that the T18 and V38 codons are base pairing within the RRE-stem-IIA, an important domain involved in Rev binding. While a structural RRE impairment in the presence of V38A alone was found, a restoration of the original RRE structure occurred in co-presence of V38A+T18A. By mutagenetic tree analysis, a compensatory evolution confirming our hypothesis on the structural modification mechanism was observed. Conclusion: We show that enfuvirtide pressure may also affect specific RRE domains involved in Rev binding, thus requiring a compensatory evolution able to preserve the secondary structure of the RRE.