Valentina Svicher

Involvement of Novel Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutations in the Regulation of Resistance to Nucleoside Inhibitors

ABSTRACT We characterized 16 additional mutations in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) whose role in drug resistance is still unknown by analyzing 1,906 plasma-derived HIV-1 subtype B pol sequences from 551 drug-naïve patients and 1,355 nucleoside RT inhibitor (NRTI)-treated patients. Twelve mutations positively associated with NRTI treatment strongly correlated both in pairs and in clusters with known NRTI resistance mutations on divergent evolutionary pathways. In particular, T39A, K43E/Q, K122E, E203K, and H208Y clustered with the nucleoside analogue mutation 1 cluster (NAM1; M41L+L210W+T215Y). Their copresence in this cluster was associated with an increase in thymidine analogue resistance. Moreover, treatment failure in the presence of K43E, K122E, or H208Y was significantly associated with higher viremia and lower CD4 cell count. Differently, D218E clustered with the NAM2 pathway (D67N+K70R+K219Q+T215F), and its presence in this cluster determined an increase in zidovudine resistance. In contrast, three mutations (V35I, I50V, and R83K) negatively associated with NRTI treatment showed negative correlations with NRTI resistance mutations and were associated with increased susceptibility to specific NRTIs. In particular, I50V negatively correlated with the lamivudine-selected mutation M184V and was associated with a decrease in M184V/lamivudine resistance, whereas R83K negatively correlated with both NAM1 and NAM2 clusters and was associated with a decrease in thymidine analogue resistance. Finally, the association pattern of the F214L polymorphism revealed its propensity for the NAM2 pathway and its strong negative association with the NAM1 pathway. Our study provides evidence of novel RT mutational patterns that regulate positively and/or negatively NRTI resistance and strongly suggests that other mutations beyond those currently known to confer resistance should be considered for improved prediction of clinical response to antiretroviral drugs.

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.

Safety of complete and sustained prophylaxis withdrawal in patients liver-transplanted for HBV-related cirrhosis at low risk of HBV recurrence

BACKGROUND & AIMS HBV reactivation after liver transplantation may be related to persistence of covalently closed circular (ccc) DNA. We investigated the safety of HBV prophylaxis withdrawal in selected HBV transplanted patients. METHODS Thirty patients transplanted 64-195months earlier (23 males, median age 56yrs), HBsAg-positive, HBeAg, and HBV-DNA negative at transplant (43% HCV/HDV co-infected), with undetectable intrahepatic total and ccc-DNA were enrolled. All patients underwent HBIg withdrawal and continued lamivudine with monthly HBsAg and HBV-DNA monitoring and sequential liver biopsies. Those with confirmed intrahepatic total and ccc-DNA undetectability 24weeks after stopping HBIg, also underwent lamivudine withdrawal and were followed-up without prophylaxis. RESULTS Twenty-five patients did not exhibit signs of HBV recurrence after prophylaxis withdrawal (median follow-up 28.7months, range 22-42). Five patients became HBsAg-positive: one early after HBIg withdrawal, the other four after HBIG and lamivudine withdrawal. None of these patients experienced clinically relevant events. In the first patient, HBIg were reinstituted with prompt HBsAg negativization. Of the other four, one remained HBsAg-positive with detectable HBV-DNA and mild ALT elevation and was successfully treated with tenofovir. In the remaining three, HBsAg positivity was transient and followed by anti-HBs seroconversion, thus no antiviral treatment was needed. CONCLUSIONS Patients with undetectable HBV viremia at transplant and no evidence of intrahepatic total and cccDNA may safely undergo cautious weaning of prophylaxis, showing low rate of HBV recurrence after a 2 year follow-up. Undetectability of intrahepatic ccc-DNA may help to identify patients at low-risk of recurrence, yet studies with longer follow-up are needed.

Characterization and Structural Analysis of Novel Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Involved in the Regulation of Resistance to Nonnucleoside Inhibitors

ABSTRACT Resistance to antivirals is a complex and dynamic phenomenon that involves more mutations than are currently known. Here, we characterize 10 additional mutations (L74V, K101Q, I135M/T, V179I, H221Y, K223E/Q, and L228H/R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase which are involved in the regulation of resistance to nonnucleoside reverse transcriptase inhibitors (NNRTIs). These mutations are strongly associated with NNRTI failure and strongly correlate with the classical NNRTI resistance mutations in a data set of 1,904 HIV-1 B-subtype pol sequences from 758 drug-naïve patients, 592 nucleoside reverse transcriptase inhibitor (NRTI)-treated but NNRTI-naïve patients, and 554 patients treated with both NRTIs and NNRTIs. In particular, L74V and H221Y, positively correlated with Y181C, were associated with an increase in Y181C-mediated resistance to nevirapine, while I135M/T mutations, positively correlated with K103N, were associated with an increase in K103N-mediated resistance to efavirenz. In addition, the presence of the I135T polymorphism in NNRTI-naïve patients significantly correlated with the appearance of K103N in cases of NNRTI failure, suggesting that I135T may represent a crucial determinant of NNRTI resistance evolution. Molecular dynamics simulations show that I135T can contribute to the stabilization of the K103N-induced closure of the NNRTI binding pocket by reducing the distance and increasing the number of hydrogen bonds between 103N and 188Y. H221Y also showed negative correlations with type 2 thymidine analogue mutations (TAM2s); its copresence with the TAM2s was associated with a higher level of zidovudine susceptibility. Our study reinforces the complexity of NNRTI resistance and the significant interplay between NRTI- and NNRTI-selected mutations. Mutations beyond those currently known to confer resistance should be considered for a better prediction of clinical response to reverse transcriptase inhibitors and for the development of more efficient new-generation NNRTIs.

Novel Human Immunodeficiency Virus Type 1 Protease Mutations Potentially Involved in Resistance to Protease Inhibitors

ABSTRACT Plasma-derived sequences of human immunodeficiency virus type 1 (HIV-1) protease from 1,162 patients (457 drug-naïve patients and 705 patients receiving protease inhibitor [PI]-containing antiretroviral regimens) led to the identification and characterization of 17 novel protease mutations potentially associated with resistance to PIs. Fourteen mutations were positively associated with PIs and significantly correlated in pairs and/or clusters with known PI resistance mutations, suggesting their contribution to PI resistance. In particular, E34Q, K43T, and K55R, which were associated with lopinavir treatment, correlated with mutations associated with lopinavir resistance (E34Q with either L33F or F53L, or K43T with I54A) or clustered with multi-PI resistance mutations (K43T with V82A and I54V or V82A, V32I, and I47V, or K55R with V82A, I54V, and M46I). On the other hand, C95F, which was associated with treatment with saquinavir and indinavir, was highly expressed in clusters with either L90M and I93L or V82A and G48V. K45R and K20T, which were associated with nelfinavir treatment, were specifically associated with D30N and N88D and with L90M, respectively. Structural analysis showed that several correlated positions were within 8 Å of each other, confirming the role of the local environment for interactions among mutations. We also identified three protease mutations (T12A, L63Q, and H69N) whose frequencies significantly decreased in PI-treated patients compared with that in drug-naïve patients. They never showed positive correlations with PI resistance mutations; if anything, H69N showed a negative correlation with the compensatory mutations M36I and L10I. These mutations may prevent the appearance of PI resistance mutations, thus increasing the genetic barrier to PI resistance. Overall, our study contributes to a better definition of protease mutational patterns that regulate PI resistance and strongly suggests that other (novel) mutations beyond those currently known to confer resistance should be taken into account to better predict resistance to antiretroviral drugs.

High Sequence Conservation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase under Drug Pressure despite the Continuous Appearance of Mutations

ABSTRACT To define the extent of sequence conservation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in vivo, the first 320 amino acids of RT obtained from 2,236 plasma-derived samples from a well-defined cohort of 1,704 HIV-1-infected individuals (457 drug naïve and 1,247 drug treated) were analyzed and examined in structural terms. In naïve patients, 233 out of these 320 residues (73%) were conserved (<1% variability). The majority of invariant amino acids clustered into defined regions comprising between 5 and 29 consecutive residues. Of the nine longest invariant regions identified, some contained residues and domains critical for enzyme stability and function. In patients treated with RT inhibitors, despite profound drug pressure and the appearance of mutations primarily associated with resistance, 202 amino acids (63%) remained highly conserved and appeared mostly distributed in regions of variable length. This finding suggests that participation of consecutive residues in structural domains is strictly required for cooperative functions and sustainability of HIV-1 RT activity. Besides confirming the conservation of amino acids that are already known to be important for catalytic activity, stability of the heterodimer interface, and/or primer/template binding, the other 62 new invariable residues are now identified and mapped onto the three-dimensional structure of the enzyme. This new knowledge could be of help in the structure-based design of novel resistance-evading drugs.

Specific HIV-1 integrase polymorphisms change their prevalence in untreated versus antiretroviral-treated HIV-1-infected patients, all naive to integrase inhibitors

OBJECTIVES To define whether the prevalence of mutations associated with integrase inhibitor (INI) resistance is different in untreated versus antiretroviral-treated HIV-1-infected individuals (all INI naive). METHODS Gene sequences of the integrase (IN) and reverse transcriptase (RT) obtained from plasma samples of a well-defined cohort of 448 HIV-1-infected individuals (134 drug naive and 314 antiretroviral treated) were analysed. Docking simulations, using RT and IN models, were also performed. RESULTS Primary mutations and the majority of secondary mutations for raltegravir or elvitegravir were completely absent (or rarely found, <1%) in INI-naive patients, either drug naive or antiretroviral treated. Specific IN polymorphisms increased their frequency in antiretroviral-treated patients, and showed positive associations with specific RT resistance mutations. M154I and V165I IN polymorphisms occurred at a frequency of 6% in untreated patients, reaching 21.3% and 13.4%, respectively, in antiretroviral-treated patients. The mutation M154L, absent in drug-naive patients, was prevalent at 5.7% in antiretroviral-treated patients, and was positively associated with RT resistance mutations F227L and T215Y. Similarly, V165I and G163R mutations were associated with the RT resistance mutations F227L and M230L, respectively, and the T206S polymorphism was associated with the RT resistance mutation L210W. Docking simulations showed several favourable contacts between IN and RT residues. CONCLUSIONS Overall, results confirm that primary and secondary INI-associated mutations are absent or extremely rare in INI-naive patients. Conversely, a few specific IN polymorphisms found in INI-naive patients increased their frequency in antiretroviral-failing patients and/or are associated with RT resistance mutations. The potential contribution of such polymorphisms to the evolution of resistance under the pressure of INIs needs further investigation.

Understanding HIV Compartments and Reservoirs

The spectrum of HIV-1 cellular reservoirs is highly diversified, and their role varies according to the milieu of the anatomical sites in which the virus replicates. In this light, mechanisms underlying HIV-1 persistence in anatomical compartments may be profoundly different from what is observed in peripheral blood. This scenario is further complicated by sub-optimal drug penetration in tissues allowing persistent and cryptic HIV-1 replication in body districts despite undetectable viremia. On this basis, this review aims at providing recent insights regarding the critical role of HIV-1 cellular reservoirs in different anatomical compartments, and their relationship with the pathogenesis of HIV-1 infection. A comprehensive definition of the complex interplay between the virus and its reservoir is critical in order to set up prophylactic and therapeutic strategies aimed at achieving the maximal virological suppression and hopefully in the near future the cure of HIV-1 infection (either functional or biological).

Mechanisms underlying activity of antiretroviral drugs in HIV-1-infected macrophages: new therapeutic strategies

Monocyte‐derived macrophages (M/M) are considered the second cellular target of HIV‐1 and a crucial virus reservoir. M/M are widely distributed in all tissues and organs, including the CNS, where they represent the most common HIV‐infected cells. Differently from activated CD4+ T lymphocytes, M/M are resistant to the cytopathic effect of HIV and survive HIV infection for a long time. Moreover, HIV‐1 replication in M/M is a key pathogenetic event during the course of HIV‐1 infection. Overall findings strongly support the clinical relevance of anti‐HIV drugs in M/M. Nucleoside RT inhibitors (NRTIs) are more active against HIV in M/M than in CD4+ T lymphocytes. Their activity is further boosted by the presence of an additional monophosphate group (i.e., a phosphonate group, as in the case of Tenofovir), thus overcoming the bottleneck of the low phosphorylation ability of M/M. In contrast, the antiviral activity of non‐NRTIs (not affecting the DNA chain elongation) in M/M is similar to that in CD4+ T lymphocytes. Protease inhibitors are the only clinically approved drugs acting at a late stage of the HIV lifecycle. They are able to interfere with HIV replication in HIV‐1 chronically infected M/M, even if at concentrations greater than those observed in HIV‐1 chronically infected CD4+ T lymphocytes. Finally, several new drugs have been shown to interfere efficiently with HIV replication in M/M, including entry inhibitors. A better understanding of the activity of the anti‐HIV drugs in M/M may represent a key element for the design of effective anti‐HIV chemotherapy.

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.