High real-world effectiveness of 12-week elbasvir/grazoprevir without resistance testing in the treatment of patients with HCV genotype 1a infection in Norway.

BACKGROUND AND AIMS: The recommended treatment duration of hepatitis C virus (HCV) genotype 1a (GT1a) infection with elbasvir/grazoprevir (EBR/GZR) in the presence of a high baseline viral load and resistance associated substitutions (RAS) is 16 weeks with ribavirin added. The objective of this study was to evaluate the real-world effectiveness of 12 weeks of EBR/GZR without ribavirin and regardless of baseline viral load and RAS testing. METHOD: This retrospective, observational cohort study was performed at five Norwegian hospitals that did not systematically utilize RAS testing. All adult patients with chronic HCV GT1a and compensated liver disease who had received 12 weeks of EBR/GZR without ribavirin and baseline RAS testing, were included. The primary endpoint was sustained virologic response at week 12 (SVR12), or if not available, at week 4 (SVR4). RESULTS: We included 433 patients and attained SVR data on 388. The mean age was 45.7 years (22-73 years). 67.2% were male. HIV co-infection was present in 3.8% (16/424) and cirrhosis in 4% (17/424). The viral load was >800 000 IU/mL in 55.0% (235/427) of patients. Overall SVR was achieved in 97.2% (377/388). SVR was achieved in 98.3% (169/172) of those with viral load ≤800 000 IU/mL and in 96.2% (202/210) of those with viral load >800 000 IU/mL. CONCLUSION: We observed high SVR rates among patients with HCV GT1a infection treated with EBR/GZR for 12 weeks without ribavirin, with no regard to baseline viral load and no RAS testing.

Treatment outcomes in hepatitis C virus genotype 1a infected patients with and without baseline NS5A resistance-associated substitutions.

BACKGROUND&AIMS: The presence of baseline resistance-associated substitutions (RASs) reduced sustained virologic response (SVR) rates in chronic hepatitis C virus (HCV) genotype 1a infected patients treated with Elbasvir/Grazoprevir (EBR/GZR). This study aimed to evaluate the frequency of NS5A RASs and treatment outcomes in patients for whom EBR/GZR was intended. METHODS: We sequenced NS5A in 832 samples from German genotype1a-infected DAA-naïve patients population-based, which were collected in the European Resistance Database. Treatment outcomes and clinical parameters were evaluated in 519 of these patients retrospectively. RESULTS: Overall, 6.5% of patients harbored EBR-specific NS5A RASs at baseline, including Q30H/R (3.3%), L31M (1.8%), Y93H (1.6%) and other individual variants. Antiviral treatment, including EBR/GZR, was initiated in 88% of patients. In the absence of RASs, the majority of patients received EBR/GZR for 12 weeks (57%) and the SVR rate was 97% compared to 99% SVR achieved using other DAA regimens (LDV/SOF±RBV, G/P, PrOD+RBV, VEL/SOF). Various regimens were used in the presence of RASs and SVR rates were high following treatment with LDV/SOF (100%), G/P (83%), PrOD/RBV (100%), VEL/SOF (100%), SMV/SOF (100%) and EBR/GZR+RBV for 16 weeks (100%). However, two patients received EBR/GZR for 16 weeks without RBV and one relapsed. CONCLUSIONS: EBR/GZR treatment with or without RBV for 12 or 16 weeks according to a baseline RAS analysis was highly effective with ≥97% SVR in patients with genotype 1a. EBR/GZR without RBV should be avoided in patients with RASs. High SVR rates were also achieved using other 8 or 12 weeks DAA regimens.

Efficacy and safety of ombitasvir/paritaprevir/ritonavir and dasabuvir with low-dose ribavirin in patients with chronic hepatitis C virus genotype 1a infection without cirrhosis.

Patients infected with hepatitis C virus (HCV) treated with interferon-free direct-acting antivirals may still require ribavirin. However, ribavirin is associated with adverse events that can limit its use. This open-label, multicentre, Phase 3 study evaluated the safety and efficacy of ombitasvir/paritaprevir/ritonavir + dasabuvir (OBV/PTV/r + DSV) with low-dose ribavirin for 12 weeks in genotype 1a-infected patients without cirrhosis. The primary efficacy endpoint was sustained virologic response at post-treatment Week 12 (SVR12). The primary safety endpoint was haemoglobin <10 g/dL during treatment and decreased from baseline. Overall, 105 patients enrolled. The SVR12 rate was 89.5% (n/N = 94/105; 95% CI, 83.7-95.4). The study did not achieve noninferiority versus the historic SVR12 rate for OBV/PTV/r + DSV plus weight-based ribavirin. Five patients experienced virologic failure, four discontinued, and two had missing SVR12 data. Excluding nonvirologic failures, the SVR12 rate was 94.9% (n/N = 94/99). One patient met the primary safety endpoint. OBV/PTV/r + DSV plus low-dose ribavirin offers an alternative option for patients in whom full-dose ribavirin may compromise tolerability, although noninferiority to the weight-based ribavirin regimen was not met.

Prevalence of NS5A resistance associated substitutions in patients with hepatitis C virus genotypes 1a and 3: Impact on current therapeutic strategies.

The presence of resistance-associated substitutions (RASs) at NS5A region might compromise the efficacy of Direct Acting Antiviral agents (DAAs). HCV resistance at NS5A region is mainly focused on patients with hepatitis C virus (HCV) genotypes 1a (G1a) and 3 (G3) with other factors of poor treatment response (ie cirrhosis, prior treatment-exposure, or HCV-RNA >800 000 IU/mL). Herein, we evaluated in a cohort of HCV G1a and G3 infected patients the prevalence of RASs at domain I NS5A using population-based sequencing and the impact of RASs on the optimization of current therapeutic strategies. The RASs considered as clinically relevant were: M28A/G/T, Q30D/E/H/G/K/L/R, L31M/V/F, H58D, and Y93C/H/N/S for G1a and Y93H for G3. A total of 232 patients naïve to NS5A inhibitors were included (166 G1a, 66 G3). The overall prevalence of NS5A RASs for G1a and G3 patients was low (5.5%) or null, respectively. A high proportion of patients harbored, at least, one factor of poor response (78.9% for G1a, and 75.8% for G3). Overall, the rates of patients harboring NS5A RASs in combination with any of the other factors were low and the vast majority of patients (G1a> 94% and G3 100%) could be treated with standard treatments of 12 weeks without ribavirin. In conclusion, testing NS5A RASs in specific HCV-infected populations (ie G1a & G3, cirrhosis, prior treatment experienced, HCV-RNA >800 000 IU/mL) might be useful to optimize current NS5A-based therapies avoiding ribavirin-related toxicities, and shortening treatment duration in the majority of patients.

NS3 Variability in Hepatitis C Virus Genotype 1A Isolates from Liver Tissue and Serum Samples of Treatment-Naïve Patients with Chronic Hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) NS3 resistance-associated substitutions (RASs) reduce HCV susceptibility to protease inhibitors. Little is known about NS3 RASs in viral isolates from the liver of chronic hepatitis C (CHC) patients infected with HCV genotype-1a (G1a). AIM: The objective of this work was to study NS3 variability in isolates from the serum and liver of HCV-G1a-infected patients naïve to direct-acting antivirals (DAAs). METHODS: NS3 variability of HCV-G1a isolates from the serum and liver of 11 naïve CHC patients, and from sera of an additional 20 naïve CHC patients, was investigated by next-generation sequencing. RESULTS: At a cutoff of 1%, NS3 RASs were detected in all the samples examined. At a cutoff of 15%, they were found in 54.5% (6/11) and 27.3% (3/11) of the paired liver and serum samples, respectively, and in 22.5% (7/31) of the overall serum samples examined. Twenty-six out of thirty-one (84%) patients showed NS3 variants with multiple RASs. Phylogenetic analysis showed that NS3 sequences clustered within 2 clades, with 10/31 (32.2%) patients infected by clade I, 15/31 (48.8%) by clade II, and 6/31 (19.3%) by both clades. CONCLUSIONS: Though the number of patients examined was limited, NS3 variants with RASs appear to be major components of both intrahepatic and circulating viral quasispecies populations in DAA-naïve patients.

Detection of Genotype 1a and 1f of Hepatitis E Virus in Patients Treated at Tertiary Care Hospitals in Central India.

BACKGROUND AND AIMS: An acute hepatitis, caused by hepatitis E virus (HEV), is a significant global health concern, especially in developing countries. HEV has 1 serotype and 8 genotypes, which are further divided into subtypes. Despite the availability of sporadic cases and outbreak data from India, specific information regarding HEV epidemiological data is lacking from central India. This study was conducted to understand epidemiological and molecular features of HEV in central India. METHODS: In this 3-year study conducted from July 2012 to June 2015, IgM ELISA was used for the diagnosis of suspected HEV cases. For identifying the genotype, nRT PCR was conducted and the PCR products were sequenced and analysed. Clinical and demographic data were analysed using statistical tools to highlight the trends. RESULT: Out of 1,369 suspected cases, 341 (24.9%) were positive for HEV. The positivity was significantly higher in males (69.2%) and in the age group of > 15-45 years (72.5%). The HEV cases peaked during the summer. Subtypes 1a and 1f of genotype 1 were detected in the area during the study period. CONCLUSION: HEV is a major aetiological agent of viral hepatitis in central India with adults and males at higher risk of infection. Two subtypes of the virus were detected in the region. Continuous serological surveillance and molecular monitoring will help to understand the epidemiology of HEV infection, outbreak mitigation and aid in providing treatment.

HCV Council--critical appraisal of data: recommendations for clinical practice in a rapidly evolving therapeutic landscape.

BACKGROUND & AIMS: HCV Council 2014, like its predecessor HCV Council 2011, assembled leading clinicians and researchers in the field of hepatitis C to critically evaluate current data regarding best practices for managing patients with chronic hepatitis C virus (HCV). METHODS: Clinical practice statements were developed that reflect the areas of potential controversy with high clinical impact. Faculty members were responsible for reviewing the literature to support or reject these statements. After a review and comprehensive discussion of the data, the HCV Council faculty voted on the nature of the evidence and the level of support for each statement. RESULTS: The results of the detailed analysis with expert opinion are summarized in this article. CONCLUSION: Numerous questions regarding optimal management of certain populations and clinical scenarios remain unanswered. The discussion in the article provides a summary of evidenced-based expert opinion that may help guide clinicians as additional information is developed.

[Significance of hepatitis C virus baseline polymorphism during the antiviral therapy]. / A hepatitis C-vírus-bázispolimorfizmus jelentosége a kezelésben.

The treatment of chronic hepatitis C has developed significantly during the last 25 years. In patients with genotype 1 infection 40-50% sustained virologic response could be achieved using pegylated interferon and ribavirin dual combination, which could be increased significantly with the introduction of direct acting antivirals. Three major groups of direct acting antivirals are known, which directly inhibit different phases of viral life cycle, by inhibiting the function of several non-structural proteins (NS3/4A protease, NS5A protein and NS5B polymerase). Due to the rapid replication rate of hepatitis C virus and the error-prone NS5B polymerase activity, mutant virions are generated, which might have reduced susceptibility to direct acting antiviral therapy. Since these resistance associated variants might exist before the antiviral therapy, they are still able to replicate during the direct acting antiviral treatment. Due to this selection pressure, the resistant virus will replace the wild type. This was especially detected during monotherapy, therefore, the first generation of direct acting antivirals have been combined with pegylated interferon and ribavirin, while recently interferon-free combinations are being developed including 2 or 3 direct acting antivirals. Using the first generation protease inhibitors boceprevir and telaprevir, it could have been seen, that the rate of resistance associated variants is higher and the therapeutic outcome is worse in patients with hepatitis C virus genotype 1a, than in 1b. Similar phenomenon was seen with the second generation of NS3/4A protease inhibitors as well as with NS5A or NS5B polymerase. This is due to the lower genetic barrier to resistance, ie. usually fewer mutations are enough for the emergence of resistance in genotype 1a. The selection of resistance associated variants is one of the most important challenges during the interferon-free therapy.

Pharmacokinetics, Food Effect, Ketoconazole Interaction, and Safety of JTK-853, a Novel Nonnucleoside HCV Polymerase Inhibitor, After Ascending Single and Multiple Doses in Healthy Subjects.

Pharmacokinetics, safety, and tolerability of JTK-853, a novel HCV polymerase inhibitor, were evaluated in single and multiple ascending dose (SAD, MAD) studies, with food- and ketoconazole-related effects on exposure to JTK-853 and its active (CYP3A4 mediated) metabolite M2. JTK-853 was safe and well tolerated in both studies. In the SAD study, at doses >1600 mg (with standard breakfast [SBF]), JTK-853 exposure did not increase further, was substantially higher (AUCinf increase: 3- to 8-fold) with SBF (vs fasted), with a moderate increase in AUCinf (approximately 1.5-fold [1600 mg]) with a high-fat breakfast (vs SBF). In the SAD study (400-1600 mg, SBF), mean effective half-life (t1/2(eff) ) of JTK-853 was 8.3 to 10.9 hours, and 20.3 to 27.3 hours in the MAD study (twice daily dosing, fed condition), with 2- to 3-fold accumulation in exposure (AUCtau ). At steady-state, AUCtau increased dose proportionally, and was approximately 2-fold higher with ketoconazole coadministration. Metabolite M2 (equipotent to JTK-853 in vitro) did not contribute significantly to parent drug exposure and decreased with increase in dose, repeated dosing, and ketoconazole coadministration. Trial simulation-based ratios (n = 1000/dose level) of trough JTK-853 plasma concentrations to the in vitro EC90 for HCV genotype 1b were assessed for dose selection in a separate proof-of-concept study in patients. The studies enabled delineation of key drug attributes for further assessments in the target population.

An outbreak of hepatitis E virus infection caused by genotype 1 in an urban setting in eastern India: a probe into risk factors for transmission.

The study was undertaken to find out the cause and etiology of an outbreak presumed to be due to water contamination that caused high morbidity in the western part of the state of Odisha during May, 2014. In this investigation 56 blood samples were collected and tested for HEV IgM through ELISA. Blood sample of 22 patients collected within 1 weeks post onset of symptoms and were subjected to RT-PCR and sequencing followed by phylogenetic analysis. Water samples were also analyzed for viral and bacterial contamination. A total of 290 individuals were examined for suspected jaundice. Out of 56 blood samples in 41 (73.2%) IgM for HEV was found. 12 samples out of 22 early phase samples were positive for RT-PCR and through phylogenetic analysis all were found to be of Genotype 1 and subtype A. This large outbreak was confirmed due to Hepatitis E virus and transmission was fast due to contamination of drinking water sources and lack of hygienic practices. The outcome of this investigation has created alertness among state health and municipal authorities to be prepared for the similar kind of situation for other part of the state.

Development of robust genotype 1a hepatitis C replicons harboring adaptive mutations for facilitating the antiviral drug discovery and study of virus replication.

The hepatitis C virus (HCV) subgenomic replicon is a valuable tool for studying virus replication and HCV drug development. Despite the fact that HCV genotype 1a (HCV1a) is the most prevalent genotype in the United States, few HCV1a reporter replicon constructs have been reported, and their replication capacities are not as efficient as those of HCV1b or 2a, especially in transient expression. In this study, we selected efficient HCV1a replicons and characterized the novel adaptive mutations derived from stable HCV1a (strain H77) replicon cells after G418 selection. These novel adaptive mutations were scored in NS3 (A1065V, C1073S, N1227D, D1431Y, and E1556G), NS4A (I1694T and E1709V), and NS4B (G1871C). The D1431Y mutation alone or combinations of other adaptive mutations introduced into the parental HCV1a replicon construct was observed to differentially enhance either transient or stable expression of replicon. In particular, two replicon mutants VDYG (A1065V, N1227D, D1431Y, and E1556G within NS3) and VDYGRG, VDYG with two additional adaptive mutations (NS4A-K1691R and NS4B-E1726G), displayed robust replication and exhibited no impairment in the susceptibility of replicon activity to various known HCV inhibitors.

A large healthcare-associated outbreak of hepatitis C virus genotype 1a in a clinic in Korea.

BACKGROUND: In November 2015, reuse of needles and syringes in conjunction with an increase in cases of HCV at a clinic in Korea was reported and investigated by public health authorities. Patients who received injections at the clinic from the first time this infection control breach may have occurred in 2008 through 2015 when the practice was stopped were offered screening for HCV and other blood-borne pathogens such as HIV, HTLV, HBV, syphilis, and malaria. OBJECTIVES: The aim of this study was to assess whether an outbreak of hepatitis C had occurred among the potentially exposed clinic patients due to this infection control breach. STUDY DESIGN: We performed hepatitis C viral RNA load tests and genotyping using plasma from hepatitis C antibody-positive individuals who had visited the clinic between May 2008 and November 2015. We analyzed the core-E2 and NS5B regions of the virus from RNA-positive samples by constructing a phylogenetic tree based on maximum likelihood analysis. To identify transmission risk factors and epidemiological relationships among the patients, we reviewed their medical records, assessed staff infection control practices and performed environmental inspection of the clinic. Environmental samples from medication room surfaces and medication vial contents were tested for HCV RNA. RESULTS AND CONCLUSIONS: Among the 1721 patients tested, 96 were IgG-positive and 70 were viral RNA-positive. Among the 61 patients whose viral loads were greater than the detection limit, 41 (67.2%) were classified as genotype 1a, 1 (1.6%) as genotype 1b, 18 (29.5%) as genotype 1, and one (1.6%) as genotype 2. After sequencing, 12 genotype 1 cases were further classified as genotype 1a (11) or 1b (1). The sequences of the core-E2 and NS5B regions of 45 patients formed a monophyletic cluster distinct from genotype 1a. The hepatitis C virus sequences from patients and environmental specimens were well-matched in the partial E1 gene region. We detected genotype 1a RNA in environmental specimens, indicating a healthcare-associated outbreak caused by reuse of syringes and contaminated multi-dose vials. Our molecular epidemiological investigation of hepatitis C genotype 1a, rare in Korea, will aid investigations of infection sources during future pathogen outbreaks.

NS5A resistance leading to failure of 24-week therapy with sofosbuvir/ledipasvir and ribavirin for the treatment of hepatitis C genotype 1a infection in a HIV-1 co-infected patient.

Herein we report a previously undescribed case of treatment-emergent non-structural protein 5A (NS5A) resistance mutations, Q30H and Y93C, leading to a failure of 24-week course of sofosbuvir/ledipasvir+ribavirin therapy for the treatment of hepatitis C virus (HCV) genotype 1a in interferon-experienced, human immunodeficiency virus type 1 (HIV-1) co-infected patient with cirrhosis.

Distribution of natural resistance to NS3 protease inhibitors in hepatitis C genotype 1a separated into clades 1 and 2 and in genotype 1b of HIV-infected patients.

Naturally occurring resistance-associated variants (RAVs) within the protease domain of hepatitis C virus (HCV) genotype (G) 1a separated into clades 1 and 2, and G1b were investigated in 59 HIV/HCV coinfected patients. RAVs were detected in 10/23 G1a/clade 1 and 1/19 G1b (p 0.0059). A similar frequency of RAVs was found when comparing G1a/clade 2 and G1b (p 0.1672). A cross-resistance to the macrocyclic compounds simeprevir and paritaprevir was detected in two G1a/clade 2 and 1 G1b sequences and none of G1a/clade 1 sequences. The simultaneous characterization of subtype and natural RAVs by population analysis of the NS3 domain by may add important information for anti-HCV treatment strategies including protease inhibitors.

Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors.

Background. Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods. Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results. All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions. Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I.

Inter and intra-host variability of hepatitis C virus genotype 1a hypervariable envelope coding domains followed for a 4-11 year of human immunodeficiency virus coinfection and highly active antiretroviral therapy.

The evolution of hepatitis C virus (HCV) quasispecies in patients with HIV-1 coinfection is not fully understood. The HCV-1a quasispecies heterogeneity was analyzed at inter and intra-host levels along 7.6 years in 21 coinfected patients that showed different virological and immunological responses to highly active antiretroviral therapy (HAART). Two to nine serial samples were subjected to direct and clonal sequence analyses of the envelope glycoprotein 2 (E2) gene. E2-based phylogenies, intra-host HCV evolution and evolutionary rates, as well as dynamics of the quasispecies heterogeneity parameters were evaluated. Bayesian coalescent phylogenies indicated complex evolutionary histories, revealing some viral lineages that persisted along the follow up and others that were detectable at a single or some sampling times, suggesting the occurrence of emergence-extinction cycles. HCV quasispecies underwent very rapid evolution in HAART-treated patients (~3.1 × 10(-2) sub/site/year) following the recovery of the host immunocompetence irrespectively of the virological response to HAART.

The crystal structure of NS5A domain 1 from genotype 1a reveals new clues to the mechanism of action for dimeric HCV inhibitors.

New direct acting antivirals (DAAs) such as daclatasvir (DCV; BMS-790052), which target NS5A function with picomolar potency, are showing promise in clinical trials. The exact nature of how these compounds have an inhibitory effect on HCV is unknown; however, major resistance mutations appear in the N-terminal region of NS5A that include the amphipathic helix and domain 1. The dimeric symmetry of these compounds suggests that they act on a dimer of NS5A, which is also consistent with the presence of dimers in crystals of NS5A domain 1 from genotype 1b. Genotype 1a HCV is less potently affected by these compounds and resistance mutations have a greater effect than in the 1b genotypes. We have obtained crystals of domain 1 of the important 1a NS5A homologue and intriguingly, our X-ray crystal structure reveals two new dimeric forms of this domain. Furthermore, the high solvent content (75%) makes it ideal for ligand-soaking. Daclatasvir (DCV) shows twofold symmetry suggesting NS5A dimers may be of physiological importance and serve as potential binding sites for DCV. These dimers also allow for new conformations of a NS5A expansive network which could explain its operation on the membranous web. Additionally, sulfates bound in the crystal structure may provide evidence for the previously proposed RNA binding groove, or explain regulation of NS5A domain 2 and 3 function and phosphorylation, by domain 1.

Strong HCV NS3/4a, NS4b, NS5a, NS5b-specific cellular immune responses induced in Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine.

Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study the immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million peripheral blood mononuclear cells (PBMCs) as measured by Interferon-γ ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD45RA- memory-like T cells remained detectable in peripheral blood. Data presented in this manuscript support the notion that vaccine immunogenicity studies using a macaque model can be used to depict key anti-HCV nonstructural antigenic cellular immune responses and support the development of DNA-based prophylactic HCV vaccines.

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.