Hepatitis C Virus Antigens Research Articles

Abstract 2498: The MVA viral platform for the treatment of cancer and chronic infectious diseases: Clinical experience from four randomized controlled phase II studies

Abstract The modified vaccinia strain Ankara (MVA) is attenuated and non-propagative but retains infectivity and immunogenicity, its large DNA genome allows the insertion of several full-length coding sequences for disease associated antigens or other transgenes of interest. MVA based targeted immunotherapeutics are designed to induce a cytolytic cellular immune response. TG4010 expresses the full-length sequences of MUC1 and IL2 and was tested in combination with first line platin-based chemotherapy in stage IV non-small cell lung cancer (NSCLC) versus chemotherapy alone (NCT00415818 and NCT01383148); 108 plaque forming units (pfu) was given S/C weekly for 6 weeks then every three weeks up to progression. TG4001 expressing E6 and E7 of HPV16, and IL2 was assessed versus placebo in women with cervical intra-epithelial neoplasia (CIN2/3) (NCT01022346) at the dose of 5.107 pfu, three S/C injections a week apart. TG4040 expresses the HCV antigens NS3, NS4 and NS5B and was evaluated in combination with peg-interferon-α and ribavirin versus the same treatment alone in treatment-naïve patients with chronic hepatitis C, (107 pfu). Two distinct schedules of administration were evaluated: with or without immunotherapy run-in phase (NCT01055821). A total of 729 patients were included and randomized in these four studies: 443 in active arms (185 NSCLC, 136 CIN2/3, 122 HCV) and 286 in control arms. In all studies the repeated S/C administration of the MVA vectors alone or in combination therapies appeared feasible and well tolerated, neither dose reduction nor modification of the schedules of administration have been necessary. Injection site reactions have been the most frequent adverse events associated with treatment, mild to moderate in the majority of cases. Despite lower doses they seemed more prevalent for vectors expressing xenoantigens (TG4001 99% pts, TG4040 42% pts) than for TG4010 (31%). The phase II studies with TG4010 met their primary endpoints based respectively on 6 month progression free survival (PFS) and overall PFS. The primary endpoint of improvedcomplete early viral response (cEVR) was also achieved in the study with TG4040. These studies had in common 1/ to combine the MVA from the beginning of standard of care and 2/ to use a schedule of administration with a run-in phase of 6 or more weekly injections followed by at least monthly injections. The study with TG4001 did not meet its threshold-based primary endpoint of complete response but significantly more women in the experimental arm had a clearance of their cervical lesions at a conization performed 6 months later. Three MVA-based immunotherapeutics have shown meaningful clinical activity in different settings. Their favorable safety profile allows the combination of these products with standard of care therapies and also with immune checkpoint inhibitors (ongoing). Citation Format: Jean-Marc LIMACHER, Elisabeth QUOIX, Heiner WEDEMEYER, Francisco GARCIA, Pekka NIEMINEN, Gisèle LACOSTE, Delphine AGATHON, Geraldine HONNET, Elizabeth CALLEJA, Isabelle DIDILLON, Berangère MARIE-BASTIEN. The MVA viral platform for the treatment of cancer and chronic infectious diseases: Clinical experience from four randomized controlled phase II studies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2015-2498

Hepatitis C virus seroprevalence in the general female population from 8 countries

BackgroundHepatitis C virus (HCV) infection is a significant global health issue because it is widespread and persistent and can cause serious liver diseases. ObjectivesThe aim of this study is to estimate HCV prevalence in women from the general population in different geographical areas worldwide and to assess the potential role of sexual behaviour in the virus transmission. Study designEach participating centre recruited a random sample of women from the general population aged from less than 20 to more than 75 years. The study included 8130 women from 8 countries with information on sociodemographic factors, reproductive and sexual behaviour, smoking habit and HPV DNA through individual interviews. A blood sample was also collected to perform serological tests. We estimated the prevalence ratios associated to HCV to evaluate the effect of sexual behaviour in viral transmission. ResultsWomen were reactive to a minimum of two HCV antigens, including at least one non structural protein were considered as positive (33% of the samples were classified as positive, 40% as negative, and 27% as indeterminate (N=402), that were considered as not positive). The age-adjusted HCV seroprevalence varied significantly by regions (0.3% in Argentina to 21.1% in Nigeria). We found no association between HCV prevalence and age, educational level, smoking habit and any of the available variables for sexual behaviour and reproductive history. ConclusionsThis large study showed heterogeneous distribution of HCV seroprevalence in female and provides evidence of the null impact of sexual behaviour in HCV transmission.

Perinatal transmission of hepatitis C antigens: envelope 1, envelope 2 and non-structural 4

Background: Perinatal exposure to hepatitis C virus (HCV) antigens during pregnancy may affect the developing immune system in the fetus. We aimed to study the perinatal transmission of HCV structural and non-structural antigens. Methods: Sera from 402 pregnant mothers were tested for anti-HCV antibody and HCV RNA. HCV antigens were determined in sera from 101 HCV-infected mothers and their cord blood. Results: In both serum and cord blood samples, HCV NS4 (non-structural 4) at 27 kDa, E1 (envelope 1) at 38 kDa and E2 (envelope 2) at 40 kDa were identified, purified and quantified using western blotting, electroelution and ELISA. Maternal sera and neonate cord blood samples had similar detection rates for NS4 (94.1%), E1 (90.1%) and E2 (90.1%). The mean maternal serum levels (optical density, OD) of HCV NS4 (0.87 ± 0.01), E1 (0.86 ± 0.01) and E2 (0.85 ± 0.01) did not differ significantly (p > 0.05) from those of neonatal cord blood (0.83 ± 0.01, 0.87 ± 0.01 and 0.85 ± 0.01, respectively). Also, strong correlations (p < 0.0001) were shown between sera and cord blood sample levels of HCV NS4, r = 0.77; E1, r = 0.76 and E2, r = 0.80. The vertical transmission of these antigens in vaginal delivery did not differ significantly (p > 0.05) from those in caesarean section. Conclusions: These findings indicate that vertical transmission of HCV NS4, E1 and E2 antigens was very high. Thus, exposure to these antigens may influence the developing immune responses to natural infection or future vaccination.

Peculiarity of Clinical Course of Childhood Hepatitis C Depending on the Viral Genotype

Hepatitis C is a serious problem for Russian Federation. Determination of HCV genotype/subtype is needed for epidemic control, development of antiviral drugs, vaccines, and planning expenditures for treatment. The aims of the study were to establish the structure of HCV genotype in infected children in Moscow region and to analyse the features of hepatitis, induced by different viral subtypes. Methods. HCV RNA was detected by RT-PCA with a high sensitivity (15 ME/ml). Viral genotyping and determination of intergenotype recombination were done by sequencing HCV genome regions of 5'-NTR-core and NS5B. HCV nucleocapsid antigen (core-Ag) revealing and quantifing in serum were done by ELISA and immunochemioluminesent method. Antibodies to individual structural and nonstructural HCV antigens were determined by ELISA. The level of specific antibodies was determined by titration. Routes of HCV transmission was established by a survey by of parents using the standard protocol. Results. The average age of 63 infected children was 11.3 ± 0.78 years. The main HCV subtypes were 3a (46.03%), 1b (33.3%), and 1a (11.6%). Three children (4.8%) showed intergenotype recombinant RF_2k/1b. Most children have been infected by vertical transmission (69.8%). In order to identify significant differences in hepatitis features we analyzed three groups of children with chronic hepatitis C: the first group - 10 children with subtype 3a, the second group - 10 patients with subtype 1b, and the third group - 7 participants with subtype 1a. There are no significant differences in the symptoms, syndromes and biochemical parameters in these groups. The value of viral load and core-Ag were significantly higher in patients with subtype 3a (P < 0.1) than in the groups of children infected with subtype 1a or 1b. Anti-HCV IgM, anti-NS4 IgG and anti-NS5 IgG in patients, chronically infected by subtype 3a, were detected less often (p < 0.05) than in the other groups. conclusion. The proportion of HCV subtype 3a has increased in infected children in the Moscow region. Intergenotype recombinant RF_2k/1b was firstly detected in children. Patients, infected by HCV subtype 3a, displayed higher viral load and often lack of anti-HCV IgM, anti-NS4ab IgG, and anti-NS5a IgG. Children, infected by HCV subtypes 1a, 1b, and 3a, have no significant differences in the symptoms, syndromes and liver biochemical parameters.

Minimum target prices for production of direct-acting antivirals and associated diagnostics to combat hepatitis C virus.

Combinations of direct-acting antivirals (DAAs) can cure hepatitis C virus (HCV) in the majority of treatment-naïve patients. Mass treatment programs to cure HCV in developing countries are only feasible if the costs of treatment and laboratory diagnostics are very low. This analysis aimed to estimate minimum costs of DAA treatment and associated diagnostic monitoring. Clinical trials of HCV DAAs were reviewed to identify combinations with consistently high rates of sustained virological response across hepatitis C genotypes. For each DAA, molecular structures, doses, treatment duration, and components of retrosynthesis were used to estimate costs of large-scale, generic production. Manufacturing costs per gram of DAA were based upon treating at least 5 million patients per year and a 40% margin for formulation. Costs of diagnostic support were estimated based on published minimum prices of genotyping, HCV antigen tests plus full blood count/clinical chemistry tests. Predicted minimum costs for 12-week courses of combination DAAs with the most consistent efficacy results were: US$122 per person for sofosbuvir+daclatasvir; US$152 for sofosbuvir+ribavirin; US$192 for sofosbuvir+ledipasvir; and US$115 for MK-8742+MK-5172. Diagnostic testing costs were estimated at US$90 for genotyping US$34 for two HCV antigen tests and US$22 for two full blood count/clinical chemistry tests. Conclusions: Minimum costs of treatment and diagnostics to cure hepatitis C virus infection were estimated at US$171-360 per person without genotyping or US$261-450 per person with genotyping. These cost estimates assume that existing large-scale treatment programs can be established. (Hepatology 2015;61:1174–1182)

Prevalence of hepatitis C virus in mothers and their children in Malawi.

ObjectivesHepatitis C virus (HCV) prevalence is poorly mapped in the East African region; with the advent of novel HCV therapies, better epidemiological data are required to target the infection. We sought to estimate HCV prevalence in healthy Malawian mothers and assess mother-to-child transmission (MTCT); context is provided by reviewing previously published HCV prevalence data from the region.MethodsUsing ELISA screening and confirmatory blot, serological testing of 418 healthy Malawian mothers for HCV was performed. To examine MTCT, the children of any positive women were also tested for HCV; all children had malignant disease unrelated to hepatocellular carcinoma. We compared our results to published literature on HCV prevalence in Malawi and its neighbouring countries.ResultsThree of 418 women were HCV reactive by ELISA; two were confirmed positive by immunoblot (0.5%). One child of an HCV-infected mother was HCV seropositive. The literature review revealed HCV prevalence ranging from 0 to 7.2% in the region, being highest in Tanzania and specifically for cohorts of inpatients and HIV-co-infected people. The overall estimated prevalence of HCV in Malawi was 1.0% (95%CI 0.7–1.4) when all studies were included (including this one), but lower in healthy cohorts alone at 0.3% (95%CI 0.1–1.2).ConclusionsThis is the first study using confirmatory tests to examine HCV prevalence in healthy Malawian mothers; the prevalence was low. Future studies need to address the source of infection in healthy women.

Is Anti-Hepatitis C Virus Antibody Level an Appropriate Marker to Preclude the Need for Supplemental Testing?

Objectives: In the present study, we aimed to determine whether signal-to-cutoff (S/Co) ratios of reactive anti-HCV samples could be used as a basis for avoiding the need for supplemental testing in our study population. Methods: We analyzed 901 anti-HCV-positive sera from 8 institutions in China. The Ortho VITROS anti-HCV assay and Monolisa Plus anti-HCV version 2 were used as screening assays to detect anti-HCV antibodies. Recombinant immunoblot assay (RIBA) and quantitative tests for HCV RNA were performed to validate confirmed HCV infection status. Results: Receiver operating characteristic curve analyses demonstrated that 41.5% (114/275) of true-positive samples with S/Co ratios ≤3.0 would be missed and the negative predictive value was 63.9 and 87.06%, using real-time polymerase chain reaction (RT-PCR) and RIBA as supplemental testing, respectively. 29.8% (90/302) of those who tested positive by RIBA samples were missed when only RT-PCR was used as supplemental testing. Conclusions: We determined that very low anti-HCV levels (S/Co ≤3.0), as determined by chemiluminescence immunoassay, was not an appropriate marker to preclude the need for supplemental testing in our study population. A screening strategy employing a secondary HCV antibody assay using different HCV antigens from the first assay as the supplemental testing method should be studied further. The immunoblot assay, as a supplemental testing method, is still necessary.

Monoclonal antibodies: Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus.

Hepatitis C virus (HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies (mAb) of mice are predominantly used for the immunodiagnosis of several viral, bacterial, and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mAb are used in the immunotherapy of several blood malignancies, such as lymphoma and leukemia, as well as for autoimmune diseases. In this review article, we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mAb in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV, which is a major health problem throughout the world, particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mAb. Finally, we will discuss available and new trends to produce antibodies, such as egg yolk-based antibodies (IgY), production in transgenic plants, and the synthetic antibody mimics approach.

Evaluation of a hepatitis C virus (HCV) antigen assay for routine HCV screening among men who have sex with men infected with HIV

BackgroundFor detection of early HCV infection and reinfection, commercial HCV-RNA tests are available. However, these tests are relatively time-consuming and expensive. A commercially available test that may supplement current screening methods, targets the HCV core protein. MethodsDuring five waves of anonymous surveys at the Amsterdam STI clinic between 2009-2012, all HIV-infected MSM (N=439) were tested for HCV-antibodies (AxSYM HCV 3.0, Abbott), and HCV-RNA (TMA Versant, Siemens). To evaluate the potential value of the ARCHITECT HCV antigen (HCV-Ag) assay (Abbott), all HCV-RNA-positive sera (N=31) were tested with this assay, as well as two HIV-infected HCV-RNA-negative controls. In addition, all included samples were tested for alanine aminotransferase (ALT). ResultsAmong 439HIV-infected MSM, 31 (7.1%) tested positive for HCV-RNA; the HCV-Ag assay showed concordant positive results for 31/31 (100%). A substantial number of MSM, i.e., 5/31 (16.1%), had detectable HCV-RNA but were HCV-seronegative at the time of screening and were presumed to have been recently infected. Concordant HCV-RNA-negative results were obtained in 57/60 control-samples. Specificity was 95.0% (95% CI: 86.1-99.0). The detection limit was between 3.0 and 3.7 Log10 IU/mL, irrespective of HCV genotype/subtype. ALT concentrations were elevated (i.e.,>40 U/L) in 9/31 (29.0%) HCV-RNA positive MSM, including 1/5 (20.0%) MSM with recent HCV-infection. ConclusionsThe HCV-Ag assay proved a valuable screening tool for detection of active HCV infection among HIV-infected MSM with and without anti-HCV. Adding ALT to current screening methods would improve case finding marginally. We therefore recommend implementation of routine HCV-Ag screening for populations at risk for HCV-(re)infection.

Genetic Variants of TNFAIP3 in Patients with HCV Related Lymphoma Are Associated with the Presence of Rheumatoid Factor (RF)

Background: HCV chronic infection is associated with an increased risk of non-Hodgkin lymphoma (NHL) occurrence. HCV-associated NHL share homologies with primary Sjögren Syndrome (pSS)-associated NHL, and especially an association with chronic antigenic stimulation and with auto-antibody presence, especially rheumatoid factor (RF) and mixed cryoglobulinemia (MC). TNFAIP3 encodes the A20 protein that plays a key role in controlling NF-kB activation. We have previously demonstrated that germline or tumor genetic impairment of A20 plays a role in lymphomagenesis in the context of pSS[1]. The aim of this study was to assess the role of variants of TNFAIP3 in patients with HCV related NHL.Methods: Sixty-one cases of HCV-associated lymphoma with available germline DNA were drawn from the 116 patients included in the LymphoC study. Total exon sequencing of TNFAIP3was performed in a discovery set of 31 cases. Then 30 additional cases and 53 controls (HCV patients without NHL) were used for extension (ie genotyping of the rs2230926 and the TT>A dinucleotide). All our cases and controls were European. Case-only associations were tested with Fischer’s exact test. Differences in lymphoma histologic type and immunological status were assessed using Fisher’s exact test.Results:Among the 61 cases, histology subsets were 23 diffuse large B cell lymphomas (DLBCL), 17 marginal zone lymphomas (MZL), 6 splenic marginal zone lymphoma (SMZL), 5 mantle cell lymphomas, 8 follicular lymphomas, 1 chronic lymphoid leukemia and 1 chronic EBV-related lymphoproliferation. RF and MC were present in 30/61 (49.2%) and 25/43 (58.1%) of the patients respectively. Among the 53 controls, RF and MC were present in 31/53 (58.5%) and 28/53 (52.8%) of the patients respectively. We found the rs2230926G variant in 6/61 (9.8%) patients with NHL and in 7/53 (13.2%) patients without NHL meaning that there was no association between this SNP and HCV-associated NHL (OR=0.72 [95%CI 0.22– 2.28] p=0.77). We did not find any association between the variant and the marginal zone subtype of the lymphoma.However, we found that, among NHL patients, the rs2230926G allele was associated with the positivity of RF (6/30 (20%) in RF+ patients compared to 0/31 (0%) in RF- patients, OR=16.7 [95%CI 0.9 – 311.5], p=0.01). We did not find any association between the rs2230926 variant and the presence of MC patients probably due to the amount of missing data and the variability of the technic of detection. Last, we previously showed that the rs2230926G was functional and able to impair the control of NF-kB activation[1].Conclusion:This study demonstrates that a germline coding mutation of TNFAIP3leading to a small functional defect of A20 function and of control of activation of NK-kB, plays a key role in lymphomagenesis in the context of chronic antigenic stimulation of RF+ B cells. It extends the scenario already demonstrated in Sjögren’s syndrome-associated lymphomas, a concept which is both novel and paradigm-shifting in the area of lymphomagenesis and autoimmunity. Interestingly, this coding mutation is associated only with HCV-associated lymphoma and presence of RF, which clearly supports different types of lymphomagenesis pathways in patients with HCV, one of them linked to the continuous stimulation of RF+ B cells by the immune complexes between HCV antigens and anti-HCV antibodies.Reference:1. Nocturne, G., et al., Germline and somatic genetic variations of TNFAIP3 in lymphoma complicating primary Sjogren’s syndrome. Blood, 2013 122(25): p. 4068-76. DisclosuresNo relevant conflicts of interest to declare.

Single Amino Acid Repeats Connect Viruses to Neurodegeneration

We report on a high level of octapeptide matching between HCV, HIV-2, MPV, MUV, EBV, HHV-6, and CMV, and human brain antigens that, when altered, have been specifically associated with neuropathologies such as amyotrophic lateral sclerosis, spinocerebellar ataxia, frontotemporal degeneration, Huntington disease, Parkinson disease, cognitive impairment, aphasia and oculomotor apraxia. Quantitatively, the extent of the viral octapeptide sharing with neurodegeneration- associated proteins is in excess when analyzed in a stochastic expectation context. Qualitatively, two main features characterize the peptide matching: 1) many common sequences are single amino acid repeats, and 2) mostly, the shared octapeptides are part of experimentally validated epitopes, thus suggesting an immune crossreactive potential of the viral peptides shared with brain antigens involved in neurodegeneration. The present study may have relevance for peptidebased therapeutic approaches to block potential autoimmune crossreactions in neurological diseases and dysfunctional behavior.

CD127 expression, exhaustion status and antigen specific proliferation predict sustained virologic response to IFN in HCV/HIV co-infected individuals.

Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality in the HIV co-infected population. Interferon-alpha (IFN-α) remains a major component of anti-HCV therapy despite its deleterious effects on the immune system. Furthermore, IFN-α was recently shown to diminish the size of the latent HIV reservoir. The objectives of this study were to monitor the impact of IFN-α on T cell phenotype and proliferation of HIV and HCV-specific T cells during IFN therapy, and to identify immune markers that can predict the response to IFN in HICV/HIV co-infected patients. We performed longitudinal analyses of T cell numbers, phenotype and function in co-infected patients undergoing IFN-α therapy with different outcomes including IFN-α non-responders (NR) (n = 9) and patients who achieved sustained virologic response (SVR) (n = 19). We examined the expression of activation (CD38, HLA-DR), functional (CD127) and exhaustion markers (PD1, Tim-3, CD160 and CD244) on total CD4 and CD8 T cells before, during and after therapy. In addition, we examined the HIV- and HCV-specific proliferative responses against HIV-p24 and HCV-NS3 proteins. Frequencies of CD127+ CD4 T cells were higher in SVR than in NR patients at baseline. An increase in CD127 expression on CD8 T cells was observed after IFN-α therapy in all patients. In addition, CD8 T cells from NR patients expressed a higher exhaustion status at baseline. Finally, SVR patients exhibited higher proliferative response against both HIV and HCV antigens at baseline. Altogether, SVR correlated with higher expression of CD127, lower T cell exhaustion status and better HIV and HCV proliferative responses at baseline. Such factors might be used as non-invasive methods to predict the success of IFN–based therapies in co-infected individuals.

Hepatitis C diagnosics: clinical evaluation of the HCV-core antigen determination

Results The determination of HCV antigen highly correlates with the quantitative measurement of HCV RNA (r=0.73), p=0.0003). The diagnostic window is comparable with that of the HCV PCR (27.1 ± 12.8 d vs. 23.9 ± 9.2 d, p=0.11). The sensitivity of the HCV antigen assay was 99.0 % with a specificity of 99.2 %. 54.3 % of the confirmed antibody positive samples were also antigen positive. Only in 3 of 560 HCV-RNA positive samples HCV antigen was not detectable, but 3 samples without HCV antibodies were confirmed positive for HCV antigen.

A polyantigenic genotype 1a/1b consensus hepatitis C virus DNA vaccine induces broadly reactive HCV-specific cellular immune responses in both mice and non-human primates (VAC7P.985)

Abstract Chronic HCV is a surreptitious disease that can lead to cirrhosis and cancer decades following infection. Each year, 3-4 million individuals are infected. However, there are currently no vaccines available to prevent chronic HCV infection. From patients who acutely resolve (A.R.) infection, a strong and broadly reactive cytotoxic T lymphocyte (CTL) response is associated with viral clearance. With in vivo electroporation (E.P.), DNA vaccines induce strong CTL responses, making them well suited for an HCV vaccine. Our vaccine strategy maximizes recipients' opportunity to induce a broad T cell response with genotype 1a/1b consensus sequences of multiple HCV antigens. Immunogenicity of the HCV vaccine was assessed in mice and non-human primates (NHP). Animals were immunized three times, two weeks apart for mice and six weeks apart for NHP as an intramuscular injection followed by E.P. In mice, interferon-γ (IFNγ) responses spanned all HCV antigens and with total values exceeding 6,000 spot-forming units per million mononuclear cells (SFU) within both the spleen and liver by IFNγ ELIspot. Similarly in NHP, immune responses up to 4,680 SFU per million PBMCs were detectable by IFNγ ELISpot. Flow cytometry revealed HCV-specific CD4+ and CD8+ T cell responses by cytokine staining and significant frequencies of cytolytic CTL compared to baseline. Here, we have demonstrated the magnitude and breadth of HCV-specific immune responses elicited by an HCV DNA vaccine in mice and NHP.

Comparison of two diagnostic algorithms for the identification of patients with HCV viremia using a new HCV Antigen test

Background. Patients exposed to hepatitis C virus (HCV) may develop chronic infection with viremia. The diagnosis of this condition requires the use of several laboratory tests in algorithms tailored to the population and resources available for each laboratory.Aim. We compared the diagnostic efficacy of two diagnostic algorithms for the identification of viremic patients with HCV. One based on confirmation of reactive antibody results with molecular techniques (reverse transcription polymerase chain reaction, RT-PCR) and the other based on the use of a new HCV core antigen test (HCV Ag).Material and methods. We measured levels of anti-HCV, HCV Ag and viral load (trough RT-PCR) in parallel, in 211 samples (53 antibody positive, 158 antibody negative). Using the three results available for each sample we simulated the diagnostic performance of the two algorithms and compared them to the results of RT-PCR as gold standard.Results. Both algorithms showed a high degree of concordance for viremic patients. The percentage of correctly classified patients was 99.05% for the algorithm based on RT-PCR and 98.10% for the HCV Ag algorithm. The HCV core Ag test showed a clinical sensitivity of 0.917 and showed a good correlation to the results of molecular biology. Spearman rank correlation coefficient (ρ) of 0.97 (95% CI 0.95 to 0.99, p < 0.0001). Conclusion. An algorithm incorporating HCV Ag as confirmatory test for anti-HCV results is a feasible alternative to the use of molecular techniques in laboratories that do not have access to them or require faster turn around times.

A novel challenge model to evaluate the efficacy of hepatitis C virus vaccines in mice

An effective hepatitis C virus (HCV) vaccine should elicit robust humoral and cell mediated immunity (CMI). A small animal challenge model is required to assess the efficacy of vaccines which elicit CMI. In this study, HCV proteins were expressed in hepatocytes of immunocompetent mice after hydrodynamic injection of a plasmid encoding the HCV NS3/4A protein. This vector, constructed as the “challenge”, was optimized for long term, specific gene expression in hepatocytes. To monitor HCV antigen expression in transfected hepatocytes, the plasmid also encoded secreted alkaline phosphatase (SEAP), which was detected in the mouse serum. The design of this novel challenge plasmid was based on studies using luciferase and SEAP as reporter molecules to examine the kinetics of the proteins expressed in hepatocytes and secreted into blood. We designed two constructs to control SEAP expression. In one construct, SEAP expression was controlled by the EMCV IRES, while in the other, a SEAP and luciferase polyprotein was cleaved by the FMDV2A proteinase. We found that SEAP expressed after FMDV2A self cleavage was more sensitive and showed a higher correlation with luciferase expressed in liver. The NS3/4A challenge model using the FMDV2A design provided a window period of 50 days to monitor changes in SEAP expression after hydrodynamic injection of DNA. In a challenge experiment, mice which received an adenovirus-based HCV vaccine showed accelerated clearance of SEAP and thus, of NS3/4A positive hepatocytes compared with a mock vaccinated group, that coincided with an increased number of CD8+ lymphocytes in the liver.

B-cell receptors expressed by lymphomas of hepatitis C virus (HCV)–infected patients rarely react with the viral proteins

AbstractChronic hepatitis C virus (HCV) infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this derives from clinical observations of tumor regressions upon antiviral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon antiviral treatments. We expressed the lymphoma B-cell receptors as soluble immunoglobulin Gs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus.

Perioperative blood transfusion affects hepatitis C virus (HCV)-specific immune responses and outcome following liver transplantation in HCV-infected patients

ObjectivesPerioperative factors can affect outcomes of liver transplantation (LT) in recipients with hepatitis C virus (HCV) infection. This study was conducted to investigate whether the immunomodulatory effects of packed red blood cells (PRBC) and platelets administered in the perioperative period might affect immune responses to HCV and thus outcomes in LT recipients. MethodsData for a total of 257 HCV LT recipients were analysed. Data on clinical demographics including perioperative transfusion (during and within the first 24h), serum cytokine concentration, HCV-specific interferon-γ (IFN-γ) and interleukin-17 (IL-17) producing cells, and outcomes including graft and patient survival were analysed. ResultsPatient survival was higher in HCV LT recipients who did not receive transfusions (Group 1, n = 65) than in those who did (Group 2, n = 192). One-year patient survival was 95% in Group 1 and 88% in Group 2 (P = 0.02); 5-year survival was 77% in Group 1 and 66% in Group 2 (P = 0.05). Group 2 had an increased post-transplant viral load (P = 0.032) and increased incidence of advanced fibrosis at 1 year (P = 0.04). After LT, Group 2 showed increased IL-10, IL-17, IL-1β and IL-6, and decreased IFN-γ, and a significantly increased rate of IL-17 production against HCV antigen. Increasing donor age (P = 0.02), PRBC transfusion (P < 0.01) and platelets administration were associated with worse survival. ConclusionsTransfusion had a negative impact on LT recipients with HCV. The associated early increase in pro-HCV IL-17 and IL-6, with decreased IFN-γ, suggests that transfusion may be associated with the modulation of HCV-specific responses, increased fibrosis and poor transplant outcomes.

Hepatitis C virus in the new era: perspectives in epidemiology, prevention, diagnostics and predictors of response to therapy.

Despite the great successes achieved in the fields of virology and diagnostics, several difficulties affect improvements in hepatitis C virus (HCV) infection control and eradication in the new era. New HCV infections still occur, especially in some of the poorest regions of the world, where HCV is endemic and long-term sequelae have a growing economic and health burden. An HCV vaccine is still no available, despite years of researches and discoveries about the natural history of infection and host-virus interactions: several HCV vaccine candidates have been developed in the last years, targeting different HCV antigens or using alternative delivery systems, but viral variability and adaption ability constitute major challenges for vaccine development. Many new antiviral drugs for HCV therapy are in preclinical or early clinical development, but different limitations affect treatment validity. Treatment predictors are important tools, as they provide some guidance for the management of therapy in patients with chronic HCV infection: in particular, the role of host genomics in HCV infection outcomes in the new era of direct-acting antivirals may evolve for new therapeutic targets, representing a chance for modulated and personalized treatment management, when also very potent therapies will be available. In the present review we discuss the most recent data about HCV epidemiology, the new perspectives for the prevention of HCV infection and the most recent evidence regarding HCV diagnosis, therapy and predictors of response to it.

HCV Antigen Testing for the Diagnosis of Hepatitis C Infection: A Cost-Efficient Algorithm

In North America, diagnosis of active hepatitis C virus (HCV) infection is currently performed using RNA testing which is highly sensitive and specific but is associated with three major limitations: lability of RNA molecules, higher costs, and longer turn-around time as compared with commercially-available HCV core antigen testing. In the current study, a new HCV core antigen assay product was evaluated for the diagnosis of HCV infection and its cost reducing potential. Ninety plasma specimens positive for HCV RNA along with 25 negative HCV specimens were used for HCV antigen assay. Twenty-four specimens positive for a panel of agents were used for possible cross-reactivity. Sixty-four HCV antibody-positive specimens with negative HCV RNA and indeterminate HCV immunoblot results were also employed. In the first group, 78/90 (86.6%) tested positive for HCV antigen with regression analysis showing no significant deviation from linearity. None of the prenatal specimens tested positive for HCV antigen. Non-specific reactions were not observed. In the HCV antibody-indeterminate group, only 2/64 (3.1%) were antigen positive. In the last group, none of the HCV antibody very-low-positive specimens tested positive for HCV antigen. Both inter- and intra-run reproducibility of 100% were noted. The cost analysis showed a minimum of 52.13% reduction in costs associated with qualitative RNA testing. Considering the complexity of HCV infection diagnosis and the significant cost and turn-around time burden it imposes on clinical laboratories, HCV antigen testing seems an attractive adjunct to the current battery of laboratory diagnosis that demands more attention.