Animal models to study hepatitis C virus infection

With more than 71 million chronically infected people, the hepatitis C virus (HCV) is a major global health concern. Although new direct acting antivirals have significantly improved the rate of HCV cure, high therapy cost, potential emergence of drug-resistant viral variants, and unavailability of a protective vaccine represent challenges for complete HCV eradication. Relevant animal models are required, and additional development remains necessary, to effectively study HCV biology, virus-host interactions and for the evaluation of new antiviral approaches and prophylactic vaccines. The chimpanzee, the only non-human primate susceptible to experimental HCV infection, has been used extensively to study HCV infection, particularly to analyze the innate and adaptive immune response upon infection. However, financial, practical, and especially ethical constraints have urged the exploration of alternative small animal models. These include different types of transgenic mice, immunodeficient mice of which the liver is engrafted with human hepatocytes (humanized mice) and, more recently, immunocompetent rodents that are susceptible to infection with viruses that are closely related to HCV. In this review, we provide an overview of the currently available animal models that have proven valuable for the study of HCV, and discuss their main benefits and weaknesses

Impact of lipids and lipoproteins on hepatitis C virus infection and virus neutralization

Hepatitis C virus (HCV) infections represent a major global health problem. End-stage liver disease caused by chronic HCV infection is a major indication for liver transplantation. However, after transplantation the engrafted liver inevitably becomes infected by the circulating virus. Direct acting antivirals are not yet approved for use in liver transplant patients, and limited efficacy and severe side effects hamper the use of pegylated interferon combined with ribavirin in a post-transplant setting. Therefore, alternative therapeutic options need to be explored. Viral entry represents an attractive target for such therapeutic intervention. Understanding the mechanisms of viral entry is essential to define the viral and cellular factors involved. The HCV life cycle is dependent of and associated with lipoprotein physiology and the presence of lipoproteins has been correlated with altered antiviral efficacy of entry inhibitors. In this review, we summarise the current knowledge on how lipoprotein physiology influences the HCV life cycle. We focus especially on the influence of lipoproteins on antibodies that target HCV envelope proteins or antibodies that target the cellular receptors of the virus. This information can be particularly relevant for the prevention of HCV re-infection after liver transplantation

Vascular endothelial growth factor: A novel marker for torsion-induced incomplete cervical dilatation in Egyptian buffaloes

Background: Uterine torsion, the most complicated cause of maternal dystocia recorded in bovine, usually followed by incomplete cervical dilatation after successful detorsion, which interfere with vaginal fetal delivery. Aim: This study aimed to evaluate the histopathological changes, variable collagen concentrations, and vascular endothelial growth factor expressions in uterine and cervical tissues following successful detorsion. Methods: Animals were classified into four groups; normally calved cases (group A), cases that respond successfully to detorsion and followed by vaginal fetal delivery without (group B) or with (group C) cervical laceration, and cases that suffered from failure of complete cervical dilatation after successful detorsion (group D). Results: Histopathological findings revealed variable changes in all uterine torsion affected groups, which were characteristic and marked in animals that suffered from failure of complete cervical dilatation following successful detorsion. Moreover, failure of cervical dilatation was associated with the highest collagen concentrations as shown by Masson trichrome stain. On the other hand, immunohistochemical findings showed that the normally calved cases have the highest vascular endothelial growth factor expression compared with animals that suffered from failure of complete cervical dilatation. Conclusion: Our results showed that the vascular endothelial growth factor is essential for cervical dilatation and its lower expression is accompanied by incomplete cervical dilatation following successful detorsion

Development and characterization of a human monoclonal antibody for prevention of HCV recurrence in liver transplant patients

More than 170 million people worldwide are chronically infected with hepatitis C virus (HCV) and are at risk of developing liver fibrosis, cirrhosis and hepatocellular carcinoma. Liver transplantation is the only option for patients with HCV-induced end-stage liver diseases. Nevertheless, infection of the newly grafted liver occurs immediately and universally after transplantation. Despite the recent progress in HCV therapy, a prophylactic vaccine is still not available. The role of neutralizing monoclonal antibodies (mAbs) in protection from different viral infections including HCV, HIV and Ebola has been reported. In the last few years, several mAbs with neutralizing activity have been described but only few mAbs have been evaluated in vivo. In the present study, we describe the development of a mAb, designated 2A5, isolated from HCV genotype 1b chronic patient. ELISA results indicated high affinity of mAb 2A5 towards HCV envelope glycoprotein (E1E2). The binding activity was completely lost against denatured E1E2 protein indicating that it targets a conformational epitope within the envelope region. Epitope mapping using alanine mutants of E1E2 proteins defined critical binding residues within the regions 419-447 and 612-617. Results of pseudoparticles (HCVpp) and cell culture produced virus (HCVcc) neutralization showed broad neutralizing activity of mAb 2A5 against all HCV genotypes. The efficacy study of mAb 2A5 in immune-deficient mice of which the liver is repopulated with human hepatocytes (humanized mice) showed complete protection from HCV challenge for genotypes 1a and 4a, while partial protection was achieved for genotypes 1b and 6a. Sequence analysis of E1E2 protein from non-protected mice did not revealed resistance mutations at interaction residues of mAb 2A5. In conclusion, mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence provide an effective strategy to prevent HCV recurrence in chronically infected HCV liver transplant patients. In addition, the broad neutralizing activity of this mAb presents a valuable epitope for the design of HCV vaccine with cross-protection activity

Targeting a host-cell entry factor barricades antiviral-resistant HCV variants from on-therapy breakthrough in human-liver mice

Objective: Direct-acting antivirals (DAAs) inhibit hepatitis C virus (HCV) infection by targeting viral proteins that play essential roles in the replication process. However, selection of resistance-associated variants (RAVs) during DAA therapy has been a cause of therapeutic failure. In this study, we wished to address whether such RAVs could be controlled by the co-administration of host-targeting entry inhibitors that prevent intrahepatic viral spread. Design: We investigated the effect of adding an entry inhibitor (the anti-scavenger receptor class B type I mAb1671) to a DAA monotherapy (the protease inhibitor ciluprevir) in human-liver mice chronically infected with HCV of genotype 1b. Clinically relevant non-laboratory strains were used to achieve viraemia consisting of a cloud of related viral variants (quasispecies) and the emergence of RAVs was monitored at high resolution using next-generation sequencing. Results: HCV-infected human-liver mice receiving DAA monotherapy rapidly experienced on-therapy viral breakthrough. Deep sequencing of the HCV protease domain confirmed the manifestation of drug-resistant mutants upon viral rebound. In contrast, none of the mice treated with a combination of the DAA and the entry inhibitor experienced on-therapy viral breakthrough, despite detection of RAV emergence in some animals. Conclusions: This study provides preclinical in vivo evidence that addition of an entry inhibitor to an anti-HCV DAA regimen restricts the breakthrough of DAA-resistant viruses. Our approach is an excellent strategy to prevent therapeutic failure caused by on-therapy rebound of DAA-RAVs. Inclusion of an entry inhibitor to the newest DAA combination therapies may further increase response rates, especially in difficult-to-treat patient populations

Development and characterization of a human monoclonal antibody targeting the N-terminal region of hepatitis C virus envelope glycoprotein E1

Monoclonal antibodies (mAbs) targeting the hepatitis C virus (HCV) envelope have been raised mainly against envelope protein 2 (E2), while the antigenic epitopes of envelope protein 1 (E1) are not fully identified. Here we describe the detailed characterization of a human mAb, designated A6, generated from an HCV genotype 1b infected patient. ELISA results showed reactivity of mAb A6 to full-length HCV E1E2 of genotypes 1a, 1b and 2a. Epitope mapping identified a region spanning amino acids 230-239 within the N-terminal region of E1 as critical for binding. Antibody binding to this epitope was not conformation dependent. Neutralization assays showed that mAb A6 lacks neutralizing capacity and does not interfere with the activity of known neutralizing antibodies. In summary, mAb A6 is an important tool to study the structure and function of E1 within the viral envelope, a crucial step in the development of an effective prophylactic HCV vaccine

A novel neutralizing human monoclonal antibody broadly abrogates hepatitis C virus infection in vitro and in vivo

Infections with hepatitis C virus (HCV) represent a worldwide health burden and a prophylactic vaccine is still not available. Liver transplantation (LT) is often the only option for patients with HCV-induced end-stage liver disease. However, immediately after transplantation, the liver graft becomes infected by circulating virus, resulting in accelerated progression of liver disease. Although the effi cacy of HCV treatment using direct-acting antivirals has improved significantly, immune compromised LT-patients and patients with advanced liver disease remain difficult to treat. As an alternative approach, interfering with viral entry could prevent infection of the donor liver. We generated a human monoclonal antibody (mAb), designated 2A5, which targets the HCV envelope. The neutralizing activity of mAb 2A5 was assessed using multiple prototype and patient-derived HCV pseudoparticles (HCVpp), cell culture produced HCV (HCVcc), and a human-liver chimeric mouse model. Neutralization levels observed for mAb 2A5 were generally high and mostly superior to those obtained with AP33, a well-characterized HCV-neutralizing monoclonal antibody. Using humanized mice, complete protection was observed after genotype 1a and 4a HCV challenge, while only partial protection was achieved using gt1b and 6a isolates. Epitope mapping revealed that mAb 2A5 binding is conformation-dependent and identified the E2-region spanning amino acids 434 to 446 (epitope II) as the predominant contact domain. Conclusion : mAb 2A5 shows potent anti-HCV neutralizing activity both in vitro and in vivo and could hence represent a valuable candidate to prevent HCV recurrence in LT-patients. In addition, the detailed identification of the neutralizing epitope can be applied for the design of prophylactic HCV vaccines

Mortality of emergency abdominal surgery in high-, middle- and low-income countries

Background: Surgical mortality data are collected routinely in high-income countries, yet virtually no low- or middle-income countries have outcome surveillance in place. The aim was prospectively to collect worldwide mortality data following emergency abdominal surgery, comparing findings across countries with a low, middle or high Human Development Index (HDI). Methods: This was a prospective, multicentre, cohort study. Self-selected hospitals performing emergency surgery submitted prespecified data for consecutive patients from at least one 2-week interval during July to December 2014. Postoperative mortality was analysed by hierarchical multivariable logistic regression. Results: Data were obtained for 10 745 patients from 357 centres in 58 countries; 6538 were from high-, 2889 from middle- and 1318 from low-HDI settings. The overall mortality rate was 1⋅6 per cent at 24 h (high 1⋅1 per cent, middle 1⋅9 per cent, low 3⋅4 per cent; P < 0⋅001), increasing to 5⋅4 per cent by 30 days (high 4⋅5 per cent, middle 6⋅0 per cent, low 8⋅6 per cent; P < 0⋅001). Of the 578 patients who died, 404 (69⋅9 per cent) did so between 24 h and 30 days following surgery (high 74⋅2 per cent, middle 68⋅8 per cent, low 60⋅5 per cent). After adjustment, 30-day mortality remained higher in middle-income (odds ratio (OR) 2⋅78, 95 per cent c.i. 1⋅84 to 4⋅20) and low-income (OR 2⋅97, 1⋅84 to 4⋅81) countries. Surgical safety checklist use was less frequent in low- and middle-income countries, but when used was associated with reduced mortality at 30 days. Conclusion: Mortality is three times higher in low- compared with high-HDI countries even when adjusted for prognostic factors. Patient safety factors may have an important role. Registration number: NCT02179112 (http://www.clinicaltrials.gov)

Induction of Oxidative Stress and Mitochondrial Dysfunction by Juglone Affects the Development of Bovine Oocytes

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus&ndash;oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 &micro;M). In addition, RT&ndash;qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8&ndash;16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 &micro;M) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5&ndash;50.0 &micro;M of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction