Rajiv G. Tawar

A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission.

Background and Aims Hepatitis C virus (HCV) infection is a challenge to prevent and treat because of the rapid development of drug resistance and escape. Viral entry is required for initiation, spread, and maintenance of infection, making it an attractive target for antiviral strategies. Methods Using genetic immunization, we produced four monoclonal antibodies (mAbs) against the HCV host entry factor CD81. The effects of antibodies on inhibition of HCV infection and dissemination were analyzed in HCV permissive human liver cell lines. Results The anti-CD81 mAbs efficiently inhibited infection by HCV of different genotypes as well as a HCV escape variant selected during liver transplantation and re-infecting the liver graft. Kinetic studies indicated that anti-CD81 mAbs target a post-binding step during HCV entry. In addition to inhibiting cell-free HCV infection, one antibody was also able to block neutralizing antibody-resistant HCV cell-cell transmission and viral dissemination without displaying any detectable toxicity. Conclusion A novel anti-CD81 mAb generated by genetic immunization efficiently blocks HCV spread and dissemination. This antibody will be useful to further unravel the role of virus-host interactions during HCV entry and cell-cell transmission. Furthermore, this antibody may be of interest for the development of antivirals for prevention and treatment of HCV infection.

CD81-Receptor Associations — Impact for Hepatitis C Virus Entry and Antiviral Therapies

Tetraspanins are integral transmembrane proteins organized in microdomains displaying specific and direct interactions with other tetraspanins and molecular partners. Among them, CD81 has been implicated in a variety of physiological and pathological processes. CD81 also plays a crucial role in pathogen entry into host cells, including hepatitis C virus (HCV) entry into hepatocytes. HCV is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV entry into hepatocytes is a complex process that requires the coordinated interaction of viral and host factors for the initiation of infection, including CD81, scavenger receptor BI, claudin-1, occludin, membrane-bound host cell kinases, Niemann-Pick C1 Like 1, Harvey rat sarcoma viral oncogene homolog (HRas), CD63 and transferrin receptor 1. Furthermore, recent data in HCV model systems have demonstrated that targeting critical components of tetraspanins and associated cell membrane proteins open new avenues to prevent and treat viral infection.

Virus-specific CD4+ T Cells Have Functional and Phenotypic Characteristics of Follicular T-helper Cells in Patients With Acute and Chronic HCV Infections.

BACKGROUND & AIMS Follicular T-helper (Tfh) cells contribute to pathogen-specific antibody responses by providing maturation signals to B cells. In mice with viral infections, virus-specific Tfh cells expand and are required to contain the infection. However, less is known about human virus-specific Tfh cells or their functions during infection. We investigated whether virus-specific CD4+ T cells from patients with hepatitis C virus (HCV) infection had phenotypic or functional features of Tfh cells and contribute to the production of HCV-specific antibodies. METHODS We collected blood samples from patients with acute and chronic HCV infection and healthy individuals (controls). We performed MHC class II tetramer analyses, assays to detect intracellular cytokines in response to HCV exposure, and analyses to quantify HCV-specific antibodies. In addition, we collected liver tissues from patients with chronic HCV infection or nonviral liver disease to analyze markers of Tfh cells. RESULTS HCV-specific CD4+ T cells from patients with acute HCV infection expressed markers of Tfh cells and secreted interleukin 21 in response to HCV exposure. Longitudinal analyses of HCV-specific T-cell responses and antibody responses showed an association between expression of inducible T-cell co-stimulator and induction of virus-specific antibodies in patients with acute HCV infection. Markers of Tfh cells were barely detectable in the peripheral blood samples from patients with chronic HCV infection, but were detected in liver tissues. CONCLUSIONS Virus-specific Tfh cells can be detected in blood samples from patients with acute HCV infection; inducible T-cell co-stimulator expression correlates with production of HCV-specific antibodies. In patients with chronic infection, Tfh cells seem to disappear from the blood but are detectable in the liver.

Claudins and pathogenesis of viral infection

Since their discovery, tremendous progress has been made in our understanding of the roles of claudins in tight junction physiology. In addition, interactions between claudins and other cellular proteins have highlighted their novel roles in cell physiology. Moreover, the importance of claudins is becoming apparent in the pathophysiology of several diseases, including viral infections. Notable is the discovery of CLDN1 as an essential host factor for hepatitis C virus (HCV) entry, which led to detailed characterization of CLDN1 and its association with tetraspanin CD81 for the initiation of HCV infection. CLDN1 has also been shown to facilitate dengue virus entry. Furthermore, owing to the roles of claudins in forming anatomical barriers, several viruses have been shown to alter claudin expression at the tight junction. This review summarizes the role of claudins in viral infection, with particular emphasis on HCV.

Humanisation of a claudin-1-specific monoclonal antibody for clinical prevention and cure of HCV infection without escape

Objective HCV infection is a leading cause of chronic liver disease and a major indication for liver transplantation. Although direct-acting antivirals (DAAs) have much improved the treatment of chronic HCV infection, alternative strategies are needed for patients with treatment failure. As an essential HCV entry factor, the tight junction protein claudin-1 (CLDN1) is a promising antiviral target. However, genotype-dependent escape via CLDN6 and CLDN9 has been described in some cell lines as a possible limitation facing CLDN1-targeted therapies. Here, we evaluated the clinical potential of therapeutic strategies targeting CLDN1. Design We generated a humanised anti-CLDN1 monoclonal antibody (mAb) (H3L3) suitable for clinical development and characterised its anti-HCV activity using cell culture models, a large panel of primary human hepatocytes (PHH) from 12 different donors, and human liver chimeric mice. Results H3L3 pan-genotypically inhibited HCV pseudoparticle entry into PHH, irrespective of donor. Escape was likely precluded by low surface expression of CLDN6 and CLDN9 on PHH. Co-treatment of a panel of PHH with a CLDN6-specific mAb did not enhance the antiviral effect of H3L3, confirming that CLDN6 does not function as an entry factor in PHH from multiple donors. H3L3 also inhibited DAA-resistant strains of HCV and synergised with current DAAs. Finally, H3L3 cured persistent HCV infection in human-liver chimeric uPA-SCID mice in monotherapy. Conclusions Overall, these findings underscore the clinical potential of CLDN1-targeted therapies and describe the functional characterisation of a humanised anti-CLDN1 antibody suitable for further clinical development to complement existing therapeutic strategies for HCV.

Broad neutralization of hepatitis C virus-resistant variants by Civacir hepatitis C immunoglobulin.

Hepatitis C virus (HCV)‐induced end‐stage liver disease is the major indication for liver transplantation (LT). However, reinfection of the liver graft is still common, especially in patients with detectable viral load at the time of LT. Limited data are available on direct‐acting antivirals in the transplant setting for prevention of graft infection. The human hepatitis C immunoglobulin (HCIG) Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing its safety and efficacy at preventing HCV recurrence after liver transplantation (LT) in the United States. Using well‐characterized patient‐derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles (HCVpp) and cell culture‐derived HCV (HCVcc) containing patient‐derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after LT. The human hepatitis C immune globulin Civacir is an investigational drug that is currently being developed in an ongoing phase 3 clinical trial assessing safety and efficacy to prevent HCV recurrence after LT in the United States. Using well‐characterized patient‐derived HCV variants selected during LT, we studied the molecular mechanism of action of Civacir. Inhibition of HCV infection was studied using infectious HCV models including HCV pseudoparticles and cell culture‐derived HCV containing patient‐derived viral envelope glycoproteins from 22 HCV variants isolated from patients before and after liver transplantation. Additionally, we studied neutralization of different HCV genotypes and of direct‐acting antiviral‐resistant viruses. Our results indicate that Civacir potently, broadly, and dose‐dependently neutralizes all tested patient variants in HCV pseudoparticles and cell culture‐derived HCV assays including variants displaying resistance to host neutralizing antibodies and antiviral monoclonal antibodies. The half‐maximal inhibitory concentrations were independent of the phenotype of the viral variant, indicating that virus neutralization by Civacir is not affected by viral selection. Furthermore, Civacir is equally active against tested direct‐acting antiviral‐resistant HCV isolates in cell culture. Conclusion: Collectively, these results demonstrate broad neutralizing activity of Civacir against resistant viruses, likely due to synergy between anti‐HCV antibodies derived from different plasma donors, and support its further clinical development for prevention of liver graft infection. (Hepatology 2016;64:1495‐1506)

Acute hepatitis C virus infection induces anti‐host cell receptor antibodies with virus‐neutralizing properties

Hepatitis C virus (HCV) causes persistent infection in the majority of infected individuals. The mechanisms of persistence and clearance are only partially understood. Antibodies (Abs) against host cell entry receptors have been shown to inhibit HCV infection in cell culture and animal models. In this study, we aimed to investigate whether anti‐receptor Abs are induced during infection in humans in vivo and whether their presence is associated with outcome of infection. We established an enzyme‐linked immunosorbant assay using a recombinant CD81‐claudin‐1 (CLDN1) fusion protein to detect and quantify Abs directed against extracellular epitopes of the HCV CD81‐CLDN1 coreceptor complex. The presence of anti‐receptor Abs was studied in serum of patients from a well‐defined cohort of a single‐source HCV outbreak of pregnant women and several control groups, including uninfected pregnant women, patients with chronic hepatitis B and D virus (HBV/HDV) infection, and healthy individuals. Virus‐neutralizing activity of Abs was determined using recombinant cell culture–derived HCV (HCVcc). Our results demonstrate that HCV‐infected patients have statistically significantly higher anti‐CD81/CLDN1 Ab titers during the early phase of infection than controls. The titers were significantly higher in resolvers compared to persisters. Functional studies using immunoadsorption and HCV cell culture models demonstrate that HCV‐neutralizing anti‐receptor Abs are induced in the early phase of HCV infection, but not in control groups. Conclusion: The virus‐neutralizing properties of these Abs suggest a role for control of viral infection in conjunction with antiviral responses. Characterization of these anti‐receptor Abs opens new avenues to prevent and treat HCV infection. (Hepatology 2015;62:726–736)

HCV Receptors and Virus Entry

Hepatitis C virus (HCV) enters its target cells in a tightly regulated process that relies on several host factors. These host factors can be classified into three distinct categories depending on their role in the entry process, namely attachment factors, entry factors and facilitators. HCV entry is the target of host neutralizing anti-HCV antibody response. Furthermore, HCV entry is the first step of virus-host interaction and thus, in-part determines the hepato- and species tropism of the virus. Understanding HCV entry is, thus, not only crucial for development of novel entry-inhibitors and vaccine but also for developing new animal models to evaluate novel therapeutics and study disease pathogenesis. Research in the last 25 years has given unprecedented insights into the molecular aspects of HCV entry and its intricate association with the host. This review summarizes our current knowledge of the host factors involved in HCV entry and the molecular mechanisms determining the entry process.

A new HCV mouse model on the block

The investigation of virus-induced liver disease and hepatocellular carcinoma needs small animal models modeling hepatitis C virus (HCV) infection and liver disease biology. A recent study published in Cell Research reports a novel mouse model which is permissive for chronic HCV infection and shows chronic liver injury including inflammation, steatosis and fibrosis.

P0677 : Civacir hepatitis C immune globulin (HCIG) potently neutralizes infection of hepatitis C virus transplant escape variants

Figure 3. Civacir/HCIG neutralizes HCVpp entry of escape and non-escape variants in primary human hepatocytes. (A) HCVpp derived from representative escape (blue line) and non-escape (black line) variants from liver transplant patients were incubated with different concentrations of Civacir/HCIG or control IgG preparation at 37° C for 1 hour and subsequently inoculated on primary human hepatocytes (PHH). The level of Infection was determined after 72 hours by measuring luciferase activity expressed as relative light unit (RLU). The level of HCVpp entry is shown as percentage of the control. (B) IC50 values of Civacir/HCIG against HCVpp derived from patient variants P2VJ, P1VL and P1VA. The IC50 values (μg/ml) were calculated by non-linear three parameter least squares analysis. Rajiv G. Tawar1,2, Laura Heydmann1, 2, Jörg Schüttrumpf3, Shailesh Chavan4, Mirjam B. Zeisel1,2 and Thomas F. Baumert1,2,5 1Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France ; 2University of Strasbourg, Strasbourg, France; 3Biotest AG, Dreieich, Germany; 4Clinical Research, Medical Affairs and Drug Safety, Biotest Pharmaceuticals, Boca Raton, Florida, United States; 5Pôle Hépato-digestif, Strasbourg University Hospital, Strasbourg, France