Thomas Vanwolleghem

Antigen sparing and cross-reactive immunity with an adjuvanted rH5N1 prototype pandemic influenza vaccine: a randomised controlled trial.

BACKGROUND Antigen sparing is regarded as crucial for pandemic vaccine development because worldwide influenza vaccine production capacity is limited. Adjuvantation is an important antigen-sparing strategy. We assessed the safety and immunogenicity of a recombinant H5N1 split-virion vaccine formulated with a proprietary adjuvant system and investigated whether it can induce cross-reactive immunity. METHODS Two doses of an inactivated split A/Vietnam/1194/2004 NIBRG-14 (recombinant H5N1 engineered by reverse genetics) vaccine were administered 21 days apart to eight groups of 50 volunteers aged 18-60 years. We studied four antigen doses (3.8 microg, 7.5 microg, 15 microg, and 30 microg haemagglutinin) given with or without adjuvant. Blood samples were collected to analyse humoral immune response. Adverse events were recorded up through study day 51. Safety analyses were of the whole vaccinated cohort and immunogenicity analyses per protocol. This trial is registered with the ClinicalTrials.gov, number NCT00309634. FINDINGS All eight vaccine formulations had a good safety profile. No serious adverse events were reported. The adjuvanted vaccines induced more injection-site symptoms and general symptoms than did the non-adjuvanted vaccines, but most were mild to moderate in intensity and transient in nature. The adjuvanted formulations were significantly more immunogenic than the non-adjuvanted formulations at all antigen doses. At the lowest antigenic dose (3.8 microg), immune responses for the adjuvanted vaccine against the recombinant homologous vaccine strain (A/Vietnam/1194/2004 NIBRG-14, clade 1) met or exceeded all US Food and Drug Administration and European Union licensure criteria. Furthermore, 37 of 48 (77%) participants receiving 3.8 microg of the adjuvanted vaccine seroconverted for neutralising antibodies against a strain derived by reverse genetics from a drifted H5N1 isolate (A/Indonesia/5/2005, clade 2). INTERPRETATION Adjuvantation conferred significant antigen sparing that could increase the production capacity of pandemic influenza vaccine. Moreover, the cross-clade neutralising antibody responses recorded imply that such a vaccine could be deployed for immunisation before a pandemic.

Anti‐CD81 antibodies can prevent a hepatitis C virus infection in vivo

The viral life cycle of the hepatitis C virus (HCV) has been studied mainly using different in vitro cell culture models. Studies using pseudoviral particles (HCVpp) and more recently cell culture–derived virus (HCVcc) suggest that at least three host cell molecules are important for HCV entry in vitro: the tetraspanin CD81, the scavenger receptor class B member I, and the tight junction protein Claudin‐1. Whether these receptors are equally important for an in vivo infection remains to be demonstrated. We show that CD81 is indispensable for an authentic in vivo HCV infection. Prophylactic treatment with anti‐CD81 antibodies completely protected human liver‐uPA‐SCID mice from a subsequent challenge with HCV consensus strains of different genotypes. Administration of anti‐CD81 antibodies after viral challenge had no effect. Conclusion: Our experiments provide evidence for the critical role of CD81 in a genuine HCV infection in vivo and open new perspectives for the prevention of allograft reinfection after orthotopic liver transplantation in chronically infected HCV patients. (HEPATOLOGY 2008;48:1761–1768.)

Scavenger Receptor BI and BII Expression Levels Modulate Hepatitis C Virus Infectivity

ABSTRACT Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.

Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver–chimeric mice from infection with a homologous hepatitis C virus strain

The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these IgG can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver–urokinase‐type plasminogen activator (uPA)+/+ severe combined immune deficient (SCID) mice. Chimeric mice were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG‐treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C‐HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve chimeric mice, the inoculum was pre‐incubated in vitro at an IgG concentration normally observed in humans. Conclusion: Polyclonal IgG from a patient with a long‐standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the ancestral HCV strain in vivo, using the human liver–chimeric mouse model. Both experimental systems will be useful tools to identify neutralizing antibodies for future clinical use. (HEPATOLOGY 2008.)

In Vivo Evaluation of the Cross-Genotype Neutralizing Activity of Polyclonal Antibodies Against Hepatitis C Virus

Control of hepatitis C virus (HCV) infection remains a huge challenge of global medical importance. Using a variety of in vitro approaches, neutralizing antibodies (nAbs) have been identified in patients with acute and chronic hepatitis C. The exact role these nAbs play in the resolution of acute HCV infection still remains elusive. We have previously shown that purified polyclonal antibodies isolated from plasma obtained in 2003 from a chronic HCV patient (Patient H) can protect human liver chimeric mice from a subsequent challenge with the autologous HCV strain isolated from Patient H in 1977 (H77). In this study we investigated whether polyclonal antibodies isolated from Patient H in 2006 (H06), which display high cross‐genotype neutralizing activity in both the HCV pseudoparticle (HCVpp) and HCV cell culture (HCVcc) systems, were also able to prevent HCV infection of different genotypes (gt) in vivo. Following passive immunization with H06‐antibodies, chimeric mice were challenged with the consensus strains H77C (gt1a), ED43 (gt4a), or HK6a (gt6a). In accordance with previous results, H06‐antibodies prevented infection of chimeric mice with the autologous virus. However, the outcome of a homologous challenge is highly influenced by the amount of challenge virus injected. Depending on the viral genotype used, H06‐antibodies were able to protect up to 50% of chimeric mice from a heterologous challenge. Animals in which the antibody pretreatment failed displayed a clear delay in the kinetics of viral infection. Sequence analysis of the recovered viruses did not suggest antibody‐induced viral escape. Conclusion: Polyclonal anti‐HCV antibodies isolated from a chronic HCV patient can protect against an in vivo challenge with different HCV genotypes. However, the in vivo protective efficacy of cross‐genotype neutralizing antibodies was less than predicted by cell culture experiments. (HEPATOLOGY 2011)

Experimental models for hepatitis C viral infection.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease. The majority of infected individuals develop a persistent infection, which is associated with a high risk of liver cirrhosis and hepatocellular carcinoma. Since its discovery 20 years ago, progress in our understanding of this virus has been suboptimal due to the lack of good model systems. However, in the past decade this has greatly accelerated with the development of various in vitro cell culture systems and in vivo small‐animal models. These systems have made a major impact on the field of HCV research, and have provided important breakthroughs in our understanding of HCV infection and replication. Importantly, the in vitro cell culture systems and the small‐animal models have allowed preclinical testing of numerous novel antiviral compounds for the treatment of chronic HCV infection. In this article, we give an overview of current models, discuss their limitations, and provide future perspectives for research directed at the prevention and cure of hepatitis C. (HEPATOLOGY 2009.)

Factors determining successful engraftment of hepatocytes and susceptibility to hepatitis B and C virus infection in uPA-SCID mice

BACKGROUND & AIMS The human liver-uPA(+/+)-SCID mouse is currently the best small animal model available for viral hepatitis infection studies. METHODS We identify critical factors affecting animal survival, engraftment efficacy, kinetics of liver repopulation and virological outcome by analysing the data from 400 human hepatocyte transplantations and 115 subsequent HBV and/or HCV inoculations in this mouse model. RESULTS Almost one third of animals succumbed during the first week after hepatocyte transplantation. Only during this critical period, liver necrosis due to embolization of donor cells in the portal vein was observed. This may have caused a fatal acute liver failure that complicated the pre-existing chronic liver disease. From the second week onwards, confluent hepatocyte clusters repopulated the liver and restored its synthetic functions as evidenced by increasing human albumin levels in plasma. Xenogenic repopulation by human cells proceeded approximately 4-times slower compared to allogenic mouse hepatocytes. All HBV inoculations were successful, even in animals with low graft take. HCV infection rate varied substantially, although every donor cell type yielded infectable animals. A reproducible 100% HCV infectivity was reached with high quality inocula in animals with human albumin plasma levels >1 mg/ml. Superior animal survival, adequate liver engraftment and a high viral infection rate were observed after transplanting cryopreserved commercial human hepatocytes. CONCLUSIONS Our findings favour the use of commercially available, cryopreserved human hepatocytes for the humanization of the uPA(+/+)-SCID liver. While HBV infectivity criteria are less stringent, human albumin plasma levels exceeding 1 mg/ml are required for a consistent HCV infection in chimeric mice.

Shear-wave elastography of the liver and spleen identifies clinically significant portal hypertension: A prospective multicentre study.

Clinically significant portal hypertension (CSPH) is associated with severe complications and decompensation of cirrhosis. Liver stiffness measured either by transient elastography (TE) or Shear‐wave elastography (SWE) and spleen stiffness by TE might be helpful in the diagnosis of CSPH. We recently showed the algorithm to rule‐out CSPH using sequential liver‐ (L‐SWE) and spleen‐Shear‐wave elastography (S‐SWE). This study investigated the diagnostic value of S‐SWE for diagnosis of CSPH.

Hepatitis E Virus (HEV) Genotype 3 Infection of Human Liver Chimeric Mice as a Model for Chronic HEV Infection

ABSTRACT Genotype 3 (gt3) hepatitis E virus (HEV) infections are emerging in Western countries. Immunosuppressed patients are at risk of chronic HEV infection and progressive liver damage, but no adequate model system currently mimics this disease course. Here we explore the possibilities of in vivo HEV studies in a human liver chimeric mouse model (uPA+/+Nod-SCID-IL2Rγ−/−) next to the A549 cell culture system, using HEV RNA-positive EDTA-plasma, feces, or liver biopsy specimens from 8 immunocompromised patients with chronic gt3 HEV. HEV from feces- or liver-derived inocula showed clear virus propagation within 2 weeks after inoculation onto A549 cells, compared to slow or no HEV propagation of HEV RNA-positive, EDTA-plasma samples. These in vitro HEV infectivity differences were mirrored in human-liver chimeric mice after intravenous (i.v.) inoculation of selected samples. HEV RNA levels of up to 8 log IU HEV RNA/gram were consistently present in 100% of chimeric mouse livers from week 2 to week 14 after inoculation with human feces- or liver-derived HEV. Feces and bile of infected mice contained moderate to large amounts of HEV RNA, while HEV viremia was low and inconsistently detected. Mouse-passaged HEV could subsequently be propagated for up to 100 days in vitro. In contrast, cell culture-derived or seronegative EDTA-plasma-derived HEV was not infectious in inoculated animals. In conclusion, the infectivity of feces-derived human HEV is higher than that of EDTA-plasma-derived HEV both in vitro and in vivo. Persistent HEV gt3 infections in chimeric mice show preferential viral shedding toward mouse bile and feces, paralleling the course of infection in humans. IMPORTANCE Hepatitis E virus (HEV) genotype 3 infections are emerging in Western countries and are of great concern for immunosuppressed patients at risk for developing chronic HEV infection. Lack of adequate model systems for chronic HEV infection hampers studies on HEV infectivity and transmission and antiviral drugs. We compared the in vivo infectivity of clinical samples from chronic HEV patients in human liver chimeric mice to an in vitro virus culture system. Efficient in vivo HEV infection is observed after inoculation with feces- and liver-derived HEV but not with HEV RNA-containing plasma or cell culture supernatant. HEV in chimeric mice is preferentially shed toward bile and feces, mimicking the HEV infection course in humans. The observed in vivo infectivity differences may be relevant for the epidemiology of HEV in humans. This novel small-animal model therefore offers new avenues to unravel HEVs pathobiology.

Inflammatory monocytes recruited to the liver within 24 hours after virus-induced inflammation, resemble Kupffer cells, but are functionally distinct

ABSTRACT Due to a scarcity of immunocompetent animal models for viral hepatitis, little is known about the early innate immune responses in the liver. In various hepatotoxic models, both pro- and anti-inflammatory activities of recruited monocytes have been described. In this study, we compared the effect of liver inflammation induced by the Toll-like receptor 4 ligand lipopolysaccharide (LPS) with that of a persistent virus, lymphocytic choriomeningitis virus (LCMV) clone 13, on early innate intrahepatic immune responses in mice. LCMV infection induces a remarkable influx of inflammatory monocytes in the liver within 24 h, accompanied by increased transcript levels of several proinflammatory cytokines and chemokines in whole liver. Importantly, while a single LPS injection results in similar recruitment of inflammatory monocytes to the liver, the functional properties of the infiltrating cells are dramatically different in response to LPS versus LCMV infection. In fact, intrahepatic inflammatory monocytes are skewed toward a secretory phenotype with impaired phagocytosis in LCMV-induced liver inflammation but exhibit increased endocytic capacity after LPS challenge. In contrast, F4/80high-Kupffer cells retain their steady-state endocytic functions upon LCMV infection. Strikingly, the gene expression levels of inflammatory monocytes dramatically change upon LCMV exposure and resemble those of Kupffer cells. Since inflammatory monocytes outnumber Kupffer cells 24 h after LCMV infection, it is highly likely that inflammatory monocytes contribute to the intrahepatic inflammatory response during the early phase of infection. Our findings are instrumental in understanding the early immunological events during virus-induced liver disease and point toward inflammatory monocytes as potential target cells for future treatment options in viral hepatitis. IMPORTANCE Insights into how the immune system deals with hepatitis B virus (HBV) and HCV are scarce due to the lack of adequate animal model systems. This knowledge is, however, crucial to developing new antiviral strategies aimed at eradicating these chronic infections. We model virus-host interactions during the initial phase of liver inflammation 24 h after inoculating mice with LCMV. We show that infected Kupffer cells are rapidly outnumbered by infiltrating inflammatory monocytes, which secrete proinflammatory cytokines but are less phagocytic. Nevertheless, these recruited inflammatory monocytes start to resemble Kupffer cells on a transcript level. The specificity of these cellular changes for virus-induced liver inflammation is corroborated by demonstrating opposite functions of monocytes after LPS challenge. Overall, this demonstrates the enormous functional and genetic plasticity of infiltrating monocytes and identifies them as an important target cell for future treatment regimens.