We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2±CD81 interaction is isolate speci®c. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identi®ed the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2±SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-speci®c manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody. Keywords: hepatitis C virus/hypervariable region 1/ scavenger receptor class B type I/second envelope glycoprotein

Several cell surface molecules have been proposed as receptor candidates, mediating cell entry of hepatitis C virus (HCV) on the basis of their physical association with virions or with soluble HCV E2 glycoproteins. However, due to the lack of infectious HCV particles, evidence that these receptor candidates support infection was missing. Using our recently described infectious HCV pseudotype particles (HCVpp) that display functional E1E2 glycoprotein complexes, here we show that HCV is a pH-dependent virus, implying that its receptor component(s) mediate virion internalization by endocytosis. Expression of the CD81 tetraspanin in non-permissive CD81-negative hepato-carcinoma cells was sufficient to restore susceptibility to HCVpp infection, confirming its critical role as a cell attachment factor. As a cell surface molecule likely to mediate endosomal trafficking, we demonstrate that the human scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein-internalization molecule that we previously proposed as a novel HCV receptor candidate due to its affinity with E2 glycoproteins, is required for infection of CD81-expressing hepatic cells. By receptor competition assays, we found that SR-B1 antibodies that blocked binding of soluble E2 could prevent HCVpp infectivity. Furthermore, we establish that the hyper-variable region 1 of the HCV E2 glycoprotein is a critical determinant mediating entry in SR-B1-positive cells. Finally, by correlating expression of HCV receptors and infectivity, we suggest that, besides CD81 and SR-B1, additional hepatocyte-specific co-factor(s) are necessary for HCV entry. Hepatitis C virus (HCV)1 is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1).About 170 million individuals are infected with HCV. The predominant site of replication and probably infection is the liver, yet several studies have suggested that HCV may infect other cell types, such as B cells, monocytes/macrophages, and dendritic cells (2, 3) where it could induce immune dysfunctions. Understanding the molecular basis by which HCV targets liver cells and extra-hepatic sites is critical to unravel the mechanisms of its pathogenesis and disease chronicity. Although substantial progresses have been made on the molecular knowledge of HCV proteins and genomic replication, there is still no efficient and reliable cell culture system available to amplify this virus (4). This represents a major hurdle for the characterization of some steps of HCV lifecycle, such as viral assembly and cell entry. Several surrogate models of HCV virions have been developed to facilitate the study of the steps of cell entry. Production of HCV virus-like particles in insect cells has already been reported (5-8). Yet, although useful to analyze interactions with the cell surface, these particles were not infectious and did not permit functional investigation of the putative HCV receptors. Alternatively, generation of viral pseudotypes is one of the most widely used methods for assaying function...

The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.

Neoantigens (nAgs) are promising tumor antigens for cancer vaccination with the potential of inducing robust and selective T cell responses. Genetic vaccines based on Adenoviruses derived from non-human Great Apes (GAd) elicit strong and effective T cell-mediated immunity in humans. Here, we investigate for the first time the potency and efficacy of a novel GAd encoding multiple neoantigens. Prophylactic or early therapeutic vaccination with GAd efficiently control tumor growth in mice. In contrast, combination of the vaccine with checkpoint inhibitors is required to eradicate large tumors. Gene expression profile of tumors in regression shows abundance of activated tumor infiltrating T cells with a more diversified TCR repertoire in animals treated with GAd and anti-PD1 compared to anti-PD1. Data suggest that effectiveness of vaccination in the presence of high tumor burden correlates with the breadth of nAgs-specific T cells and requires concomitant reversal of tumor suppression by checkpoint blockade.

Respiratory syncytial virus (RSV) causes respiratory infection in annual epidemics, with infants and the elderly at particular risk of developing severe disease and death. However, despite its importance, no vaccine exists. The chimpanzee adenovirus, PanAd3-RSV, and modified vaccinia virus Ankara, MVA-RSV, are replication defective viral vectors encoding the RSV proteins F, N and M2-1 for the induction of humoral and cellular responses. We performed an open-label, doseescalation, phase 1 clinical trial in 42 healthy adults in which four different combinations of prime/boost vaccinations were investigated for safety and immunogenicity, including both intramuscular and intra-nasal administration of the adenoviral vectored vaccine. The vaccines were safe and well tolerated, with the most common reported adverse events being mild injection site reactions. No vaccine-related serious adverse events occurred. RSV neutralising antibody titres rose in response to intramuscular (IM) prime with PanAd3-RSV, and after IM boost for individuals primed by the intra-nasal (IN) route. Circulating anti-F IgG and IgA antibody secreting cells (ASCs) were observed after IM prime and IM boost. RSV-specific T-cell responses were increased after IM PanAd3-RSV prime and were most efficiently boosted by IM MVA-RSV.Correspondence: christopher.green@paediatrics.ox.uk, tel/fax; 0044 +1865 857 420. Competing interests AJP has previously conducted clinical trials of vaccines on behalf of Oxford University funded by GlaxoSmithKline Biologicals SA and ReiThera S.r.l, but does not receive any personal payments from them. AJP is chair of the UK Department of Health's (DH) Joint Committee on Vaccination and Immunisation (JCVI), but the views expressed in this manuscript do not necessarily represent the views of JCVI or DH. AV, RC, and AN are named inventors on patent applications covering RSV antigen expression system (WO 2012/089833). The remaining authors declare they have no competing interests. IFNγ secretion after boost was from both CD4+ and CD8+ T-cells, without detectable Th2 cytokines that have been previously associated with immune pathogenesis following exposure to RSV after formalin inactivated RSV vaccine. In conclusion, PanAd3-RSV and MVA-RSV are safe and immunogenic in healthy adults. These vaccine candidates warrant further clinical evaluation of efficacy to assess their potential to reduce the burden of RSV disease. Data and materials availability Europe PMC Funders Group