Hepatitis C virus (HCV) establishes a chronic infection in the majority of exposed individuals and can cause cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. Here, we demonstrate that three broadly nAbs, AR3A, AR3B and AR4A, delivered with adeno-associated viral (AAV) vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a novel therapeutic approach to interfere with HCV infection exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes in order to sustain chronicity.

SummaryInterferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.

An estimated 71 million people worldwide are infected with hepatitis C virus (HCV). The lack of small animal models has impeded studies of antiviral immune mechanisms. Here we show that an HCV-related hepacivirus discovered in Norway rats can establish high titer hepatotropic infections in laboratory mice with immunological features resembling those seen in human viral hepatitis. While immune-compromised mice developed persistent infection, immune-competent mice cleared the virus within 3–5 weeks. Acute clearance was T cell dependent and associated with liver injury. Transient depletion of CD4+ T cells prior to infection resulted in chronic infection, characterized by high levels of intrahepatic regulatory T cells and expression of inhibitory molecules on intrahepatic CD8+ T cells. Natural killer cells controlled early infection but were not essential for viral clearance. This model may provide mechanistic insights into hepatic antiviral immunity, a prerequisite for the development of HCV vaccines.

Umbilical cord blood (UCB) is a rich source of hematopoetic stem cells (HSCs). We have isolated a novel cell line population of stem cells from human UCB that exhibit properties of self-renewal, but do not have cell-surface markers that are typically found on HSCs. Analysis of transcripts revealed that these cells express transcription factors Oct-4, Rex-1, and Sox-2 that are typically expressed by stem cells. We refer to these novel cells as nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs). Previous studies have shown that the intravenous infusion of UCBCs can ameliorate neurological deficits arising from ischemic brain injury. The identity of the cells that mediate this restorative effect, however, has yet to be determined. We postulate that nh-UCBSCs may be a source of the UCB cells that can mediate these effects. To test this hypothesis, we intravenously injected one million human nh-UCBSCs into rats 48 h after transient unilateral middle cerebral artery occlusion. Animals in other experimental groups received either saline injections or injections of RN33b neural stem cells. Animals were tested for neurological function before the infusion of nh-UCBSCs and at various time periods afterwards using a battery of behavioral tests. In limb placement tests, animals treated with nh-UCBSCs exhibited mean scores that were significantly better than animals treated with RN33b neural stem cells or saline. Similarly, in stepping tests, nh-UCBSC-treated animals again exhibited significantly better performance than the other experimental groups of animals. Analysis of infarct volume revealed that ischemic animals treated with nh-UCBSCs exhibited a 50% reduction in lesion volume in comparison to saline-treated controls. Histological analysis of brain tissue further revealed the presence of cells that stained for human nuclei. Some human nuclei-positive cells were also co-labeled for NeuN, indicating that the transplanted cells expressed markers of a neuronal phenotype. Cells expressing the human nuclei marker within the brain, however, were rather scant, suggesting that the restorative effects of nh-UCBSCs may be mediated by mechanisms other than cell replacement. To test this hypothesis, nh-UCBSCs were directly transplanted into the brain parenchyma after ischemic brain injury. Sprouting of nerve fibers from the nondamaged hemisphere into the ischemically damaged side of the brain was assessed by anterograde tracing using biotinylated dextran amine (BDA). Animals with nh-UCBSC transplants exhibited significantly greater densities of BDA-positive cells in the damaged side of the brain compared to animals with intraparenchymal saline injections. These results suggest that restorative effects observed with nh-UCBSC treatment following ischemic brain injury may be mediated by trophic actions that result in the reorganization of host nerve fiber connections within the injured brain.

In spite of the vast collective experience in tissue engineering, control of both tissue architecture and scale are fundamental translational roadblocks. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. Here, we introduce an approach called ‘SEEDs’ (‘in Situ Expansion of Engineered Devices’), in which we fabricate a structurally organized engineered tissue unit that expands in response to regenerative cues after implantation. We find that tissues containing pre-patterned human primary hepatocytes, endothelial cells, and stromal cells in degradable hydrogel expand over 50-fold over the course of 11 weeks in animals with liver injury, with concomitant increased function as characterized by the production of multiple human liver proteins. Histologically, we observe the emergence of stereotypical microstructure via coordinated growth of hepatocytes in close juxtaposition with a perfused, chimeric vasculature. Importantly, we demonstrate the utility of this platform for probing the impact of multicellular geometric architecture on tissue expansion in response to regenerative cues. This approach represents a hybrid strategy that harnesses both biology and engineering to deploy a limited cell mass more efficiently than either approach could do in isolation, and thus offers a new convergent paradigm for tissue engineering.