The role of Januskinases Jak1 and Tyk2 in monocyte-induced growth inhibition of smooth muscle cells

Das Wechselspiel von Monozyten und glatten Gefäßmuskelzellen (VSMC) spielt eine bedeutende Rolle bei den Vorgängen nach Gefäßwandverletzung. Vor allem geschieht dies über eine Modulation des funktionellen Verhaltens der VSMC, die über eine Interaktion von monozytärem urokinase Plasminogenaktivator (uPA) und ihrem uPA Rezeptor (uPAR) realisiert wird. Es konnte gezeigt werden, dass der Jak/Stat-Signalweg die uPA/uPAR-induzierte Migration und Proliferation von VSMC vermittelt. Die vorliegende Arbeit untersucht molekulare Mechanismen, die bisher noch nicht vollständig verstanden waren. Im Einzelnen hatte diese Arbeit die Identifizierung der Kinase zum Ziel, die innerhalb der Signalkaskade zur Phosphorylierung von Stat1 durch monozytäres uPA und den damit verbundenen Einfluss auf das Zellwachstum führt. VSMC, die die kinasedomäne-defiziente Mutante von Jak1 und Tyk2 exprimieren wurden in einem Monozyten-Kokulturmodell verwendet, das die Vorgänge nach Gefäßwandverletzung nachahmte. Die Arbeit zeigt auf, dass Tyk2, nicht hingegen Jak1, die monozyten-induzierte uPA-vermittelte Phosphorylierung von Stat1 und die VSMC- Wachstumshemmung bewirkt und legt eine neuartige Funktion für Tyk2 als wichtigen Modulator der monozyteninduzierten Funktionsänderung von VSMC am Ort der Gefäßwandverletzung nahe.The interaction between monocytes and vascular smooth muscle cells (VSMC) plays an important role in the response of the vessel wall to injury, presumably by modulating VSMC functional behaviour via an interaction of monozytic urokinase plasminogen (uPA) and its uPA receptor (uPAR). The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR- directed cell migration and proliferation in VSMC. This work investigates the underlying molecular mechanisms, which remained not completely understood. In particular, this work aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by monozytic uPA and its impact on VSMC growth. VSMC expressing kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 were used in a monocyte coculture model imitating the response to vascular injury. This work provides evidence that Tyk2, but not Jak1, mediates moncyte-induced Stat1 phosphorylation and VSMC growth inhibition via secretion of uPA and suggests a novel function for Tyk2 as an important modulator of the monocyte-directed VSMC functional behaviour at the place of injury

Visualizing hepatitis C virus infection in humanized mice

Hepatitis C virus (HCV) establishes frequently persistent infections. Chronic carriers can develop severe liver disease. HCV has been intensely studied in a variety of cell culture systems. However, commonly used cell lines and primary hepatocyte cultures do not or only in part recapitulate the intricate host environment HCV faces in the liver. HCV infects readily only humans and chimpanzees, which poses challenges in studying HCV infection in vivo. Consequently, tractable small animal models are needed that are not only suitable for analyzing HCV infection but also for testing novel therapeutics. Here, we will focus our discussion on humanized mice, i.e. mice engrafted with human tissues or expressing human genes, which support HCV infection. We will further highlight novel methods that can be used to unambiguously detect HCV infected cells in situ, thereby facilitating a spatio-temporal dissection of HCV infection in the three dimensional context of the liver

Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo. IMPORTANCE At least 150 million individuals are chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can result in progressive liver disease and liver cancer. New antiviral treatments can cure HCV in the majority of patients, but a vaccine remains elusive. To gain a better understanding of the processes culminating in liver failure and cancer and to prioritize vaccine candidates more efficiently, small-animal models are needed. Here, we describe the characterization of a new mouse model in which the parts of two host factors that are essential for HCV uptake, CD81 and occludin (OCLN), which differ between mice and humans, were humanized. We demonstrate that such minimally humanized mice develop normally, express the modified genes at physiological levels, and support HCV uptake. This model is of considerable utility for studying viral entry in the three-dimensional context of the liver and to test approaches aimed at preventing HCV entry

Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo

Hepatitis C virus (HCV) causes chronic infections in at least 150 million individuals worldwide. HCV has a narrow host range and robustly infects only humans and chimpanzees. The underlying mechanisms for this narrow host range are incompletely understood. At the level of entry, differences in the amino acid sequences between the human and mouse orthologues of two essential host factors, the tetraspanin CD81 and the tight junction protein occludin (OCLN), explain, at least in part, HCV's limited ability to enter mouse hepatocytes. We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo. In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake. We demonstrate that the humanized CD81 and OCLN were expressed at physiological levels in a tissue-appropriate fashion. Mice bearing the humanized alleles formed normal tight junctions and did not exhibit any immunologic abnormalities, indicating that interactions with their physiological ligands were intact. HCV entry factor knock-in mice take up HCV with an efficiency similar to that in mice expressing HCV entry factors transgenically or adenovirally, demonstrating the utility of this model for studying HCV infection in vivo

Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice

Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines

Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice

Hepatitis C virus (HCV) infection is a global health problem for which no vaccine is available. HCV has a highly heterogeneous RNA genome and can be classified into seven genotypes. Due to the high genetic and resultant antigenic variation among the genotypes, inducing antibodies capable of neutralizing most of the HCV genotypes by experimental vaccination has been challenging. Previous efforts focused on priming humoral immune responses with recombinant HCV envelope E2 protein produced in mammalian cells. Here, we report that a soluble form of HCV E2 (sE2) produced in insect cells possesses different glycosylation patterns and is more immunogenic, as evidenced by the induction of higher titers of broadly neutralizing antibodies (bNAbs) against cell culture-derived HCV (HCVcc) harboring structural proteins from a diverse array of HCV genotypes. We affirm that continuous and discontinuous epitopes of well-characterized bNAbs are conserved, suggesting that sE2 produced in insect cells is properly folded. In a genetically humanized mouse model, active immunization with sE2 efficiently protected against challenge with a heterologous HCV genotype. These data not only demonstrate that sE2 is a promising HCV vaccine candidate, but also highlight the importance of glycosylation patterns in developing subunit viral vaccines. IMPORTANCE A prophylactic vaccine with high efficacy and low cost is urgently needed for global control of HCV infection. Induction of broadly neutralizing antibodies against most HCV genotypes has been challenging due to the antigenic diversity of the HCV genome. Here, we refined a high-yield subunit HCV vaccine that elicited broadly neutralizing antibody responses in preclinical trials. We found that soluble HCV E2 protein (sE2) produced in insect cells is distinctly glycosylated and is more immunogenic than sE2 produced in mammalian cells, suggesting that glycosylation patterns should be taken into consideration in efforts to generate antibody-based recombinant vaccines against HCV. We further showed that sE2 vaccination confers protection against HCV infection in a genetically humanized mouse model. Thus, our work identified a promising broadly protective HCV vaccine candidate that should be considered for further preclinical and clinical development

Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4

Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis

Identification, Molecular Cloning, and Analysis of Full-Length Hepatitis C Virus Transmitted/Founder Genotypes 1, 3, and 4

Hepatitis C virus (HCV) infection is characterized by persistent replication of a complex mixture of viruses termed a “quasispecies.” Transmission is generally associated with a stringent population bottleneck characterized by infection by limited numbers of “transmitted/founder” (T/F) viruses. Characterization of T/F genomes of human immunodeficiency virus type 1 (HIV-1) has been integral to studies of transmission, immunopathogenesis, and vaccine development. Here, we describe the identification of complete T/F genomes of HCV by single-genome sequencing of plasma viral RNA from acutely infected subjects. A total of 2,739 single-genome-derived amplicons comprising 10,966,507 bp from 18 acute-phase and 11 chronically infected subjects were analyzed. Acute-phase sequences diversified essentially randomly, except for the poly(U/UC) tract, which was subject to polymerase slippage. Fourteen acute-phase subjects were productively infected by more than one genetically distinct virus, permitting assessment of recombination between replicating genomes. No evidence of recombination was found among 1,589 sequences analyzed. Envelope sequences of T/F genomes lacked transmission signatures that could distinguish them from chronic infection viruses. Among chronically infected subjects, higher nucleotide substitution rates were observed in the poly(U/UC) tract than in envelope hypervariable region 1. Fourteen full-length molecular clones with variable poly(U/UC) sequences corresponding to seven genotype 1a, 1b, 3a, and 4a T/F viruses were generated. Like most unadapted HCV clones, T/F genomes did not replicate efficiently in Huh 7.5 cells, indicating that additional cellular factors or viral adaptations are necessary for in vitro replication. Full-length T/F HCV genomes and their progeny provide unique insights into virus transmission, virus evolution, and virus-host interactions associated with immunopathogenesis