Timothy Sheahan

Modeling hepatitis C virus infection using human induced pluripotent stem cells

Human pathogens impact patient health through a complex interplay with the host, but models to study the role of host genetics in this process are limited. Human induced pluripotent stem cells (iPSCs) offer the ability to produce host-specific differentiated cells and thus have the potential to transform the study of infectious disease; however, no iPSC models of infectious disease have been described. Here we report that hepatocyte-like cells derived from iPSCs support the entire life cycle of hepatitis C virus, including inflammatory responses to infection, enabling studies of how host genetics impact viral pathogenesis.

Hepatitis C virus induces interferon-λ and interferon-stimulated genes in primary liver cultures

Hepatitis C virus (HCV) replication in primary liver cells is less robust than that in hepatoma cell lines, suggesting that innate antiviral mechanisms in primary cells may limit HCV replication or spread. Here we analyzed the expression of 47 genes associated with interferon (IFN) induction and signaling following HCV infection of primary human fetal liver cell (HFLC) cultures from 18 different donors. We report that cell culture‐produced HCV (HCVcc) induced expression of Type III (λ) IFNs and of IFN‐stimulated genes (ISGs). Little expression of Type I IFNs was detected. Levels of IFNλ and ISG induction varied among donors and, often, between adapted and nonadapted HCV chimeric constructs. Higher levels of viral replication were associated with greater induction of ISGs and of λ IFNs. Gene induction was dependent on HCV replication, as ultraviolet light‐inactivated virus was not stimulatory and an antiviral drug, 2′‐C‐methyladenosine, reduced induction of λ IFNs and ISGs. The level of IFNλ protein induced was sufficient to inhibit HCVcc infection of naïve cultures. Conclusion: Together, these results indicate that despite its reported abilities to blunt the induction of an IFN response, HCV infection is capable of inducing antiviral cytokines and pathways in primary liver cell cultures. Induction of ISGs and λ IFNs may limit the growth and spread of HCV in primary cell cultures and in the infected liver. HCV infection of HFLC may provide a useful model for the study of gene induction by HCV in vivo. (HEPATOLOGY 2011;)

Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice

Defining prospective pathways by which zoonoses evolve and emerge as human pathogens is critical for anticipating and controlling both natural and deliberate pandemics. However, predicting tenable pathways of animal-to-human movement has been hindered by challenges in identifying reservoir species, cultivating zoonotic organisms in culture, and isolating full-length genomes for cloning and genetic studies. The ability to design and recover pathogens reconstituted from synthesized cDNAs has the potential to overcome these obstacles by allowing studies of replication and pathogenesis without identification of reservoir species or cultivation of primary isolates. Here, we report the design, synthesis, and recovery of the largest synthetic replicating life form, a 29.7-kb bat severe acute respiratory syndrome (SARS)-like coronavirus (Bat-SCoV), a likely progenitor to the SARS-CoV epidemic. To test a possible route of emergence from the noncultivable Bat-SCoV to human SARS-CoV, we designed a consensus Bat-SCoV genome and replaced the Bat-SCoV Spike receptor-binding domain (RBD) with the SARS-CoV RBD (Bat-SRBD). Bat-SRBD was infectious in cell culture and in mice and was efficiently neutralized by antibodies specific for both bat and human CoV Spike proteins. Rational design, synthesis, and recovery of hypothetical recombinant viruses can be used to investigate mechanisms of transspecies movement of zoonoses and has great potential to aid in rapid public health responses to known or predicted emerging microbial threats.

A human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo.

Endstage liver disease caused by chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation in the Western world. However, immediate reinfection of the grafted donor liver by circulating virus is inevitable and liver disease progresses much faster than the original disease. Standard antiviral therapy is not well tolerated and usually ineffective in liver transplant patients, whereas anti‐HCV immunotherapy is hampered by the extreme genetic diversity of the virus and its ability to spread by way of cell‐cell contacts. We generated a human monoclonal antibody against scavenger receptor class B type I (SR‐BI), monoclonal antibody (mAb)16‐71, which can efficiently prevent infection of Huh‐7.5 hepatoma cells and primary hepatocytes by cell‐culture‐derived HCV (HCVcc). Using an Huh7.5 coculture system we demonstrated that mAb16‐71 interferes with direct cell‐to‐cell transmission of HCV. Finally we evaluated the in vivo efficacy of mAb16‐71 in “human liver urokinase‐type plasminogen activator, severe combined immune deficiency (uPA‐SCID) mice” (chimeric mice). A 2‐week anti‐SR‐BI therapy that was initiated 1 day before viral inoculation completely protected all chimeric mice from infection with serum‐derived HCV of different genotypes. Moreover, a 9‐day postexposure therapy that was initiated 3 days after viral inoculation (when viremia was already observed in the animals) suppressed the rapid viral spread observed in untreated control animals. After cessation of anti‐SR‐BI‐specific antibody therapy, a rise of the viral load was observed. Conclusion: Using in vitro cell culture and human liver‐chimeric mouse models, we show that a human mAb targeting the HCV coreceptor SR‐BI completely prevents infection and intrahepatic spread of multiple HCV genotypes. This strategy may be an efficacious way to prevent infection of allografts following liver transplantation in chronic HCV patients, and may even hold promise for the prevention of virus rebound during or following antiviral therapy. (HEPATOLOGY 2012)

Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003/2004 by an independent animal-to-human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the human monoclonal antibodies (hmAbs) 80R and S3.1, which can potently neutralize isolates from the first outbreak. Here we report that two hmAbs, m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani or recombinant viruses bearing the GD03 and SZ16 spike (S) glycoproteins. Both antibodies competed with the SARS-CoV receptor, ACE2, for binding to the receptor-binding domain (RBD), suggesting a mechanism of neutralization that involves interference with the SARS-CoV–ACE2 interaction. Two putative hot-spot residues in the RBD (Ile-489 and Tyr-491) were identified within the SARS-CoV spike that likely contribute to most of the m396-binding energy. Residues Ile-489 and Tyr-491 are highly conserved within the SARS-CoV spike, indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS-CoV isolates with known sequences, except strains derived from bats. These antibodies exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets and could have potential applications for diagnosis, prophylaxis, and treatment of SARS-CoV infections.

Expression of Paramyxovirus V Proteins Promotes Replication and Spread of Hepatitis C Virus in Cultures of Primary Human Fetal Liver Cells

Here we demonstrate that primary cultures of human fetal liver cells (HFLC) reliably support infection with laboratory strains of hepatitis C virus (HCV), although levels of virus replication vary significantly between different donor cell preparations and frequently decline in a manner suggestive of active viral clearance. To investigate possible contributions of the interferon (IFN) system to control HCV infection in HFLC, we exploited the well‐characterized ability of paramyxovirus (PMV) V proteins to counteract both IFN induction and antiviral signaling. The V proteins of measles virus (MV) and parainfluenza virus 5 (PIV5) were introduced into HFLC using lentiviral vectors encoding a fluorescent reporter for visualization of HCV‐infected cells. V protein‐transduced HFLC supported enhanced (10 to 100‐fold) levels of HCV infection relative to untransduced or control vector‐transduced HFLC. Infection was assessed by measurement of virus‐driven luciferase, by assays for infectious HCV and viral RNA, and by direct visualization of HCV‐infected hepatocytes. Live cell imaging between 48 and 119 hours postinfection demonstrated little or no spread of infection in the absence of PMV V protein expression. In contrast, V protein‐transduced HFLC showed numerous HCV infection events. V protein expression efficiently antagonized the HCV‐inhibitory effects of added IFNs in HFLC. In addition, induction of the type III IFN, IL29, following acute HCV infection was inhibited in V protein‐transduced cultures. Conclusion: These studies suggest that the cellular IFN response plays a significant role in limiting the spread of HCV infection in primary hepatocyte cultures. Strategies aimed at dampening this response may be key to further development of robust HCV culture systems, enabling studies of virus pathogenicity and the mechanisms by which HCV spreads in its natural host cell population. (HEPATOLOGY 2011;)

MyD88 is required for protection from lethal infection with a mouse-adapted SARS-CoV.

A novel human coronavirus, SARS-CoV, emerged suddenly in 2003, causing approximately 8000 human cases and more than 700 deaths worldwide. Since most animal models fail to faithfully recapitulate the clinical course of SARS-CoV in humans, the virus and host factors that mediate disease pathogenesis remain unclear. Recently, our laboratory and others developed a recombinant mouse-adapted SARS-CoV (rMA15) that was lethal in BALB/c mice. In contrast, intranasal infection of young 10-week-old C57BL/6 mice with rMA15 results in a nonlethal infection characterized by high titer replication within the lungs, lung inflammation, destruction of lung tissue, and loss of body weight, thus providing a useful model to identify host mediators of protection. Here, we report that mice deficient in MyD88 (MyD88−/−), an adapter protein that mediates Toll-like receptor (TLR), IL-1R, and IL-18R signaling, are far more susceptible to rMA15 infection. The genetic absence of MyD88 resulted in enhanced pulmonary pathology and greater than 90% mortality by day 6 post-infection. MyD88−/− mice had significantly higher viral loads in lung tissue throughout the course of infection. Despite increased viral loads, the expression of multiple proinflammatory cytokines and chemokines within lung tissue and recruitment of inflammatory monocytes/macrophages to the lung was severely impaired in MyD88−/− mice compared to wild-type mice. Furthermore, mice deficient in chemokine receptors that contribute to monocyte recruitment to the lung were more susceptible to rMA15-induced disease and exhibited severe lung pathology similar to that seen in MyD88−/−mice. These data suggest that MyD88-mediated innate immune signaling and inflammatory cell recruitment to the lung are required for protection from lethal rMA15 infection.

Interferon lambda alleles predict innate antiviral immune responses and hepatitis C virus permissiveness.

Hepatitis C virus (HCV) infection can result in viral chronicity or clearance. Although host genetics and particularly genetic variation in the interferon lambda (IFNL) locus are associated with spontaneous HCV clearance and treatment success, the mechanisms guiding these clinical outcomes remain unknown. Using a laser capture microdissection-driven unbiased systems virology approach, we isolated and transcriptionally profiled HCV-infected and adjacent primary human hepatocytes (PHHs) approaching single-cell resolution. An innate antiviral immune signature dominated the transcriptional response but differed in magnitude and diversity between HCV-infected and adjacent cells. Molecular signatures associated with more effective antiviral control were determined by comparing donors with high and low infection frequencies. Cells from donors with clinically unfavorable IFNL genotypes were infected at a greater frequency and exhibited dampened antiviral and cell death responses. These data suggest that early virus-host interactions, particularly host genetics and induction of innate immunity, critically determine the outcome of HCV infection.

Animal Models and Vaccines for SARS-CoV Infection

Abstract We summarize findings of SARS-CoV infections in several animal models each of which support viral replication in lungs accompanied by histopathological changes and/or clinical signs of illness to varying degrees. New findings are reported on SARS-CoV replication and associated pathology in two additional strains (C57BL/6 and 129S6) of aged mice. We also provide new comparative data on viral replication and associated pathology following infection of golden Syrian hamsters with various SARS-CoV strains and report the levels of neutralizing antibody titers following these infections and the cross-protective efficacy of infection with these strains in protecting against heterologous challenge. Finally, we summarize findings of a variety of vaccine approaches and discuss the available in vitro and in vivo data addressing the potential for disease enhancement following re-infection in animals previously vaccinated against or infected with SARS-CoV.

Early Upregulation of Acute Respiratory Distress Syndrome-Associated Cytokines Promotes Lethal Disease in an Aged-Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Infection

ABSTRACT Several respiratory viruses, including influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV), produce more severe disease in the elderly, yet the molecular mechanisms governing age-related susceptibility remain poorly studied. Advanced age was significantly associated with increased SARS-related deaths, primarily due to the onset of early- and late-stage acute respiratory distress syndrome (ARDS) and pulmonary fibrosis. Infection of aged, but not young, mice with recombinant viruses bearing spike glycoproteins derived from early human or palm civet isolates resulted in death accompanied by pathological changes associated with ARDS. In aged mice, a greater number of differentially expressed genes were observed than in young mice, whose responses were significantly delayed. Differences between lethal and nonlethal virus phenotypes in aged mice could be attributed to differences in host response kinetics rather than virus kinetics. SARS-CoV infection induced a range of interferon, cytokine, and pulmonary wound-healing genes, as well as several genes associated with the onset of ARDS. Mice that died also showed unique transcriptional profiles of immune response, apoptosis, cell cycle control, and stress. Cytokines associated with ARDS were significantly upregulated in animals experiencing lung pathology and lethal disease, while the same animals experienced downregulation of the ACE2 receptor. These data suggest that the magnitude and kinetics of a disproportionately strong host innate immune response contributed to severe respiratory stress and lethality. Although the molecular mechanisms governing ARDS pathophysiology remain unknown in aged animals, these studies reveal a strategy for dissecting the genetic pathways by which SARS-CoV infection induces changes in the host response, leading to death.