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Publications

2022
R, Wang, et al. “Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate”. Proc Natl Acad Șci USA (2022). Web. Publisher's Version
T, Tamura, et al. “Generation and characterization of genetically and antigenically diverse infectious clones of dengue virus serotypes 1-4”. Emerging Microbes & Infections (2022). Web. Publisher's Version
2021
Wei, L, and Ploss A. “Mechanism of Hepatitis B Virus cccDNA Formation”. Viruses (2021). Web. Publisher's Version
Wei, L, and Ploss A. “Hepatitis B virus cccDNA is formed through distinct repair processes of each strand”. Nature Communications (2021). Web. Publisher's Version
Liu, Y, Maya S, and Ploss A. “Animal Models of Hepatitis B Virus Infection-Success, Challenges, and Future Directions”. Viruses (2021). Web. Publisher's Version
2020
Berggren K, Suzuki S, Ploss A. “Animal Models Used in Hepatitis C Virus Research”. Int J Mol Sci (2020). Web. Publisher's Version
Wei, L and Ploss, A. “Core Components of DNA Lagging Strand Synthesis Machinery Are Essential for Hepatitis B Virus cccDNA Formation”. Nature Microbiology (2020). Web. Publisher's Version
2019
Gaska, J, et al. “Conservation of cell-intrinsic immune responses in diverse nonhuman primate species.”. Life Science Alliance 2019. Web. Publisher's Version
Maya, S, and A Ploss. “Master of disguise: hepatitis delta virus packaging and spread facilitated by diverse viral envelope proteins”. Hepatology 2019. Web. Publisher's Version
LeDesma, RA, I Nimgaonkar, and A Ploss. “Hepatitis E Virus Replication”. Viruses (2019). Web. Publisher's Version
Winer, BY, et al. “Analysis of host responses to hepatitis B and delta viral infections in a micro‐scalable hepatic co‐culture system”. Hepatology (2019). Web. Publisher's Version
Gaska, JM, et al. “Differences across cyclophilin A orthologs contribute to the host range restriction of hepatitis C virus.”. Elife 8 (2019): , 8, e44436. Web. Publisher's Version
Mesev, E, R LeDesma, and A Ploss. “Decoding type I and III interferon signalling during viral infection”. Nature Microbiology (2019). Web. Publisher's Version
2018
Douam, Florian, et al. “Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses”. (2018). Web. Publisher's VersionAbstract
Mice engrafted with components of a human immune system have become widely-used models for studying aspects of human immunity and disease. However, a defined methodology to objectively measure and compare the quality of the human immune response in different models is lacking. Here, by taking advantage of the highly immunogenic live-attenuated yellow fever virus vaccine YFV-17D, we provide an in-depth comparison of immune responses in human vaccinees, conventional humanized mice, and second generation humanized mice. We demonstrate that selective expansion of human myeloid and natural killer cells promotes transcriptomic responses akin to those of human vaccinees. These enhanced transcriptomic profiles correlate with the development of an antigen-specific cellular and humoral response to YFV-17D. Altogether, our approach provides a robust scoring of the quality of the human immune response in humanized mice and highlights a rational path towards developing better pre-clinical models for studying the human immune response and disease.
Winer, BY, .., and A Ploss. “Preclinical assessment of antiviral combination therapy in a genetically humanized mouse model for hepatitis delta virus infection”. Science Translational Medicine (2018). Web. Publisher's VersionAbstract
Chronic delta hepatitis, caused by hepatitis delta virus (HDV), is the most severe form of viral hepatitis, affecting at least 20 million hepatitis B virus (HBV)–infected patients worldwide. HDV/HBV co- or superinfections are major drivers for hepatocarcinogenesis. Antiviral treatments exist only for HBV and can only suppress but not cure infection. Development of more effective therapies has been impeded by the scarcity of suitable small-animal models. We created a transgenic (tg) mouse model for HDV expressing the functional receptor for HBV and HDV, the human sodium taurocholate cotransporting peptide NTCP. Both HBV and HDV entered hepatocytes in these mice in a glycoprotein-dependent manner, but one or more postentry blocks prevented HBV replication. In contrast, HDV persistently infected hNTCP tg mice coexpressing the HBV envelope, consistent with HDV dependency on the HBV surface antigen (HBsAg) for packaging and spread. In immunocompromised mice lacking functional B, T, and natural killer cells, viremia lasted at least 80 days but resolved within 14 days in immunocompetent animals, demonstrating that lymphocytes are critical for controlling HDV infection. Although acute HDV infection did not cause overt liver damage in this model, cell-intrinsic and cellular innate immune responses were induced. We further demonstrated that single and dual treatment with myrcludex B and lonafarnib efficiently suppressed viremia but failed to cure HDV infection at the doses tested. This small-animal model with inheritable susceptibility to HDV opens opportunities for studying viral pathogenesis and immune responses and for testing novel HDV therapeutics.
Douam, F, and A Ploss. “Yellow Fever Virus: Knowledge Gaps Impeding the Fight Against an Old Foe”. Cell Press: Trends in Microbiology (2018). Web. Publisher's VersionAbstract
Yellow fever (YF) was one of the most dangerous infectious diseases of the 18th and 19th centuries, resulting in mass casualties in Africa and the Americas. The etiologic agent is yellow fever virus (YFV), and its live-attenuated form, YFV-17D, remains one of the most potent vaccines ever developed. During the first half of the 20th century, vaccination combined with mosquito control eradicated YFV transmission in urban areas. However, the recent 2016–2018 outbreaks in areas with historically low or no YFV activity have raised serious concerns for an estimated 400–500 million unvaccinated people who now live in at-risk areas. Once a forgotten disease, we highlight here that YF still represents a very real threat to human health and economies. As many gaps remain in our understanding of how YFV interacts with the human host and causes disease, there is an urgent need to address these knowledge gaps and propel YFV research forward.
Ding, Q, et al. “Species-specific disruption of STING-dependent antiviral cellular defenses by the Zika virus NS2B3 protease”. PNAS (2018). Web. Publisher's VersionAbstract
The limited host tropism of numerous viruses causing disease in humans remains incompletely understood. One example is Zika virus (ZIKV), an RNA virus that has reemerged in recent years. Here, we demonstrate that ZIKV efficiently infects fibroblasts from humans, great apes, New and Old World monkeys, but not rodents. ZIKV infection in human—but not murine—cells impairs responses to agonists of the cGMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling pathway, suggesting that viral mechanisms to evade antiviral defenses are less effective in rodent cells. Indeed, human, but not mouse, STING is subject to cleavage by proteases encoded by ZIKV, dengue virus, West Nile virus, and Japanese encephalitis virus, but not that of yellow fever virus. The protease cleavage site, located between positions 78/79 of human STING, is only partially conserved in nonhuman primates and rodents, rendering these orthologs resistant to degradation. Genetic disruption of STING increases the susceptibility of mouse—but not human—cells to ZIKV. Accordingly, expression of only mouse, not human, STING in murine STING knockout cells rescues the ZIKV suppression phenotype. STING-deficient mice, however, did not exhibit increased susceptibility, suggesting that other redundant antiviral pathways control ZIKV infection in vivo. Collectively, our data demonstrate that numerous RNA viruses evade cGAS/STING-dependent signaling and affirm the importance of this pathway in shaping the host range of ZIKV. Furthermore, our results explain—at least in part—the decreased permissivity of rodent cells to ZIKV, which could aid in the development of mice model with inheritable susceptibility to ZIKV and other flaviviruses.
Ding, Q., ..., and A. Ploss. “Identification of the Intragenomic Promoter Controlling Hepatitis E Virus Subgenomic RNA Transcription”. mbio 93 (2018). Web. Publisher's VersionAbstract
Approximately 20 million hepatitis E virus (HEV) infections occur annually in both developing and industrialized countries. Most infections are self-limiting, but they can lead to chronic infections and cirrhosis in immunocompromised patients, and death in pregnant women. The mechanisms of HEV replication remain incompletely understood due to scarcity of adequate experimental platforms. HEV undergoes asymmetric genome replication, but it produces an additional subgenomic (SG) RNA encoding the viral capsid and a viroporin in partially overlapping open reading frames. Using a novel transcomplementation system, we mapped the intragenomic subgenomic promoter regulating SG RNA synthesis. This cis-acting element is highly conserved across all eight HEV genotypes, and when the element is mutated, it abrogates particle assembly and release. Our work defines previously unappreciated viral regulatory elements and provides the first in-depth view of the intracellular genome dynamics of this emerging human pathogen.
Dragovic, SM, et al. “Immunization with AgTRIO, a Protein in Anopheles Saliva, Contributes to Protection against Plasmodium Infection in Mice.”. Cell Host and Microbe 23 (2018): , 23, 523-535. Web. Publisher's VersionAbstract
Plasmodium infection begins with the bite of an anopheline mosquito, when sporozoites along with saliva are injected into a vertebrate host. The role of the host responses to mosquito saliva components in malaria remains unclear. We observed that antisera against Anopheles gambiae salivary glands partially protected mice from mosquito-borne Plasmodium infection. Specifically, antibodies to A. gambiae TRIO (AgTRIO), a mosquito salivary gland antigen, contributed to the protection. Mice administered AgTRIO antiserum showed lower Plasmodium liver burden and decreased parasitemia when exposed to infected mosquitoes. Active immunization with AgTRIO was also partially protective against Plasmodium berghei infection. A combination of AgTRIO antiserum and antibodies against Plasmodium circumsporozoite protein, a vaccine candidate, further decreased P. berghei infection. In humanized mice, AgTRIO antiserum afforded some protection against mosquito-transmitted Plasmodium falciparum. AgTRIO antiserum reduced the movement of sporozoites in the murine dermis. AgTRIO may serve as an arthropod-based target against Plasmodium to combat malaria.
Douam, F, and A Ploss. “The use of humanized mice for studies of viral pathogenesis and immunity”. Current Opinion in Virology 29 (2018): , 29, 62-71. Web. Publisher's VersionAbstract
Humanized mice, that is, animals engrafted with human tissues and/or expressing human genes, have been instrumental in improving our understanding of the pathogenesis and immunological processes that define some of the most challenging human-tropic viruses. In particular, mice engrafted with components of a human immune system (HIS) offer unprecedented opportunities for mechanistic studies of human immune responses to infection. Here, we provide a brief overview of the current panel of HIS mouse models available and cite recent examples of how such humanized animals have been used to study immune responses and pathogenesis elicited by human-tropic viruses. Finally, we will outline some of the challenges that lay ahead and strategies to improve and refine humanized mice with the goal of more accurately recapitulating human immune responses to viral infection.
Douam, F, et al. “A protein coevolution method uncovers critical features of the Hepatitis C Virus fusion mechanism”. Plos Pathogens (2018). Web. Publisher's VersionAbstract
Amino-acid coevolution can be referred to mutational compensatory patterns preserving the function of a protein. Viral envelope glycoproteins, which mediate entry of enveloped viruses into their host cells, are shaped by coevolution signals that confer to viruses the plasticity to evade neutralizing antibodies without altering viral entry mechanisms. The functions and structures of the two envelope glycoproteins of the Hepatitis C Virus (HCV), E1 and E2, are poorly described. Especially, how these two proteins mediate the HCV fusion process between the viral and the cell membrane remains elusive. Here, as a proof of concept, we aimed to take advantage of an original coevolution method recently developed to shed light on the HCV fusion mechanism. When first applied to the well-characterized Dengue Virus (DENV) envelope glycoproteins, coevolution analysis was able to predict important structural features and rearrangements of these viral protein complexes. When applied to HCV E1E2, computational coevolution analysis predicted that E1 and E2 refold interdependently during fusion through rearrangements of the E2 Back Layer (BL). Consistently, a soluble BL-derived polypeptide inhibited HCV infection of hepatoma cell lines, primary human hepatocytes and humanized liver mice. We showed that this polypeptide specifically inhibited HCV fusogenic rearrangements, hence supporting the critical role of this domain during HCV fusion. By combining coevolution analysis and in vitro assays, we also uncovered functionally-significant coevolving signals between E1 and E2 BL/Stem regions that govern HCV fusion, demonstrating the accuracy of our coevolution predictions. Altogether, our work shed light on important structural features of the HCV fusion mechanism and contributes to advance our functional understanding of this process. This study also provides an important proof of concept that coevolution can be employed to explore viral protein mediated-processes, and can guide the development of innovative translational strategies against challenging human-tropic viruses.
Nimgaonkar, I, and A Ploss. “Hepatitis E virus: advances and challenges”. Nature Reviews Gastroenterology & Hepatology 15 (2018): , 15, 96-110. Web. Publisher's VersionAbstract
At least 20 million hepatitis E virus (HEV) infections occur annually, with >3 million symptomatic cases and ∼60,000 fatalities. Hepatitis E is generally self-limiting, with a case fatality rate of 0.5-3% in young adults. However, it can cause up to 30% mortality in pregnant women in the third trimester and can become chronic in immunocompromised individuals, such as those receiving organ transplants or chemotherapy and individuals with HIV infection. HEV is transmitted primarily via the faecal-oral route and was previously thought to be a public health concern only in developing countries. It is now also being frequently reported in industrialized countries, where it is transmitted zoonotically or through organ transplantation or blood transfusions. Although a vaccine for HEV has been developed, it is only licensed in China. Additionally, no effective, non-teratogenic and specific treatments against HEV infections are currently available. Although progress has been made in characterizing HEV biology, the scarcity of adequate experimental platforms has hampered further research. In this Review, we focus on providing an update on the HEV life cycle. We will further discuss existing cell culture and animal models and highlight platforms that have proven to be useful and/or are emerging for studying other hepatotropic (viral) pathogens.
2017
Nimgaonkar I, Ploss A. “A porcine model for chronic hepatitis E.”. Hepatology (2017). Web. Publisher's Version
Douam, F, et al. “Type III interferon-mediated signaling is critical for controlling live-attenuated yellow fever virus infection in vivo.”. mBio 8: (2017): , 8:, 00819-17. Web. Publisher's VersionAbstract

Yellow fever virus (YFV) is an arthropod-borne flavivirus, infecting ~200,000 people worldwide annually and causing about 30,000 deaths. The live-attenuated vaccine strain, YFV-17D, has significantly contributed in controlling the global burden of yellow fever worldwide. However, the viral and host contributions to YFV-17D attenuation remain elusive. Type I (IFN-α/β) and type II interferon (IFN-γ) signaling have been shown to be mutually supportive in controlling YFV-17D infection despite distinct mechanisms of action on viral infection However, it remains unclear how type III IFN (IFN-λ) integrates into this anti-viral system.  Here, we report that while wild-type (WT) and IFN-λ receptor knock-out (λR -/-) mice were largely resistant to YFV-17D, deficiency in type I IFN-signaling resulted in robust infection. Although IFN-α/β receptor knock-out (α/βR -/-) mice survived the infection, mice with a combined deficiency in both type I and III IFN-signaling were hyper-susceptible to YFV-17D and succumbed to the infection. Mortality was associated with viral neuroinvasion and increased permeability of the blood brain barrier (BBB).  α/βR -/- λR -/- mice also exhibited distinct changes in the frequencies of multiple immune cell lineages, impaired T-cell activation and severe perturbation of the pro-inflammatory cytokine balance. Taken together, our data highlight that type III IFN has critical immunomodulatory and neuroprotective functions that prevent viral neuroinvasion during active YFV-17D replication. Type III IFN thus likely represents a safeguard mechanism crucial for controlling YFV-17D infection and contributing to shaping vaccine immunogenicity.

Winer, BY, et al. “Long-term hepatitis B infection in a scalable hepatic co-culture system”. Nature Communications 8:.125 (2017). Web. Publisher's VersionAbstract
Hepatitis B virus causes chronic infections in 250 million people worldwide. Chronic hepatitis B virus carriers are at risk of developing fibrosis, cirrhosis, and hepatocellular carcinoma. A prophylactic vaccine exists and currently available antivirals can suppress but rarely cure chronic infections. The study of hepatitis B virus and development of curative antivirals are hampered by a scarcity of models that mimic infection in a physiologically relevant, cellular context. Here, we show that cell-culture and patient-derived hepatitis B virus can establish persistent infection for over 30 days in a self-assembling, primary hepatocyte co-culture system. Importantly, infection can be established without antiviral immune suppression, and susceptibility is not donor dependent. The platform is scalable to microwell formats, and we provide proof-of-concept for its use in testing entry inhibitors and antiviral compounds.The lack of models that mimic hepatitis B virus (HBV) infection in a physiologically relevant context has hampered drug development. Here, Winer et al. establish a self-assembling, primary hepatocyte co-culture system that can be infected with patient-derived HBV without further modifications.
Li, D, et al. “Immunization with a subunit hepatitis C virus vaccine elicits pan-genotypic neutralizing antibodies and intra-hepatic T-cell responses in non-human primates”. Journal of Infectious Diseases 215.12 (2017): , 215, 12, 1824–1831. Web. Publisher's VersionAbstract

Background:

The global control of hepatitis C virus (HCV) infection remains a great burden, due to the high prices and potential drug resistance of the new direct-acting antivirals (DAAs) as well as the risk of reinfection in DAA-cured patients. Thus, a prophylactic vaccine for HCV is of great importance. We previously reported a single recombinant soluble E2 (sE2) vaccine produced in insect cells was able to induce broadly neutralizing antibodies (NAbs) and prevent HCV infection in mice. Here the sE2 vaccine was evaluated in non-human primates.

Methods:

Rhesus macaques were immunized with sE2 vaccine in combination with different adjuvants. Vaccine-induced NAbs in antisera were tested for neutralization activities against a panel of cell culture-derived HCV (HCVcc), while T-cell responses were evaluated in splenocytes, PBMC and hepatic lymphocytes.

Results:

sE2 is able to elicit NAbs against HCVcc harboring structural proteins from multiple HCV genotypes in rhesus macaques. Moreover, sE2-immunized macaques developed systemic and intra-hepatic memory T cells specific for E2. A significant correlation between the sE2-specific IgG titers and neutralization spectrum was observed, highlighting the essential role of sE2 immunogenicity on achieving broad NAbs.

Conclusions:

sE2 is a promising HCV vaccine candidate that warrants further pre-clinical and clinical development.

Shirvani-Dastgerdi, E, et al. “Selection of the highly replicative and partially multidrug resistant rtS78T HBV polymerase mutation during TDF-ETV combination therapy”. Journal of Hepatology 67 (2017): , 67, 246-254. Web. Publisher's VersionAbstract

Background & Aims

Patients chronically infected with the hepatitis B virus (HBV) and receiving long-term treatment with nucleoside or nucleotide analogues are at risk of selecting HBV strains with complex mutational patterns. We herein report two cases of HBV-infected patients with insufficient viral suppression, despite dual antiviral therapy with entecavir (ETV) and tenofovir (TDF). One patient died from aggressive hepatocellular carcinoma (HCC).

Methods

Serum samples from the two patients at different time points were analyzed using ultra-deep pyrosequencing analysis. HBV mutations were identified and transiently transfected into hepatoma cells in vitro using replication-competent HBV vectors, and functionally analyzed. We assessed replication efficacy, resistance to antivirals and potential impact on HBV secretion (viral particles, exosomes).

Results

Sequencing analyses revealed the selection of the rtS78T HBV polymerase mutation in both cases that simultaneously creates a premature stop codon at sC69 and thereby deletes almost the entire small HBV surface protein. One of the patients had an additional 261 bp deletion in the preS1/S2 region. Functional analyses of the mutations in vitro revealed that the rtS78T/sC69∗ mutation, but not the preS1/S2 deletion, significantly enhanced viral replication and conferred reduced susceptibility to ETV and TDF. The sC69∗ mutation caused truncation of HBs protein, leading to impaired detection by commercial HBsAg assay, without causing intracellular HBsAg retention or affecting HBV secretion.

Conclusions

The rtS78T/sC69∗ HBV mutation, associated with enhanced replication and insufficient response to antiviral treatment, may favor long-term persistence of these isolates. In addition to the increased production of HBV transcripts and the sustained secretion of viral particles in the absence of antigenic domains of S protein, this HBV mutation may predispose patients to carcinogenic effects.

Douam, F, et al. “Single-cell tracking of flavivirus RNA uncovers species-specific interactions with the immune system dictating disease outcome.”. Nature Communications 8:.14781 (2017). Web. Publisher's VersionAbstract

Positive-sense RNA viruses pose increasing health and economic concerns worldwide. Our limited understanding of how these viruses interact with their host and how these processes lead to virulence and disease seriously hampers the development of anti-viral strategies. Here, we demonstrate the tracking of (+) and (−) sense viral RNA at single-cell resolution within complex subsets of the human and murine immune system in different mouse models. Our results provide insights into how a prototypic flavivirus, yellow fever virus (YFV-17D), differentially interacts with murine and human hematopoietic cells in these mouse models and how these dynamics influence distinct outcomes of infection. We detect (−) YFV-17D RNA in specific secondary lymphoid compartments and cell subsets not previously recognized as permissive for YFV replication, and we highlight potential virus–host interaction events that could be pivotal in regulating flavivirus virulence and attenuation.

Ding, Qiang, et al. “Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles.”. Proc Natl Acad Sci U S A 114.5 (2017): , 114, 5, 1147-1152. Web. Publisher's VersionAbstract
Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV's three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3's ion channel function is further evidenced by its ability to mediate ionic currents when expressed in Xenopus laevis oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals.
Ding, Qiang, et al. “Mice Expressing Minimally Humanized CD81 and Occludin Genes Support Hepatitis C Virus Uptake In Vivo.”. J Virol 91.4 (2017). Web.Abstract
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.
Winer, Benjamin Y, et al. “Recapitulation of treatment response patterns in a novel humanized mouse model for chronic hepatitis B virus infection.”. Virology 502 (2017): , 502, 63-72. Web.Abstract
There are ~350 million chronic carriers of hepatitis B (HBV). While a prophylactic vaccine and drug regimens to suppress viremia are available, chronic HBV infection is rarely cured. HBV's limited host tropism leads to a scarcity of susceptible small animal models and is a hurdle to developing curative therapies. Mice that support engraftment with human hepatoctyes have traditionally been generated through crosses of murine liver injury models to immunodeficient backgrounds. Here, we describe the disruption of fumarylacetoacetate hydrolase directly in the NOD Rag1(-/-) IL2RγNULL (NRG) background using zinc finger nucleases. The resultant human liver chimeric mice sustain persistent HBV viremia for >90 days. When treated with standard of care therapy, HBV DNA levels decrease below detection but rebound when drug suppression is released, mimicking treatment response observed in patients. Our study highlights the utility of directed gene targeting approaches in zygotes to create new humanized mouse models for human diseases.
2016
Winer, Benjamin Y, and Alexander Ploss. “Breaking the species barrier for hepatitis delta virus.”. Hepatology 63.1 (2016): , 63, 1, 334-6. Web.
Perin, Paula M, et al. “Flunarizine prevents hepatitis C virus membrane fusion in a genotype-dependent manner by targeting the potential fusion peptide within E1.”. Hepatology 63.1 (2016): , 63, 1, 49-62. Web.Abstract
UNLABELLED: To explore mechanisms of hepatitis C viral (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. CONCLUSION: These observations reveal novel details about HCV membrane fusion; moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as a cost-effective component of HCV combination therapies.
von Schaewen, Markus, Gabriela Hrebikova, and Alexander Ploss. “Generation of Human Liver Chimeric Mice for the Study of Human Hepatotropic Pathogens.”. Methods Mol Biol 1438 (2016): , 1438, 79-101. Web.Abstract
Human liver chimeric mice have become valuable tools for the study of human hepatotropic pathogens and for the investigation of metabolism and pharmacokinetics of novel drugs. The evolution of the underlying mouse models has been rapid in the past years. The diverse fields of applications of those model systems and their technical challenges will be discussed in this chapter.
Basta, Nicole E, et al. “Immunogenicity of a Meningococcal B Vaccine during a University Outbreak.”. N Engl J Med 375.3 (2016): , 375, 3, 220-8. Web.Abstract
BACKROUND: In December 2013, a multicomponent meningococcal serogroup B (4CMenB) vaccine was used before licensure on the basis of special consideration by the Food and Drug Administration to respond to an outbreak of Neisseria meningitidis B at a U.S. university. Data suggested that vaccination would control the outbreak because isolates expressed antigens that were closely related to the vaccine antigens (factor H-binding protein [fHbp] and neisserial heparin-binding antigen). We quantified the immune responses induced by 4CMenB during the outbreak. METHODS: We conducted a seroprevalence survey among students to assess vaccination status and collect serum specimens to quantify titers of serum bactericidal antibodies (SBA) with an assay that included human complement (hSBA). We compared the proportion of vaccinated and unvaccinated participants who were seropositive for the outbreak strain and for one closely related reference strain (44/76-SL, which included fHbp) and one mismatched reference strain (5/99, which included neisserial adhesin A), both of which were used in vaccine development. Seropositivity was defined as an hSBA titer of 4 or higher. RESULTS: Among the 499 participants who received two doses of the 4CMenB vaccine 10 weeks apart, 66.1% (95% confidence interval [CI], 61.8 to 70.3) were seropositive for the outbreak strain, although the geometric mean titer was low at 7.6 (95% CI, 6.7 to 8.5). Among a random subgroup of 61 vaccinees who also received two doses but did not have a detectable protective response to the outbreak strain, 86.9% (95% CI, 75.8 to 94.2) were seropositive for the 44/76-SL strain, for which there was a geometric mean titer of 17.4 (95% CI, 13.0 to 23.2), whereas 100% of these vaccinees (95% CI, 94.1 to 100) were seropositive for the 5/99 strain and had a higher geometric mean titer (256.3; 95% CI, 187.3 to 350.7). The response to the outbreak strain was moderately correlated with the response to the 44/76-SL strain (Pearson's correlation,0.64; P<0.001) but not with the response to the 5/99 strain (Pearson's correlation,-0.06; P=0.43). CONCLUSIONS: Eight weeks after the second dose of the 4CMenB vaccine was administered, there was no evidence of an hSBA response against the outbreak strain in 33.9% of vaccinees, although no cases of meningococcal disease caused by N. meningitidis B were reported among vaccinated students. (Funded by Princeton University and others.).
Winer, Benjamin Y, et al. “In vivo models of hepatitis B and C virus infection.”. FEBS Lett 590.13 (2016): , 590, 13, 1987-99. Web.Abstract
Globally, more than 500 million individuals are chronically infected with hepatitis B (HBV), delta (HDV), and/or C (HCV) viruses, which can result in severe liver disease. Mechanistic studies of viral persistence and pathogenesis have been hampered by the scarcity of animal models. The limited species and cellular host range of HBV, HDV, and HCV, which robustly infect only humans and chimpanzees, have posed challenges for creating such animal models. In this review, we will discuss the barriers to interspecies transmission and the progress that has been made in our understanding of the HBV, HDV, and HCV life cycles. Additionally, we will highlight a variety of approaches that overcome these barriers and thus facilitate in vivo studies of these hepatotropic viruses.
Bartucci, Monica, et al. “Personalized Medicine Approaches in Prostate Cancer Employing Patient Derived 3D Organoids and Humanized Mice.”. Front Cell Dev Biol 4 (2016): , 4, 64. Web.Abstract
Prostate cancer (PCa) is the most common malignancy and the second most common cause of cancer death in Western men. Despite its prevalence, PCa has proven very difficult to propagate in vitro. PCa represents a complex organ-like multicellular structure maintained by the dynamic interaction of tumoral cells with parenchymal stroma, endothelial and immune cells, and components of the extracellular matrix (ECM). The lack of PCa models that recapitulate this intricate system has hampered progress toward understanding disease progression and lackluster therapeutic responses. Tissue slices, monolayer cultures and genetically engineered mouse models (GEMM) fail to mimic the complexities of the PCa microenvironment or reproduce the diverse mechanisms of therapy resistance. Moreover, patient derived xenografts (PDXs) are expensive, time consuming, difficult to establish for prostate cancer, lack immune cell-tumor regulation, and often tumors undergo selective engraftments. Here, we describe an interdisciplinary approach using primary PCa and tumor initiating cells (TICs), three-dimensional (3D) tissue engineering, genetic and morphometric profiling, and humanized mice to generate patient-derived organoids for examining personalized therapeutic responses in vitro and in mice co-engrafted with a human immune system (HIS), employing adaptive T-cell- and chimeric antigen receptor- (CAR) immunotherapy. The development of patient specific therapies targeting the vulnerabilities of cancer, when combined with antiproliferative and immunotherapy approaches could help to achieve the full transformative power of cancer precision medicine.
Douam, Florian, Qiang Ding, and Alexander Ploss. “Recent advances in understanding hepatitis C.”. F1000Res 5 (2016). Web.Abstract
The past decade has seen tremendous progress in understanding hepatitis C virus (HCV) biology and its related disease, hepatitis C. Major advances in characterizing viral replication have led to the development of direct-acting anti-viral therapies that have considerably improved patient treatment outcome and can even cure chronic infection. However, the high cost of these treatments, their low barrier to viral resistance, and their inability to prevent HCV-induced liver cancer, along with the absence of an effective HCV vaccine, all underscore the need for continued efforts to understand the biology of this virus. Moreover, beyond informing therapies, enhanced knowledge of HCV biology is itself extremely valuable for understanding the biology of related viruses, such as dengue virus, which is becoming a growing global health concern. Major advances have been realized over the last few years in HCV biology and pathogenesis, such as the discovery of the envelope glycoprotein E2 core structure, the generation of the first mouse model with inheritable susceptibility to HCV, and the characterization of virus-host interactions that regulate viral replication or innate immunity. Here, we review the recent findings that have significantly advanced our understanding of HCV and highlight the major challenges that remain.
Li, Dapeng, et al. “Altered Glycosylation Patterns Increase Immunogenicity of a Subunit Hepatitis C Virus Vaccine, Inducing Neutralizing Antibodies Which Confer Protection in Mice.”. J Virol 90.23 (2016): , 90, 23, 10486-10498. Web.Abstract
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.
Bartucci, Monica, et al. “Corrigendum: Personalized Medicine Approaches in Prostate Cancer Employing Patient Derived 3D Organoids and Humanized Mice.”. Front Cell Dev Biol 4 (2016): , 4, 74. Web.Abstract
[This corrects the article DOI: 10.3389/fcell.2016.00064.].
von Schaewen, Markus, et al. “Expanding the Host Range of Hepatitis C Virus through Viral Adaptation.”. MBio 76 (2016). Web.Abstract
Hepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytes in vivo and in vitro Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytes in vivo in the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV's ability to enter murine hepatocytes in vivo, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C. IMPORTANCE: At least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV.
Shirvani-Dastgerdi, Elham, Robert E Schwartz, and Alexander Ploss. “Hepatocarcinogenesis associated with hepatitis B, delta and C viruses.”. Curr Opin Virol 20 (2016): , 20, 1-10. Web.Abstract
Globally, over half a billion people are persistently infected with hepatitis B (HBV) and/or hepatitis C viruses. Chronic HBV and HCV infection frequently lead to fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Co-infections with hepatitis delta virus (HDV), a subviral satellite requiring HBV for its propagation, accelerates the progression of liver disease toward HCC. The mechanisms by which these viruses cause malignant transformation, culminating in HCC, remain incompletely understood, partially due to the lack of adequate experimental models for dissecting these complex disease processes in vivo.
2015
Gardner, Matthew R, et al. “AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges.”. Nature 519.7541 (2015): , 519, 7541, 87-91. Web.Abstract
Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs). However, even the best bNAbs neutralize 10-50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 μg ml(-1)), suggesting that high concentrations of these antibodies would be necessary to achieve general protection. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 μg ml(-1)). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17-77 μg ml(-1) of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.
Scull, Margaret A, et al. “Hepatitis C virus infects rhesus macaque hepatocytes and simianized mice.”. Hepatology 62.1 (2015): , 62, 1, 57-67. Web.Abstract
UNLABELLED: At least 170 million people are chronically infected with hepatitis C virus (HCV). Owing to the narrow host range of HCV and restricted use of chimpanzees, there is currently no suitable animal model for HCV pathogenesis studies or the development of a HCV vaccine. To identify cellular determinants of interspecies transmission and establish a novel immunocompetent model system, we examined the ability of HCV to infect hepatocytes from a small nonhuman primate, the rhesus macaque (Macaca mulatta). We show that the rhesus orthologs of critical HCV entry factors support viral glycoprotein-dependent virion uptake. Primary hepatocytes from rhesus macaques are also permissive for HCV-RNA replication and particle production, which is enhanced when antiviral signaling is suppressed. We demonstrate that this may be owing to the diminished capacity of HCV to antagonize mitochondrial antiviral-signaling protein-dependent innate cellular defenses. To test the ability of HCV to establish persistent replication in vivo, we engrafted primary rhesus macaque hepatocytes into immunocompromised xenorecipients. Inoculation of resulting simian liver chimeric mice with either HCV genotype 1a or 2a resulted in HCV serum viremia for up to 10 weeks. CONCLUSION: Together, these data indicate that rhesus macaques may be a viable model for HCV and implicate host immunity as a potential species-specific barrier to HCV infection. We conclude that suppression of host immunity or further viral adaptation may allow robust HCV infection in rhesus macaques and creation of a new animal model for studies of HCV pathogenesis, lentivirus coinfection, and vaccine development.
Stoddard, Mark B, et al. “Identification, molecular cloning, and analysis of full-length hepatitis C virus transmitted/founder genotypes 1, 3, and 4.”. MBio 62 (2015): , 6, 2, e02518. Web.Abstract
UNLABELLED: 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. IMPORTANCE: Hepatitis C virus (HCV) infects 2% to 3% of the world's population and exhibits extraordinary genetic diversity. This diversity is mirrored by HIV-1, where characterization of transmitted/founder (T/F) genomes has been instrumental in studies of virus transmission, immunopathogenesis, and vaccine development. Here, we show that despite major differences in genome organization, replication strategy, and natural history, HCV (like HIV-1) diversifies essentially randomly early in infection, and as a consequence, sequences of actual T/F viruses can be identified. This allowed us to capture by molecular cloning the full-length HCV genomes that are responsible for infecting the first hepatocytes and eliciting the initial immune responses, weeks before these events could be directly analyzed in human subjects. These findings represent an enabling experimental strategy, not only for HCV and HIV-1 research, but also for other RNA viruses of medical importance, including West Nile, chikungunya, dengue, Venezuelan encephalitis, and Ebola viruses.
Siu, Evelyn, and Alexander Ploss. “Modeling malaria in humanized mice: opportunities and challenges.”. Ann N Y Acad Sci 1342 (2015): , 1342, 29-36. Web.Abstract
About half of the world is at risk for developing malaria, which killed over 600,000 people in 2012 alone. The limited host range of the Plasmodium species that cause malaria in humans poses challenges for studying these parasites in experimentally tractable in vivo systems. To address this challenge, humanized mice have been constructed in which the tissue compartments relevant to the mammalian stages of plasmodial parasites are humanized. The development of human liver chimeric mice, human erythroid chimeric mice, and dually engrafted mice now allows for faithful replication of the entire Plasmodium falciparum life cycle. Although refinements to these models are still needed, humanized mice provide a promising small-animal model to study development of P. falciparum and, conceivably, other human malarial parasite species, and may also aid in drug and vaccine development.
von Schaewen, Markus, and Alexander Ploss. “Novel Biomarkers Associated With the Outcome of Interferon-Based Hepatitis C Virus Therapy.”. Cell Mol Gastroenterol Hepatol 13 (2015): , 1, 3, 257-258. Web.
Douam, Florian, and Alexander Ploss. “Proteomic approaches to analyzing hepatitis C virus biology.”. Proteomics 15.12 (2015): , 15, 12, 2051-65. Web.Abstract
Hepatitis C virus (HCV) is a major cause of liver disease worldwide. Acute infection often progresses to chronicity resulting frequently in fibrosis, cirrhosis, and in rare cases, in the development of hepatocellular carcinoma. Although HCV has proven to be an arduous object of research and has raised important technical challenges, several experimental models have been developed all over the last two decades in order to improve our understanding of the virus life cycle, pathogenesis and virus-host interactions. The recent development of direct acting-agents, leading to considerable progress in treatment of patients, represents the direct outcomes of these achievements. Proteomic approaches have been of critical help to shed light on several aspect of the HCV biology such as virion composition, viral replication, and virus assembly and to unveil diagnostic or prognostic markers of HCV-induced liver disease. Here, we review how proteomic approaches have led to improve our understanding of HCV life cycle and liver disease, thus highlighting the relevance of these approaches for studying the complex interactions between other challenging human viral pathogens and their host.
Gaska, Jenna M, and Alexander Ploss. “Study of viral pathogenesis in humanized mice.”. Curr Opin Virol 11 (2015): , 11, 14-20. Web.Abstract
Many of the viral pathogens that cause infectious diseases in humans have a highly restricted species tropism, making the study of their pathogenesis and the development of clinical therapies difficult. The improvement of humanized mouse models over the past 30 years has greatly facilitated researchers' abilities to study host responses to viral infections in a cost effective and ethical manner. From HIV to hepatotropic viruses to Middle East Respiratory Syndrome coronavirus, humanized mice have led to the identification of factors crucial to the viral life cycle, served as an outlet for testing candidate therapies, and improved our abilities to analyze human immune responses to infection. In tackling both new and old viruses as they emerge, humanized mice will continue to be an indispensable tool.
Winer, Benjamin Y, and Alexander Ploss. “Determinants of hepatitis B and delta virus host tropism.”. Curr Opin Virol 13 (2015): , 13, 109-16. Web.Abstract
Hepatitis B virus (HBV) infections are a global health problem afflicting approximately 360 million patients. Of these individuals, 15-20 million are co-infected with hepatitis delta virus (HDV). Progress toward curative therapies has been impeded by the highly restricted host tropism of HBV, which is limited to productive infections in humans and chimpanzees. Here, we will discuss different approaches that have been taken to study HBV and HDV infections in vivo. The development of transgenic and humanized mice has lead to deeper insights into HBV pathogenesis. An improved understanding of the determinants governing HBV and HDV species tropism will aid in the construction of a small animal model with inheritable susceptible to HBV/HDV.
Ploss, Alexander, and Christopher Walker. “Editorial overview: Progress and challenges in modeling human viral diseases in vivo.”. Curr Opin Virol 13 (2015): , 13, v-vii. Web.
Douam, Florian, et al. “Genetic Dissection of the Host Tropism of Human-Tropic Pathogens.”. Annu Rev Genet 49 (2015): , 49, 21-45. Web.Abstract
Infectious diseases are the second leading cause of death worldwide. Although the host multitropism of some pathogens has rendered their manipulation possible in animal models, the human-restricted tropism of numerous viruses, bacteria, fungi, and parasites has seriously hampered our understanding of these pathogens. Hence, uncovering the genetic basis underlying the narrow tropism of such pathogens is critical for understanding their mechanisms of infection and pathogenesis. Moreover, such genetic dissection is essential for the generation of permissive animal models that can serve as critical tools for the development of therapeutics or vaccines against challenging human pathogens. In this review, we describe different experimental approaches utilized to uncover the genetic foundation regulating pathogen host tropism as well as their relevance for studying the tropism of several important human pathogens. Finally, we discuss the current and future uses of this knowledge for generating genetically modified animal models permissive for these pathogens.
2014
Billerbeck, Eva, et al. “Insufficient interleukin-12 signalling favours differentiation of human CD4(+) and CD8(+) T cells into GATA-3(+) and GATA-3(+) T-bet(+) subsets in humanized mice.”. Immunology 143.2 (2014): , 143, 2, 202-18. Web.Abstract
Differentiation of CD4(+) T cells into type 1 or type 2 subsets is mediated by the expression of the opposing lineage defining transcription factors T-bet and GATA-3. However, the existence of GATA-3(+) T-bet(+) CD4(+) T cells in mice suggests functional plasticity of these subsets. Little is known about type 1 and type 2 plasticity of human T-cell subsets in vivo. Here, we show that in the xenogeneic environment of humanized mice, which lacks a functional immune-regulatory network, human CD4(+) and, notably, CD8(+) T cells preferentially differentiate into interleukin (IL)-4(+) GATA-3(+) and IL-4(+) interferon-γ(+) GATA-3(+) T-bet(+) subsets. Treatment with recombinant human IL-12 or expansion of IL-12-producing human dendritic cells in vivo reverted this phenotype and led to the down-regulation of GATA-3 expression. These changes also correlated with improved antiviral immune responses in humanized mice. In conclusion, our study shows the capacity of human CD4(+) and CD8(+) T cells for stable co-expression of GATA-3 and T-bet in humanized mice and reveals a critical role for IL-12 in regulating this phenotype.
Sheahan, Timothy, et al. “Interferon lambda alleles predict innate antiviral immune responses and hepatitis C virus permissiveness.”. Cell Host Microbe 15.2 (2014): , 15, 2, 190-202. Web.Abstract
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.
Ploss, Alexander. “Mouse models for human infectious diseases.”. J Immunol Methods 410 (2014): , 410, 1-2. Web.
von Schaewen, Markus, and Alexander Ploss. “Murine models of hepatitis C: what can we look forward to?”. Antiviral Res 104 (2014): , 104, 15-22. Web.Abstract
The study of interactions between hepatitis C virus (HCV) with its mammalian host, along with the development of more effective therapeutics and vaccines has been delayed by the lack of a suitable small animal model. HCV readily infects only humans and chimpanzees, which poses logistic, economic and ethical challenges with analyzing HCV infection in vivo. Progress has been made in understanding the determinants that dictate HCV's narrow host range providing a blueprint for constructing a mouse model with inheritable susceptibility to HCV infection. Indeed, genetically humanized mice were generated that support viral uptake, replication and production of infectious virions--albeit at low levels. These efforts are complemented with attempts to select for viral variants that are inherently more capable of replicating in non-human species. In parallel, engraftment of relevant human tissues into improved xenorecipients is being continuously refined. Incorporating advances in stem-cell-biology and tissue engineering may allow the generation of patient-specific humanized mice. Construction of such mouse "avatars" may allow analyzing functionally patient-specific differences with respect to susceptibility to infection, disease progression and responses to treatment. In this review, we discuss the three, before mentioned approaches to overcome current species barriers and generate a small animal model for HCV infection, i.e. genetic modification of mice to increase their susceptibility to the virus; genetic modification of HCV, to increase its pathogenicity for mice; and the introduction of human liver and immune cells into immunodeficient mice, to create "humanized" mice. Although in the foreseeable future there will not be a single model that perfectly mimics the natural course of HCV in humans there is reason for optimism. The spectrum of murine animal models for hepatitis C provides a broad arsenal for analyzing the disease. These models may play an important role by prioritizing vaccine candidates and possibly refining combination anti-viral drug therapies. This article forms part of a symposium in Anti-viral Research on "Hepatitis C: next steps toward global eradication."
Colpitts, Che C, et al. “Turmeric curcumin inhibits entry of all hepatitis C virus genotypes into human liver cells.”. Gut 63.7 (2014): , 63, 7, 1137-49. Web.Abstract
OBJECTIVE: Hepatitis C virus (HCV) infection causes severe liver disease and affects more than 160 million individuals worldwide. People undergoing liver organ transplantation face universal re-infection of the graft. Therefore, affordable antiviral strategies targeting the early stages of infection are urgently needed to prevent the recurrence of HCV infection. The aim of the study was to determine the potency of turmeric curcumin as an HCV entry inhibitor. DESIGN: The antiviral activity of curcumin and its derivatives was evaluated using HCV pseudo-particles (HCVpp) and cell-culture-derived HCV (HCVcc) in hepatoma cell lines and primary human hepatocytes. The mechanism of action was dissected using R18-labelled virions and a membrane fluidity assay. RESULTS: Curcumin treatment had no effect on HCV RNA replication or viral assembly/release. However, co-incubation of HCV with curcumin potently inhibited entry of all major HCV genotypes. Similar antiviral activities were also exerted by other curcumin derivatives but not by tetrahydrocurcumin, suggesting the importance of α,β-unsaturated ketone groups for the antiviral activity. Expression levels of known HCV receptors were unaltered, while pretreating the virus with the compound reduced viral infectivity without viral lysis. Membrane fluidity experiments indicated that curcumin affected the fluidity of the HCV envelope resulting in impairment of viral binding and fusion. Curcumin has also been found to inhibit cell-to-cell transmission and to be effective in combination with other antiviral agents. CONCLUSIONS: Turmeric curcumin inhibits HCV entry independently of the genotype and in primary human hepatocytes by affecting membrane fluidity thereby impairing virus binding and fusion.
Frias-Staheli, Natalia, et al. “Utility of humanized BLT mice for analysis of dengue virus infection and antiviral drug testing.”. J Virol 88.4 (2014): , 88, 4, 2205-18. Web.Abstract
Dengue virus (DENV) is the cause of a potentially life-threatening disease that affects millions of people worldwide. The lack of a small animal model that mimics the symptoms of DENV infection in humans has slowed the understanding of viral pathogenesis and the development of therapies and vaccines. Here, we investigated the use of humanized "bone marrow liver thymus" (BLT) mice as a model for immunological studies and assayed their applicability for preclinical testing of antiviral compounds. Human immune system (HIS) BLT-NOD/SCID mice were inoculated intravenously with a low-passage, clinical isolate of DENV-2, and this resulted in sustained viremia and infection of leukocytes in lymphoid and nonlymphoid organs. In addition, DENV infection increased serum cytokine levels and elicited DENV-2-neutralizing human IgM antibodies. Following restimulation with DENV-infected dendritic cells, in vivo-primed T cells became activated and acquired effector function. An adenosine nucleoside inhibitor of DENV decreased the circulating viral RNA when administered simultaneously or 2 days postinfection, simulating a potential treatment protocol for DENV infection in humans. In summary, we demonstrate that BLT mice are susceptible to infection with clinical DENV isolates, mount virus-specific adaptive immune responses, and respond to antiviral drug treatment. Although additional refinements to the model are required, BLT mice are a suitable platform to study aspects of DENV infection and pathogenesis and for preclinical testing of drug and vaccine candidates. IMPORTANCE Infection with dengue virus remains a major medical problem. Progress in our understanding of the disease and development of therapeutics has been hampered by the scarcity of small animal models. Here, we show that humanized mice, i.e., animals engrafted with components of a human immune system, that were infected with a patient-derived dengue virus strain developed clinical symptoms of the disease and mounted virus-specific immune responses. We further show that this mouse model can be used to test preclinically the efficacy of antiviral drugs.
von Schaewen, Markus, Qiang Ding, and Alexander Ploss. “Visualizing hepatitis C virus infection in humanized mice.”. J Immunol Methods 410 (2014): , 410, 50-9. Web.Abstract
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.
de Jong, Ype P, et al. “Broadly neutralizing antibodies abrogate established hepatitis C virus infection.”. Sci Transl Med 6254 (2014): , 6, 254, 254ra129. Web.Abstract
In most exposed individuals, hepatitis C virus (HCV) establishes a chronic infection; this long-term infection in turn contributes to the development of liver diseases such as 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. We demonstrate that three broadly nAbs-AR3A, AR3B, and AR4A-delivered with adeno-associated viral 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 therapeutic approach to interfere with HCV infection by exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes to sustain a chronic infection.
Lacek, Krzysztof, et al. “Dramatic potentiation of the antiviral activity of HIV antibodies by cholesterol conjugation.”. J Biol Chem 289.50 (2014): , 289, 50, 35015-28. Web.Abstract
The broadly neutralizing antibodies HIV 2F5 and 4E10, which bind to overlapping epitopes in the membrane-proximal external region of the fusion protein gp41, have been proposed to use a two-step mechanism for neutralization; first, they bind and preconcentrate at the viral membrane through their long, hydrophobic CDRH3 loops, and second, they form a high affinity complex with the protein epitope. Accordingly, mutagenesis of the CDRH3 can abolish their neutralizing activity, with no change in the affinity for the peptide epitope. We show here that we can mimic this mechanism by conjugating a cholesterol group outside of the paratope of an antibody. Cholesterol-conjugated antibodies bind to lipid raft domains on the membrane, and because of this enrichment, they show increased antiviral potency. In particular, we find that cholesterol conjugation (i) rescues the antiviral activity of CDRH3-mutated 2F5, (ii) increases the antiviral activity of WT 2F5, (iii) potentiates the non-membrane-binding HIV antibody D5 10-100-fold (depending on the virus strain), and (iv) increases synergy between 2F5 and D5. Conjugation can be made at several positions, including variable and constant domains. Cholesterol conjugation therefore appears to be a general strategy to boost the potency of antiviral antibodies, and, because membrane affinity is engineered outside of the antibody paratope, it can complement affinity maturation strategies.
Ding, Qiang, Markus von Schaewen, and Alexander Ploss. “The impact of hepatitis C virus entry on viral tropism.”. Cell Host Microbe 16.5 (2014): , 16, 5, 562-8. Web.Abstract
Uptake of hepatitis C virus (HCV) into hepatocytes is an orchestrated process, involving numerous host factors, virion-associated lipoproteins, and a growing number of cell-associated factors. Several of these factors likely contribute to the hepatotropism and limited host range of this virus. Discerning the minimal set of human-specific factors required for viral uptake into nonhuman cells has facilitated the development of small animal models with inheritable HCV susceptibility. This review summarizes current knowledge of host factors required for HCV entry, the molecular mechanisms underlying HCV entry into hepatocytes, and aspects of viral entry contributing to HCV host tropism.
2013
Dorner, Marcus, Charles M Rice, and Alexander Ploss. “Study of hepatitis C virus entry in genetically humanized mice.”. Methods 59.2 (2013): , 59, 2, 249-57. Web.Abstract
Approximately 2% of the world's population is chronically infected with hepatitis C virus (HCV). Chronic hepatitis C can culminate in end stage liver disease and liver cancer if the infection is untreated. Current therapy is only partially effective and a vaccine for HCV does not exist. Since the discovery of HCV as the etiologic agent causing hepatitis C several experimental tools have been developed which have improved our understanding of the viral life cycle and the interaction of HCV with human cells. However, it remains challenging to study HCV infection in its native liver environment given its narrow species tropism, limited to humans and chimpanzees. Mice can be rendered susceptible to HCV infection by transplanting human hepatocytes into immunocompromized liver injury strains. Such human liver chimeric mice are useful as a challenge model for human hepatotropic pathogens but their utility is hampered by their inability to mount functional immune responses and practical aspects including high costs, low throughput, and donor-to-donor variability. The barriers that restrict HCV species tropism are incompletely understood. We have previously shown that expression of human CD81 and human OCLN is required for HCV uptake into mouse cells. This led to the construction of a genetically humanized mouse model for HCV infection. Here, we provide a detailed protocol for the generation of these animals and highlight some of its applications for studying HCV biology and preclinical testing of drug and vaccine candidates.
Billerbeck, Eva, et al. “Animal models for hepatitis C.”. Curr Top Microbiol Immunol 369 (2013): , 369, 49-86. Web.Abstract
Hepatitis C remains a global epidemic. Approximately 3 % of the world's population suffers from chronic hepatitis C, which is caused by hepatitis C virus (HCV)-a positive sense, single-stranded RNA virus of the Flaviviridae family. HCV has a high propensity for establishing a chronic infection. If untreated chronic HCV carriers can develop severe liver disease including fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). Antiviral treatment is only partially effective, costly, and poorly tolerated. A prophylactic or therapeutic vaccine for HCV does not exist. Mechanistic studies of virus-host interactions, HCV immunity, and pathogenesis as well as the development of more effective therapies have been hampered by the lack of a suitable small animal model. Besides humans, chimpanzees are the only species that is naturally susceptible to HCV infection. While experimentation in these large primates has yielded valuable insights, ethical considerations, limited availability, genetic heterogeneity, and cost limit their utility. In search for more tractable small animal models, numerous experimental approaches have been taken to recapitulate parts of the viral life cycle and/or aspects of viral pathogenesis that will be discussed in this review. Exciting new models and improvements in established models hold promise to further elucidate our understanding of chronic HCV infection.
Gruell, Henning, et al. “Antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal HIV-1 infection in a transgenic mouse model.”. J Virol 87.15 (2013): , 87, 15, 8535-44. Web.Abstract
The development of an effective vaccine preventing HIV-1 infection remains elusive. Thus, the development of novel approaches capable of preventing HIV-1 transmission is of paramount importance. However, this is partly hindered by the lack of an easily accessible small-animal model to rapidly measure viral entry. Here, we report the generation of a human CD4- and human CCR5-expressing transgenic luciferase reporter mouse that facilitates measurement of peritoneal and genitomucosal HIV-1 pseudovirus entry in vivo. We show that antibodies and antiretrovirals mediate preexposure protection in this mouse model and that the serum antibody concentration required for protection from cervicovaginal infection is comparable to that required to protect macaques. Our results suggest that this system represents a model for the preclinical evaluation of prophylactic or vaccine candidates. It further supports the idea that broadly neutralizing antibodies should be evaluated for use as preexposure prophylaxis in clinical trials.
Sandmann, Lisa, and Alexander Ploss. “Barriers of hepatitis C virus interspecies transmission.”. Virology 435.1 (2013): , 435, 1, 70-80. Web.Abstract
Hepatitis C virus (HCV) is a major causative agent of severe liver disease including fibrosis, cirrhosis and liver cancer. Therapy has improved over the years, but continues to be associated with adverse side effects and variable success rates. Furthermore, a vaccine protecting against HCV infection remains elusive. Development of more effective intervention measures has been delayed by the lack of a suitable animal model. Naturally, HCV infects only humans and chimpanzees. The determinants of this limited host range are poorly understood in part due to difficulties of studying HCV in cell culture. Some progress has been made elucidating the barriers for the HCV lifecycle in non-permissive species which will help in the future to construct animal models for HCV infection, immunity and pathogenesis.
Horwitz, Joshua A, et al. “Expression of heterologous proteins flanked by NS3-4A cleavage sites within the hepatitis C virus polyprotein.”. Virology 439.1 (2013): , 439, 1, 23-33. Web.Abstract
Hepatitis C virus (HCV) contributes substantially to human morbidity and mortality world-wide. The development of HCV genomes expressing heterologous proteins has enhanced the ability to study viral infection, but existing systems have drawbacks. Recombinant viruses often require adaptive mutations to compensate for reduced viral titers, or rely on an artificial genomic organization that uncouples viral protein expression from recombinant gene expression. Here, we sought to exploit the viral polyprotein processing machinery to express heterologous proteins within the context of the HCV polyprotein. We show that HCV genotypes 2a and 1b permit insertion of reporter proteins between NS5A and NS5B with minimal impact on viral fitness. Using this strategy we constructed reporter genomes exhibiting a wide dynamic range, simplifying analysis of HCV infection in primary hepatocytes. Expression of heterologous proteins within the HCV genome offers new opportunities to analyze HCV infection in experimental systems without perturbing functions of individual viral proteins.
Shi, Chao, and Alexander Ploss. “Hepatitis C virus vaccines in the era of new direct-acting antivirals.”. Expert Rev Gastroenterol Hepatol 72 (2013): , 7, 2, 171-85. Web.Abstract
Hepatitis C virus (HCV) infection is a major global health problem as it has a high propensity for establishing chronicity. Chronic HCV carriers are at risk of developing severe liver disease including fibrosis, cirrhosis and liver cancer. While treatment has considerably improved over the years, therapy is still only partially effective, and is plagued by side effects, which contribute to treatment failure and is expensive to manage. The drug development pipeline contains several compounds that hold promise to achieve the goal of a short and more tolerable therapy, and are also likely to improve treatment response rates. It remains to be seen, however, how potent antiviral drug cocktails will affect the hepatitis C burden worldwide. In resource-poor environments, considerable costs, inadequate infrastructure for medical supervision and distribution may diminish the impact of future therapies. Consequently, development of novel therapeutic and prophylactic strategies is imperative to contain HCV infection globally.
Guermonprez, Pierre, et al. “Inflammatory Flt3l is essential to mobilize dendritic cells and for T cell responses during Plasmodium infection.”. Nat Med 19.6 (2013): , 19, 6, 730-8. Web.Abstract
Innate sensing mechanisms trigger a variety of humoral and cellular events that are essential to adaptive immune responses. Here we describe an innate sensing pathway triggered by Plasmodium infection that regulates dendritic cell homeostasis and adaptive immunity through Flt3 ligand (Flt3l) release. Plasmodium-induced Flt3l release in mice requires Toll-like receptor (TLR) activation and type I interferon (IFN) production. We found that type I IFN supports the upregulation of xanthine dehydrogenase, which metabolizes the xanthine accumulating in infected erythrocytes to uric acid. Uric acid crystals trigger mast cells to release soluble Flt3l from a pre-synthesized membrane-associated precursor. During infection, Flt3l preferentially stimulates expansion of the CD8-α(+) dendritic cell subset or its BDCA3(+) human dendritic cell equivalent and has a substantial impact on the magnitude of T cell activation, mostly in the CD8(+) compartment. Our findings highlight a new mechanism that regulates dendritic cell homeostasis and T cell responses to infection.
Vogt, Alexander, et al. “Recapitulation of the hepatitis C virus life-cycle in engineered murine cell lines.”. Virology 444.1-2 (2013): , 444, 1-2, 1-11. Web.Abstract
Hepatitis C virus (HCV) remains a major medical problem. In-depth study of HCV pathogenesis and immune responses is hampered by the lack of suitable small animal models. The narrow host range of HCV remains incompletely understood. We demonstrate that the entire HCV life-cycle can be recapitulated in mouse cells. We show that antiviral signaling interferes with HCV RNA replication in mouse cells. We were able to infect mouse cells expressing human CD81 and occludin (OCLN)-the minimal set of entry factor factors required for HCV uptake into mouse cells. Infected mouse cells sustain HCV RNA replication in the presence of miR122 and release infectious particles when mouse apoE is supplied. Our data demonstrate that the barriers of HCV interspecies transmission can be overcome by engineering a suitable cellular environment and provide a blue-print towards constructing a small animal model for HCV infection.
Billerbeck, Eva, et al. “Characterization of human antiviral adaptive immune responses during hepatotropic virus infection in HLA-transgenic human immune system mice.”. J Immunol 191.4 (2013): , 191, 4, 1753-64. Web.Abstract
Humanized mice have emerged as a promising model to study human immunity in vivo. Although they are susceptible to many pathogens exhibiting an almost exclusive human tropism, human immune responses to infection remain functionally impaired. It has recently been demonstrated that the expression of HLA molecules improves human immunity to lymphotropic virus infections in humanized mice. However, little is known about the extent of functional human immune responses in nonlymphoid tissues, such as in the liver, and the role of HLA expression in this context. Therefore, we analyzed human antiviral immunity in humanized mice during a hepatotropic adenovirus infection. We compared immune responses of conventional humanized NOD SCID IL-2Rγ-deficient (NSG) mice to those of a novel NOD SCID IL-2Rγ-deficient strain transgenic for both HLA-A*0201 and a chimeric HLA-DR*0101 molecule. Using a firefly luciferase-expressing adenovirus and in vivo bioluminescence imaging, we demonstrate a human T cell-dependent partial clearance of adenovirus-infected cells from the liver of HLA-transgenic humanized mice. This correlated with liver infiltration and activation of T cells, as well as the detection of Ag-specific humoral and cellular immune responses. When infected with a hepatitis C virus NS3-expressing adenovirus, HLA-transgenic humanized mice mounted an HLA-A*0201-restricted hepatitis C virus NS3-specific CD8(+) T cell response. In conclusion, our study provides evidence for the generation of partial functional antiviral immune responses against a hepatotropic pathogen in humanized HLA-transgenic mice. The adenovirus reporter system used in our study may serve as simple in vivo method to evaluate future strategies for improving human intrahepatic immune responses in humanized mice.
Dorner, Marcus, et al. “Completion of the entire hepatitis C virus life cycle in genetically humanized mice.”. Nature 501.7466 (2013): , 501, 7466, 237-41. Web.Abstract
More than 130 million people worldwide chronically infected with hepatitis C virus (HCV) are at risk of developing severe liver disease. Antiviral treatments are only partially effective against HCV infection, and a vaccine is not available. Development of more efficient therapies has been hampered by the lack of a small animal model. Building on the observation that CD81 and occludin (OCLN) comprise the minimal set of human factors required to render mouse cells permissive to HCV entry, we previously showed that transient expression of these two human genes is sufficient to allow viral uptake into fully immunocompetent inbred mice. Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo. Blunting antiviral immunity in genetically humanized mice infected with HCV results in measurable viraemia over several weeks. In mice lacking the essential cellular co-factor cyclophilin A (CypA), HCV RNA replication is markedly diminished, providing genetic evidence that this process is faithfully recapitulated. Using a cell-based fluorescent reporter activated by the NS3-4A protease we visualize HCV infection in single hepatocytes in vivo. Persistently infected mice produce de novo infectious particles, which can be inhibited with directly acting antiviral drug treatment, thereby providing evidence for the completion of the entire HCV life cycle in inbred mice. This genetically humanized mouse model opens new opportunities to dissect genetically HCV infection in vivo and provides an important preclinical platform for testing and prioritizing drug candidates and may also have utility for evaluating vaccine efficacy.
Horwitz, Joshua A, et al. “HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice.”. Proc Natl Acad Sci U S A 110.41 (2013): , 110, 41, 16538-43. Web.Abstract
Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1-infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice.
2012
Meng, Xiangzhi, et al. “C7L family of poxvirus host range genes inhibits antiviral activities induced by type I interferons and interferon regulatory factor 1.”. J Virol 86.8 (2012): , 86, 8, 4538-47. Web.Abstract
Vaccinia virus (VACV) K1L and C7L function equivalently in many mammalian cells to support VACV replication and antagonize antiviral activities induced by type I interferons (IFNs). While K1L is limited to orthopoxviruses, genes that are homologous to C7L are found in diverse mammalian poxviruses. In this study, we showed that the C7L homologues from sheeppox virus and swinepox virus could rescue the replication defect of a VACV mutant deleted of both K1L and C7L (vK1L(-)C7L(-)). Interestingly, the sheeppox virus C7L homologue could rescue the replication of vK1L(-)C7L(-) in human HeLa cells but not in murine 3T3 and LA-4 cells, in contrast to all other C7L homologues. Replacing amino acids 134 and 135 of the sheeppox virus C7L homologue, however, made it functional in the two murine cell lines, suggesting that these two residues are critical for antagonizing a putative host restriction factor which has some subtle sequence variation in human and murine cells. Furthermore, the C7L family of host range genes from diverse mammalian poxviruses were all capable of antagonizing type I IFN-induced antiviral activities against VACV. Screening of a library of more than 350 IFN-stimulated genes (ISGs) identified interferon-regulated factor 1 (IRF1) as an inhibitor of vK1L(-)C7L(-) but not wild-type VACV. Expression of either K1L or C7L, however, rendered vK1L(-)C7L(-) resistant to IRF1-induced antiviral activities. Altogether, our data show that K1L and C7L antagonize IRF1-induced antiviral activities and that the host modulation function of C7L is evolutionally conserved in all poxviruses that can readily replicate in tissue-cultured mammalian cells.
Schoggins, John W, et al. “Dengue reporter viruses reveal viral dynamics in interferon receptor-deficient mice and sensitivity to interferon effectors in vitro.”. Proc Natl Acad Sci U S A 109.36 (2012): , 109, 36, 14610-5. Web.Abstract
Dengue virus (DENV) is a global disease threat for which there are no approved antivirals or vaccines. Establishing state-of-the-art screening systems that rely on fluorescent or luminescent reporters may accelerate the development of anti-DENV therapeutics. However, relatively few reporter DENV platforms exist. Here, we show that DENV can be genetically engineered to express a green fluorescent protein or firefly luciferase. Reporter viruses are infectious in vitro and in vivo and are sensitive to antiviral compounds, neutralizing antibodies, and interferons. Bioluminescence imaging was used to follow the dynamics of DENV infection in mice and revealed that the virus localized predominantly to lymphoid and gut-associated tissues. The high-throughput potential of reporter DENV was demonstrated by screening a library of more than 350 IFN-stimulated genes for antiviral activity. Several antiviral effectors were identified, and they targeted DENV at two distinct life cycle steps. These viruses provide a powerful platform for applications ranging from validation of vaccine candidates to antiviral discovery.
Vaughan, Ashley M, et al. “Development of humanized mouse models to study human malaria parasite infection.”. Future Microbiol 75 (2012): , 7, 5, 657-65. Web.Abstract
Malaria is a disease caused by infection with Plasmodium parasites that are transmitted by mosquito bite. Five different species of Plasmodium infect humans with severe disease, but human malaria is primarily caused by Plasmodium falciparum. The burden of malaria on the developing world is enormous, and a fully protective vaccine is still elusive. One of the biggest challenges in the quest for the development of new antimalarial drugs and vaccines is the lack of accessible animal models to study P. falciparum infection because the parasite is restricted to the great apes and human hosts. Here, we review the current state of research in this field and provide an outlook of the development of humanized small animal models to study P. falciparum infection that will accelerate fundamental research into human parasite biology and could accelerate drug and vaccine design in the future.
Scull, Margaret A, and Alexander Ploss. “Exiting from uncharted territory: hepatitis C virus assembles in mouse cell lines.”. Hepatology 55.2 (2012): , 55, 2, 645-8. Web.
Ploss, Alexander, and Matthew J Evans. “Hepatitis C virus host cell entry.”. Curr Opin Virol 21 (2012): , 2, 1, 14-9. Web.Abstract
The hepatitis C virus (HCV) is a major medical problem with at least 130 million infected individuals worldwide. Over the last decade multiple host factors required for HCV cell entry have been identified, but a detailed understanding of their mechanistic interplay remains elusive. Nonetheless, recent advances in defining species-specific barriers of HCV transmission have allowed the identification of a minimal set of entry factors that are required for HCV infection of rodent cells and has culminated in an animal model that recapitulates HCV entry in vivo. A detailed understanding of the viral uptake pathway is imperative to define new drug targets allowing for more effective intervention against this devastating disease.
Giang, Erick, et al. “Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus.”. Proc Natl Acad Sci U S A 109.16 (2012): , 109, 16, 6205-10. Web.Abstract
Hepatitis C virus (HCV) infects ∼2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.
Schwartz, Robert E, et al. “Modeling hepatitis C virus infection using human induced pluripotent stem cells.”. Proc Natl Acad Sci U S A 109.7 (2012): , 109, 7, 2544-8. Web.Abstract
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.
Ploss, Alexander, and Jean Dubuisson. “New advances in the molecular biology of hepatitis C virus infection: towards the identification of new treatment targets.”. Gut 61 Suppl 1 (2012): , 61 Suppl 1, i25-35. Web.Abstract
Hepatitis C virus (HCV) causes chronic infection in almost 2% of the world's population. If untreated, chronic carriers can develop severe liver disease including fibrosis, cirrhosis and hepatocellular carcinoma. Until recently, hepatitis C was treated with a combination of pegylated interferon and ribavirin, a treatment which was only partially effective and was plagued with side effects. In 2011 two inhibitors of the virally encoded NS3/4 protease have become part of standard therapy, which have improved treatment rates but can exacerbate the problematic side effects. While the addition of these first directly acting antivirals (DAAs) marks a milestone in anti-HCV therapy, new and improved combinations of drugs are desperately needed. New generations of drugs will have to address genetic variability of HCV and issues of viral resistance. Furthermore, combination therapies have to be tailored to effectively cure patient populations that have traditionally been hardest to treat, including patients with cirrhosis, those receiving liver transplants and individuals who are co-infected with HIV or hepatitis B virus. Since the discovery of HCV a plethora of experimental tools have been developed which enabled detailed analysis of various aspects of the viral life cycle and the interaction of HCV with its human host. Such studies have revealed a growing list of targets for therapeutic intervention, some of which will be discussed in this review.
Vaughan, Ashley M, et al. “Complete Plasmodium falciparum liver-stage development in liver-chimeric mice.”. J Clin Invest 122.10 (2012): , 122, 10, 3618-28. Web.Abstract
Plasmodium falciparum, which causes the most lethal form of human malaria, replicates in the host liver during the initial stage of infection. However, in vivo malaria liver-stage (LS) studies in humans are virtually impossible, and in vitro models of LS development do not reconstitute relevant parasite growth conditions. To overcome these obstacles, we have adopted a robust mouse model for the study of P. falciparum LS in vivo: the immunocompromised and fumarylacetoacetate hydrolase-deficient mouse (Fah-/-, Rag2-/-, Il2rg-/-, termed the FRG mouse) engrafted with human hepatocytes (FRG huHep). FRG huHep mice supported vigorous, quantifiable P. falciparum LS development that culminated in complete maturation of LS at approximately 7 days after infection, providing a relevant model for LS development in humans. The infections allowed observations of previously unknown expression of proteins in LS, including P. falciparum translocon of exported proteins 150 (PTEX150) and exported protein-2 (EXP-2), components of a known parasite protein export machinery. LS schizonts exhibited exoerythrocytic merozoite formation and merosome release. Furthermore, FRG mice backcrossed to the NOD background and repopulated with huHeps and human red blood cells supported reproducible transition from LS infection to blood-stage infection. Thus, these mice constitute reliable models to study human LS directly in vivo and demonstrate utility for studies of LS-to-blood-stage transition of a human malaria parasite.
Klein, Florian, et al. “HIV therapy by a combination of broadly neutralizing antibodies in humanized mice.”. Nature 492.7427 (2012): , 492, 7427, 118-22. Web.Abstract
Human antibodies to human immunodeficiency virus-1 (HIV-1) can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice. Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy, the longer half-life of antibodies led to control of viraemia for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in humanized mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals.
Pietzsch, John, et al. “A mouse model for HIV-1 entry.”. Proc Natl Acad Sci U S A 109.39 (2012): , 109, 39, 15859-64. Web.Abstract
Passive transfer of neutralizing antibodies against HIV-1 can prevent infection in macaques and seems to delay HIV-1 rebound in humans. Anti-HIV antibodies are therefore of great interest for vaccine design. However, the basis for their in vivo activity has been difficult to evaluate systematically because of a paucity of small animal models for HIV infection. Here we report a genetically humanized mouse model that incorporates a luciferase reporter for rapid quantitation of HIV entry. An antibody's ability to block viral entry in this in vivo model is a function of its bioavailability, direct neutralizing activity, and effector functions.
2011
Billerbeck, Eva, et al. “Development of human CD4+FoxP3+ regulatory T cells in human stem cell factor-, granulocyte-macrophage colony-stimulating factor-, and interleukin-3-expressing NOD-SCID IL2Rγ(null) humanized mice.”. Blood 117.11 (2011): , 117, 11, 3076-86. Web.Abstract
Human hematolymphoid mice have become valuable tools for the study of human hematopoiesis and uniquely human pathogens in vivo. Recent improvements in xenorecipient strains allow for long-term reconstitution with a human immune system. However, certain hematopoietic lineages, for example, the myeloid lineage, are underrepresented, possibly because of the limited cross-reactivity of murine and human cytokines. Therefore, we created a nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-γ-null (NOD-SCID IL2Rγ(null)) mouse strain that expressed human stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3, termed NSG-SGM3. Transplantation of CD34(+) human hematopoietic stem cells into NSG-SGM3 mice led to robust human hematopoietic reconstitution in blood, spleen, bone marrow, and liver. Human myeloid cell frequencies, specifically, myeloid dendritic cells, were elevated in the bone marrow of humanized NSG-SGM3 mice compared with nontransgenic NSG recipients. Most significant, however, was the increase in the CD4(+)FoxP3(+) regulatory T-cell population in all compartments analyzed. These CD4(+)FoxP3(+) regulatory T cells were functional, as evidenced by their ability to suppress T-cell proliferation. In conclusion, humanized NSG-SGM3 mice might serve as a useful model to study human regulatory T-cell development in vivo, but this unexpected lineage skewing also highlights the importance of adequate spatiotemporal expression of human cytokines for future xenorecipient strain development.
de Jong, Ype P, Charles M Rice, and Alexander Ploss. “Evaluation of combination therapy against hepatitis C virus infection in human liver chimeric mice.”. J Hepatol 54.5 (2011): , 54, 5, 848-50. Web.
Andrus, Linda, et al. “Expression of paramyxovirus V proteins promotes replication and spread of hepatitis C virus in cultures of primary human fetal liver cells.”. Hepatology 54.6 (2011): , 54, 6, 1901-12. Web.Abstract
UNLABELLED: 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.
Dorner, Marcus, et al. “A genetically humanized mouse model for hepatitis C virus infection.”. Nature 474.7350 (2011): , 474, 7350, 208-11. Web.Abstract
Hepatitis C virus (HCV) remains a major medical problem. Antiviral treatment is only partially effective and a vaccine does not exist. Development of more effective therapies has been hampered by the lack of a suitable small animal model. Although xenotransplantation of immunodeficient mice with human hepatocytes has shown promise, these models are subject to important challenges. Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach. Here we show that expression of two human genes is sufficient to allow HCV infection of fully immunocompetent inbred mice. We establish a precedent for applying mouse genetics to dissect viral entry and validate the role of scavenger receptor type B class I for HCV uptake. We demonstrate that HCV can be blocked by passive immunization, as well as showing that a recombinant vaccinia virus vector induces humoral immunity and confers partial protection against heterologous challenge. This system recapitulates a portion of the HCV life cycle in an immunocompetent rodent for the first time, opening opportunities for studying viral pathogenesis and immunity and comprising an effective platform for testing HCV entry inhibitors in vivo.
Marukian, Svetlana, et al. “Hepatitis C virus induces interferon-λ and interferon-stimulated genes in primary liver cultures.”. Hepatology 54.6 (2011): , 54, 6, 1913-23. Web.Abstract
UNLABELLED: 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.
Washburn, Michael L, et al. “A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease.”. Gastroenterology 140.4 (2011): , 140, 4, 1334-44. Web.Abstract
BACKGROUND & AIMS: Studies of hepatitis C virus (HCV) infection, immunopathogenesis, and resulting liver diseases have been hampered by the lack of a small animal model. We developed humanized mice with human immune system and liver tissues to improve the studies of hepatitis C virus pathogenesis and treatment. METHODS: To promote engraftment of human hepatocytes, we expressed a fusion protein of the FK506 binding protein (FKBP) and caspase 8 under control of the albumin promoter (AFC8), which induces liver cell death, in Balb/C Rag2(-/-) γC-null mice. Cotransplantation of human CD34(+) human hematopoietic stem cells (HSC) and hepatocyte progenitors into the transgenic mice led to efficient engraftment of human leukocytes and hepatocytes. We then infected these humanized mice (AFC8-hu HSC/Hep) with primary HCV isolates and studied HCV-induced immune responses and liver diseases. RESULTS: AFC8-hu HSC/Hep mice supported HCV infection in the liver and generated a human immune T-cell response against HCV. HCV infection induced liver inflammation, hepatitis, and fibrosis, which correlated with activation of stellate cells and expression of human fibrogenic genes. CONCLUSIONS: AFC8-hu HSC/Hep mice are a useful model of HCV infection, the immune response, and liver disease because they contain human immune system and liver cells. These mice become infected with HCV, generate a specific immune response against the virus, and develop liver diseases that include hepatitis and fibrosis. This model might also be used to develop therapeutics for HCV infection.
Dorner, Marcus, and Alexander Ploss. “Deconstructing hepatitis C virus infection in humanized mice.”. Ann N Y Acad Sci 1245 (2011): , 1245, 59-62. Web.Abstract
Hepatitis C virus is a major medical problem. Novel small-animal models are likely to accelerate the development of more effective therapeutics and a vaccine.
2010
Ploss, Alexander, et al. “Persistent hepatitis C virus infection in microscale primary human hepatocyte cultures.”. Proc Natl Acad Sci U S A 107.7 (2010): , 107, 7, 3141-5. Web.Abstract
Hepatitis C virus (HCV) remains a major public health problem, affecting approximately 130 million people worldwide. HCV infection can lead to cirrhosis, hepatocellular carcinoma, and end-stage liver disease, as well as extrahepatic complications such as cryoglobulinemia and lymphoma. Preventative and therapeutic options are severely limited; there is no HCV vaccine available, and nonspecific, IFN-based treatments are frequently ineffective. Development of targeted antivirals has been hampered by the lack of robust HCV cell culture systems that reliably predict human responses. Here, we show the entire HCV life cycle recapitulated in micropatterned cocultures (MPCCs) of primary human hepatocytes and supportive stroma in a multiwell format. MPCCs form polarized cell layers expressing all known HCV entry factors and sustain viral replication for several weeks. When coupled with highly sensitive fluorescence- and luminescence-based reporter systems, MPCCs have potential as a high-throughput platform for simultaneous assessment of in vitro efficacy and toxicity profiles of anti-HCV therapeutics.
Jones, Christopher T, et al. “Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter system.”. Nat Biotechnol 28.2 (2010): , 28, 2, 167-71. Web.Abstract
Hepatitis C virus (HCV), which infects 2-3% of the world population, is a causative agent of chronic hepatitis and the leading indication for liver transplantation. The ability to propagate HCV in cell culture (HCVcc) is a relatively recent breakthrough and a key tool in the quest for specific antiviral therapeutics. Monitoring HCV infection in culture generally involves bulk population assays, use of genetically modified viruses and/or terminal processing of potentially precious samples. Here we develop a cell-based fluorescent reporter system that allows sensitive distinction of individual HCV-infected cells in live or fixed samples. We demonstrate use of this technology for several previously intractable applications, including live-cell imaging of viral propagation and host response, as well as visualizing infection of primary hepatocyte cultures. Integration of this reporter with modern image-based analysis methods could open new doors for HCV research.
Sheahan, Timothy, Christopher T Jones, and Alexander Ploss. “Advances and challenges in studying hepatitis C virus in its native environment.”. Expert Rev Gastroenterol Hepatol 45 (2010): , 4, 5, 541-50. Web.Abstract
Approximately 2% of the worldwide population is infected with hepatitis C virus (HCV), the major causative agent of non-A, non-B hepatitis. Although substantial progress has been made in developing tools to dissect the viral life cycle, most in vitro studies rely on hepatoma cell lines, which are functionally disparate from the natural in vivo target of the virus – hepatocytes. To gain insights into virus–host interactions, there is a need for HCV-model systems that more closely mimic the physiological environment of the liver. Here, we discuss recent advances in culture and detection systems that facilitate the study of HCV in primary cells. Use of these new models may help bridge the gap between in vitro studies and clinical research.
de Jong, Ype P, Charles M Rice, and Alexander Ploss. “New horizons for studying human hepatotropic infections.”. J Clin Invest 120.3 (2010): , 120, 3, 650-3. Web.Abstract
The liver serves as a target organ for several important pathogens, including hepatitis B and C viruses (HBV and HCV, respectively) and the human malaria parasites, all of which represent serious global health problems. Because these pathogens are restricted to human hepatocytes, research in small animals has been compromised by the frailty of the current mouse xenotransplantation models. In this issue of the JCI, Bissig et al. demonstrate robust HBV and HCV infection in a novel xenotransplantation model in which large numbers of immunodeficient mice with liver injury were engrafted with significant quantities of human hepatocytes. This technical advance paves the way for more widespread use of human liver chimeric mice and forms the basis for creating increasingly complex humanized mouse models that could prove useful for studying immunopathogenesis and vaccine development against hepatotropic pathogens.
Kohaar, Indu, et al. “Splicing diversity of the human OCLN gene and its biological significance for hepatitis C virus entry.”. J Virol 84.14 (2010): , 84, 14, 6987-94. Web.Abstract
Persistent hepatitis C virus (HCV) infection is a primary etiological factor for the development of chronic liver disease, including cirrhosis and cancer. A recent study identified occludin (OCLN), an integral tight junction protein, as one of the key factors for HCV entry into cells. We explored the splicing diversity of OCLN in normal human liver and observed variable expression of alternative splice variants, including two known forms (WT-OCLN and OCLN-ex4del) and six novel forms (OCLN-ex7ext, OCLN-ex3pdel, OCLN-ex3del, OCLN-ex3-4del, OCLN-ex3p-9pdel, and OCLN-ex3p-7pdel). Recombinant protein isoforms WT-OCLN and OCLN-ex7ext, which retained the HCV-interacting MARVEL domain, were expressed on the cell membrane and were permissive for HCV infection in in vitro infectivity assays. All other forms lacked the MARVEL domain, were expressed in the cytoplasm, and were nonpermissive for HCV infection. Additionally, we observed variable expression of OCLN splicing forms across human tissues and cell lines. Our study suggests that the remarkable natural splicing diversity of OCLN might contribute to HCV tissue tropism and possibly modify the outcome of HCV infection in humans. Genetic factors crucial for regulation of OCLN expression and susceptibility to HCV infection remain to be elucidated.
Gerold, Gisa, Charles M Rice, and Alexander Ploss. “Teaching new tricks to an old foe: murinizing hepatitis C virus.”. Hepatology 52.6 (2010): , 52, 6, 2233-6. Web.
2009
Ploss, Alexander, et al. “Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.”. Nature 457.7231 (2009): , 457, 7231, 882-6. Web.Abstract
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. The development of much needed specific antiviral therapies and an effective vaccine has been hampered by the lack of a convenient small animal model. The determinants restricting HCV tropism to human and chimpanzee hosts are unknown. Replication of the viral RNA has been demonstrated in mouse cells, but these cells are not infectable with either lentiviral particles bearing HCV glycoproteins (HCVpp) or HCV produced in cell culture (HCVcc) (A.P., M.E. and C.M.R., unpublished observations), suggesting that there is a block at the level of entry. Here we show, using an iterative complementary DNA library screening approach, that human occludin (OCLN) is an essential HCV cell entry factor that is able to render murine cells infectable with HCVpp. Similarly, OCLN is required for the HCV-susceptibility of human cells, because its overexpression in uninfectable cells specifically enhanced HCVpp uptake, whereas its silencing in permissive cells impaired both HCVpp and HCVcc infection. In addition to OCLN, HCVpp infection of murine cells required expression of the previously identified HCV entry factors CD81 (ref. 4), scavenger receptor class B type I (SR-BI, also known as SCARB1) and claudin-1 (CLDN1). Although the mouse versions of SR-BI and CLDN1 function at least as well as the human proteins in promoting HCV entry, both OCLN and CD81 must be of human origin to allow efficient infection. The species-specific determinants of OCLN were mapped to its second extracellular loop. The identification of OCLN as a new HCV entry factor further highlights the importance of the tight junction complex in the viral entry process, and provides an important advance towards efforts to develop small animal models for HCV.
Legrand, Nicolas, et al. “Humanized mice for modeling human infectious disease: challenges, progress, and outlook.”. Cell Host Microbe 61 (2009): , 6, 1, 5-9. Web.Abstract
Over 800 million people worldwide are infected with hepatitis viruses, human immunodeficiency virus (HIV), and malaria, resulting in more than 5 million deaths annually. Here we discuss the potential and challenges of humanized mouse models for developing effective and affordable therapies and vaccines, which are desperately needed to combat these diseases.
Strowig, Till, et al. “Priming of protective T cell responses against virus-induced tumors in mice with human immune system components.”. J Exp Med 206.6 (2009): , 206, 6, 1423-34. Web.Abstract
Many pathogens that cause human disease infect only humans. To identify the mechanisms of immune protection against these pathogens and also to evaluate promising vaccine candidates, a small animal model would be desirable. We demonstrate that primary T cell responses in mice with reconstituted human immune system components control infection with the oncogenic and persistent Epstein-Barr virus (EBV). These cytotoxic and interferon-gamma-producing T cell responses were human leukocyte antigen (HLA) restricted and specific for EBV-derived peptides. In HLA-A2 transgenic animals and similar to human EBV carriers, T cell responses against lytic EBV antigens dominated over recognition of latent EBV antigens. T cell depletion resulted in elevated viral loads and emergence of EBV-associated lymphoproliferative disease. Both loss of CD4(+) and CD8(+) T cells abolished immune control. Therefore, this mouse model recapitulates features of symptomatic primary EBV infection and generates T cell-mediated immune control that resists oncogenic transformation.

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