Selective expansion of myeloid and NK cells in humanized mice yields human-like vaccine responses

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.National Institute of General Medical Sciences (U.S.) (Award T32GM007753)Searle Scholars ProgramArnold and Mabel Beckman Foundation (Young Investigator Program)National Institutes of Health (U.S.) (1DP2OD020839)National Institutes of Health (U.S.) (5U24AI118672)National Institutes of Health (U.S.) (1U54CA217377)National Institutes of Health (U.S.) (1R33CA202820)National Institutes of Health (U.S.) (2U19AI089992)National Institutes of Health (U.S.) (21R01HL134539)National Institutes of Health (U.S.) (2RM1HG006193)National Institutes of Health (U.S.) (2R01HL095791)National Institutes of Health (U.S.) (P01AI039671)Bill & Melinda Gates Foundation (OPP1139972

Up-regulation of the ATP-binding cassette transporter A1 inhibits hepatitis C virus infection.

International audienceHepatitis C virus (HCV) establishes infection using host lipid metabolism pathways that are thus considered potential targets for indirect anti-HCV strategies. HCV enters the cell via clathrin-dependent endocytosis, interacting with several receptors, and virus-cell fusion, which depends on acidic pH and the integrity of cholesterol-rich domains of the hepatocyte membrane. The ATP-binding Cassette Transporter A1 (ABCA1) mediates cholesterol efflux from hepatocytes to extracellular Apolipoprotein A1 and moves cholesterol within cell membranes. Furthermore, it generates high-density lipoprotein (HDL) particles. HDL protects against arteriosclerosis and cardiovascular disease. We show that the up-regulation of ABCA1 gene expression and its cholesterol efflux function in Huh7.5 hepatoma cells, using the liver X receptor (LXR) agonist GW3965, impairs HCV infection and decreases levels of virus produced. ABCA1-stimulation inhibited HCV cell entry, acting on virus-host cell fusion, but had no impact on virus attachment, replication, or assembly/secretion. It did not affect infectivity or properties of virus particles produced. Silencing of the ABCA1 gene and reduction of the specific cholesterol efflux function counteracted the inhibitory effect of the GW3965 on HCV infection, providing evidence for a key role of ABCA1 in this process. Impaired virus-cell entry correlated with the reorganisation of cholesterol-rich membrane microdomains (lipid rafts). The inhibitory effect could be reversed by an exogenous cholesterol supply, indicating that restriction of HCV infection was induced by changes of cholesterol content/distribution in membrane regions essential for virus-cell fusion. Stimulation of ABCA1 expression by GW3965 inhibited HCV infection of both human primary hepatocytes and isolated human liver slices. This study reveals that pharmacological stimulation of the ABCA1-dependent cholesterol efflux pathway disrupts membrane cholesterol homeostasis, leading to the inhibition of virus-cell fusion and thus HCV cell entry. Therefore besides other beneficial roles, ABCA1 might represent a potential target for HCV therapy

Hepatitis C virus quasispecies and pseudotype analysis from acute infection to chronicity in HIV-1 co-infected individuals

HIV-1 infected patients who acquire HCV infection have higher rates of chronicity and liver disease progression than patients with HCV mono-infection. Understanding early events in this pathogenic process is important. We applied single genome sequencing of the E1 to NS3 regions and viral pseudotype neutralization assays to explore the consequences of viral quasispecies evolution from pre-seroconversion to chronicity in four co-infected individuals (mean follow up 566 days). We observed that one to three founder viruses were transmitted. Relatively low viral sequence diversity, possibly related to an impaired immune response, due to HIV infection was observed in three patients. However, the fourth patient, after an early purifying selection displayed increasing E2 sequence evolution, possibly related to being on suppressive antiretroviral therapy. Viral pseudotypes generated from HCV variants showed relative resistance to neutralization by autologous plasma but not to plasma collected from later time points, confirming ongoing virus escape from antibody neutralization

Figure S3: Analysis of the influence of SDS or NaCl on C124 structure

Background Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. Methods Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. Results The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12), indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. Discussion Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles

Translational rodent models for research on parasitic protozoa – a review of confounders and possibilities

Rodents, in particular Mus musculus, have a long and invaluable history as models for human diseases in biomedical research, although their translational value has been challenged in a number of cases. We provide some examples in which rodents have been suboptimal as models for human biology and discuss confounders which influence experiments and may explain some of the misleading results. Infections of rodents with protozoan parasites are no exception in requiring close consideration upon model choice. We focus on the significant differences between inbred, outbred and wild animals, and the importance of factors such as microbiota, which are gaining attention as crucial variables in infection experiments. Frequently, mouse or rat models are chosen for convenience, e.g., availability in the institution rather than on an unbiased evaluation of whether they provide the answer to a given question. Apart from a general discussion on translational success or failure, we provide examples where infections with single-celled parasites in a chosen lab rodent gave contradictory or misleading results, and when possible discuss the reason for this. We present emerging alternatives to traditional rodent models, such as humanized mice and organoid primary cell cultures. So-called recombinant inbred strains such as the Collaborative Cross collection are also a potential solution for certain challenges. In addition, we emphasize the advantages of using wild rodents for certain immunological, ecological, and/or behavioral questions. The experimental challenges (e.g., availability of species-specific reagents) that come with the use of such non-model systems are also discussed. Our intention is to foster critical judgment of both traditional and newly available translational rodent models for research on parasitic protozoa that can complement the existing mouse and rat models

Les réseaux de co-évolution au sein des protéines E1 et E2 du Virus de l'Hépatite C révèlent leurs dialogues fonctionnels et proposent de nouvelles stratégies thérapeutiques

Hepatitis C Virus (HCV) infects more than 170 million people worldwide but no vaccine is available yet. HCV entry may represent a promising target for therapies and is mediated by two envelope glycoproteins, E1 and E2, assembled as heterodimer onto the virus surface. However, how E1 and E2 dialog, structurally rearrange and act together during these steps remain poorly defined. In this work, we aimed to clarify the interrelation of E1E2 during virus entry, thus opening ways to potential new therapeutic strategies. We first investigated whether a strong genetic divergence between E1E2 heterodimers may highlight distinct functions. We observed that B-cell derived E1E2 were specialized for B-cell infection, suggesting that new functions can emerge from the E1E2 conformational plasticity. In a second approach, we identified a conserved dialog between E1 and the domain III of E2 that was critical for virus binding and fusion. Moreover, a computational model predicted a strong co-evolution between E1 and E2 as well as potential structural rearrangements, suggesting that HCV E2 is likely a fusion protein able to fold over via its domain III through the mediation of E1. Altogether, these different works highlight that E1 and E2 are involved in complex dialogs that regulate the heterodimer folding and functions, suggesting that E1E2 heterodimer is more likely a single functional protein entity than an association of two proteins with specific functions.Le Virus de l’Hépatite C (VHC) infecte 170 millions de personnes dans le monde mais aucun vaccin n’est encore disponible. Le processus d’entrée du VHC dans les hépatocytes représente une cible prometteuse pour le développement de stratégie thérapeutique et est finement régulé par un nombre par les deux glycoprotéines d’envelope du VHC, E1 et E2, assemblé sous la forme d’un hétérodimère incorporé à la surface des particules virales. Cependant, comment E1 et E2 dialoguent, modifient leurs conformations et se coordonnent mutuellement au cours de l’entrée reste encore à être définit. Dans ce travail, nous avons souhaité clarifier l’interrelation entre E1 and E2 au cours de l’entrée afin d’ouvrir la voie à de potentiels stratégies thérapeutiques. Nous avons tout d’abord examiné si une importante divergence génétique entre des hétérodimères E1E2 pouvait être liée à l’existence de fonctions particulières. Nous avons observé une spécialisation des E1E2 isolé des Lymphocytes B pour l’infection de ces mêmes cellules mais pas des hépatocytes, suggérant que de nouvelles fonctions peuvent émerger de la plasticité conformationel de E1E2. Dans un second temps, nous sommes parvenus à identifier un dialogue conservé entre E1 et le domaine III de E2 (E2 DIII), critique pour les processus d’attachement et de fusion du VHC. Nous avons aussi montré grâce à une approche bio-informatique l’existence d’une co-évolution très importante entre E1 et E2. Cette approche a également prédit de potentiel changement de conformations au sein de l’hétérodimère, suggérant que E2 est sans doute une protéine de fusion capable de se replier sur elle-même via le repliement de son domaine III et l’aide de E1. Ainsi, ces différents travaux soulignent l’implication de E1 et E2 au sein de dialogues fins et complexes, qui régulent à la fois les conformations et les fonctions de l’hétérodimère. Ainsi, cela suggère que l’hétérodimère E1E2 représente plutôt une unité fonctionnelle et structurale unique, plutôt que l’association de deux protéines aux fonctions distinctes

Formation option "Conférence" !

Florian Douam, doctorant à l’ENS de Lyon et président du bureau exécutif de l’association ConférENS, revient sur l’histoire de cette association étudiante qui met en place chaque année une dizaine de conférences sur des sujets scientifiques, sociétaux ou littéraires. Il dévoile les coulisses de cette belle organisation, revient sur les motivations des participants et précise l’intérêt de la démarche. Une initiative à forte valeur ajoutée pour les étudiants !   Un manque à combler… Encore en M..

Pourquoi la science a plus que jamais besoin du web - Par Florian Douam, doctorant en biologie

Florian Douam propose un retour d'expérience, suite à la soirée “Médiation culturelle des sciences sur le web”, organisée par le service Science & Société de l’Université de Lyon à l’Atelier des Médias, dans le cadre des off du www2012, en partenariat avec le collectif Le Grand Mix, et faisant suite à la journée de rencontre et de réflexion organisée en mars 2011 (vidéos en ligne). Vous pouvez retrouver en ligne un compte-rendu multimédia (Storify) de cette soirée, effectué par l’équipe de Kn..

Les réseaux de co-évolution au sein des protéines E1 et E2 du Virus de l'Hépatite C révèlent leurs dialogues fonctionnels et proposent de nouvelles stratégies thérapeutiques

Hepatitis C Virus (HCV) infects more than 170 million people worldwide but no vaccine is available yet. HCV entry may represent a promising target for therapies and is mediated by two envelope glycoproteins, E1 and E2, assembled as heterodimer onto the virus surface. However, how E1 and E2 dialog, structurally rearrange and act together during these steps remain poorly defined. In this work, we aimed to clarify the interrelation of E1E2 during virus entry, thus opening ways to potential new therapeutic strategies. We first investigated whether a strong genetic divergence between E1E2 heterodimers may highlight distinct functions. We observed that B-cell derived E1E2 were specialized for B-cell infection, suggesting that new functions can emerge from the E1E2 conformational plasticity. In a second approach, we identified a conserved dialog between E1 and the domain III of E2 that was critical for virus binding and fusion. Moreover, a computational model predicted a strong co-evolution between E1 and E2 as well as potential structural rearrangements, suggesting that HCV E2 is likely a fusion protein able to fold over via its domain III through the mediation of E1. Altogether, these different works highlight that E1 and E2 are involved in complex dialogs that regulate the heterodimer folding and functions, suggesting that E1E2 heterodimer is more likely a single functional protein entity than an association of two proteins with specific functions.Le Virus de l’Hépatite C (VHC) infecte 170 millions de personnes dans le monde mais aucun vaccin n’est encore disponible. Le processus d’entrée du VHC dans les hépatocytes représente une cible prometteuse pour le développement de stratégie thérapeutique et est finement régulé par un nombre par les deux glycoprotéines d’envelope du VHC, E1 et E2, assemblé sous la forme d’un hétérodimère incorporé à la surface des particules virales. Cependant, comment E1 et E2 dialoguent, modifient leurs conformations et se coordonnent mutuellement au cours de l’entrée reste encore à être définit. Dans ce travail, nous avons souhaité clarifier l’interrelation entre E1 and E2 au cours de l’entrée afin d’ouvrir la voie à de potentiels stratégies thérapeutiques. Nous avons tout d’abord examiné si une importante divergence génétique entre des hétérodimères E1E2 pouvait être liée à l’existence de fonctions particulières. Nous avons observé une spécialisation des E1E2 isolé des Lymphocytes B pour l’infection de ces mêmes cellules mais pas des hépatocytes, suggérant que de nouvelles fonctions peuvent émerger de la plasticité conformationel de E1E2. Dans un second temps, nous sommes parvenus à identifier un dialogue conservé entre E1 et le domaine III de E2 (E2 DIII), critique pour les processus d’attachement et de fusion du VHC. Nous avons aussi montré grâce à une approche bio-informatique l’existence d’une co-évolution très importante entre E1 et E2. Cette approche a également prédit de potentiel changement de conformations au sein de l’hétérodimère, suggérant que E2 est sans doute une protéine de fusion capable de se replier sur elle-même via le repliement de son domaine III et l’aide de E1. Ainsi, ces différents travaux soulignent l’implication de E1 et E2 au sein de dialogues fins et complexes, qui régulent à la fois les conformations et les fonctions de l’hétérodimère. Ainsi, cela suggère que l’hétérodimère E1E2 représente plutôt une unité fonctionnelle et structurale unique, plutôt que l’association de deux protéines aux fonctions distinctes

Pourquoi la science a plus que jamais besoin du web - Par Florian Douam, doctorant en biologie

Florian Douam propose un retour d'expérience, suite à la soirée “Médiation culturelle des sciences sur le web”, organisée par le service Science & Société de l’Université de Lyon à l’Atelier des Médias, dans le cadre des off du www2012, en partenariat avec le collectif Le Grand Mix, et faisant suite à la journée de rencontre et de réflexion organisée en mars 2011 (vidéos en ligne). Vous pouvez retrouver en ligne un compte-rendu multimédia (Storify) de cette soirée, effectué par l’équipe de Kn..