Repurposing of Drugs as Novel Influenza Inhibitors From Clinical Gene Expression Infection Signatures

Influenza virus infections remain a major and recurrent public health burden. The intrinsic ever-evolving nature of this virus, the suboptimal efficacy of current influenza inactivated vaccines, as well as the emergence of resistance against a limited antiviral arsenal, highlight the critical need for novel therapeutic approaches. In this context, the aim of this study was to develop and validate an innovative strategy for drug repurposing as host-targeted inhibitors of influenza viruses and the rapid evaluation of the most promising candidates in Phase II clinical trials. We exploited in vivo global transcriptomic signatures of infection directly obtained from a patient cohort to determine a shortlist of already marketed drugs with newly identified, host-targeted inhibitory properties against influenza virus. The antiviral potential of selected repurposing candidates was further evaluated in vitro, in vivo, and ex vivo. Our strategy allowed the selection of a shortlist of 35 high potential candidates out of a rationalized computational screening of 1,309 FDA-approved bioactive molecules, 31 of which were validated for their significant in vitro antiviral activity. Our in vivo and ex vivo results highlight diltiazem, a calcium channel blocker currently used in the treatment of hypertension, as a promising option for the treatment of influenza infections. Additionally, transcriptomic signature analysis further revealed the so far undescribed capacity of diltiazem to modulate the expression of specific genes related to the host antiviral response and cholesterol metabolism. Finally, combination treatment with diltiazem and virus-targeted oseltamivir neuraminidase inhibitor further increased antiviral efficacy, prompting rapid authorization for the initiation of a Phase II clinical trial. This original, host-targeted, drug repurposing strategy constitutes an effective and highly reactive process for the rapid identification of novel anti-infectious drugs, with potential major implications for the management of antimicrobial resistance and the rapid response to future epidemic or pandemic (re)emerging diseases for which we are still disarmed

Climate-Based Models for Understanding and Forecasting Dengue Epidemics

Dengue fever is a major public health problem in the tropics and subtropics. Since no vaccine exists, understanding and predicting outbreaks remain of crucial interest. Climate influences the mosquito-vector biology and the viral transmission cycle. Its impact on dengue dynamics is of growing interest. We analyzed the epidemiology of dengue in Noumea (New Caledonia) from 1971 to 2010 and its relationships with local and remote climate conditions using an original approach combining a comparison of epidemic and non epidemic years, bivariate and multivariate analyses. We found that the occurrence of outbreaks in Noumea was strongly influenced by climate during the last forty years. Efficient models were developed to estimate the yearly risk of outbreak as a function of two meteorological variables that were contemporaneous (explicative model) or prior (predictive model) to the outbreak onset. Local threshold values of maximal temperature and relative humidity were identified. Our results provide new insights to understand the link between climate and dengue outbreaks, and have a substantial impact on dengue management in New Caledonia since the health authorities have integrated these models into their decision making process and vector control policies. This raises the possibility to provide similar early warning systems in other countries

Analyse et exploitation de signatures transcriptomiques de patients infectés par des virus respiratoires en vue du repositionnement de médicaments pour de nouvelles indications thérapeutiques antivirales

Acute respiratory infections (ARIs) are caused by many pathogens and among them, respiratory viruses hold a very privileged place. Epidemiological studies have highlighted numerous cases of infections caused by rhinoviruses, adenoviruses, pneumoviruses (Respiratory Syncytial Virus and Human Metapneumovirus) or even coronaviruses, but also influenza and parainfluenza viruses. Young children and immunocompromised or elderly people are considered at risk populations, but no age group is spared from these viral respiratory infections. These remain a major cause of consultations, hospitalizations and deaths in both developing and industrialized countries. They are the leading cause of death in young children (more than 2 million deaths per year) and their annual direct cost to our societies is estimated at 2.5 billion euros. Despite this, the therapeutic and / or prophylactic arsenal is very scarce except for influenza viruses, but remains nevertheless limited. The emergence of influenza strains resistant to the few antivirals on the market is indeed a source of major concern, as is the protection conferred by annual vaccines which is sometimes suboptimal, due to the variability of seasonal viral strains. In this context of unmet medical needs and major health and economic issues, my thesis work was part of a research program (RESPIROMIX) which proposes an innovative strategy for the development of antivirals, based on the repositioning of drugs already on the market for new anti- infectious therapeutic indications. My work focused on the characterization and exploitation of transcriptomic signatures of in vivo (bank of biological samples from infected patients) and in vitro (model of human respiratory epithelium cultivated at the air / liquid interface) infections, obtained by hybridization on Affymetrix chips and by high throughput sequencing (NGS), respectively. Our informed choice of tools and the implementation of an adapted and optimized pipeline enabled the differential and functional analyses of these virogenomic signatures, as well as their comparison with a set of chemogenomic signatures, from the Connectivity Map database (CMap). This database is a collection of expression data from human cells in culture treated or not with small bioactive molecules (more than 7 000 gene expression profiles corresponding to 1 309 compounds). My results have notably contributed to (i) the identification and repositioning of Diltiazem, a drug usually used as an antihypertensive, as an inhibitor of influenza viruses, and (ii) the characterization in experimental in vitro and murine models of diltiazem mode of action (MoA), which leads to the endogenous activation of type III interferon genes and metabolic biosynthesis pathways. These results enabled the setup of a multicentric phase II clinical trial (FLUNEXT TRIAL PHRC n°15-0442) aiming to evaluate the use of Diltiazem in the management of patients admitted to intensive care for severe influenza. In the same dynamic, the methodology and pipeline developed during my doctoral work have also led, based on signatures of infected patients, to the selection of several drugs for their therapeutic repositioning against pneumovirus infections (HRSV and HMPV). Some of these candidates are currently being validated in our in vitro and murine models of infectionsLes infections respiratoires aigües (IRAs) sont causées par de nombreux pathogènes et parmi eux, les virus respiratoires tiennent une place très privilégiée. Les études épidémiologiques mettent en lumière de nombreux cas d’infections causées par des rhinovirus, des adénovirus, des pneumovirus (Virus Respiratoire Syncytial et Métapneumovirus humain) ou encore des coronavirus, mais également des virus influenza et parainfluenza. Les jeunes enfants et les personnes immunodéprimées ou âgées sont considérés comme des populations à risque, mais aucune tranche d’âge n’est épargnée par ces infections virales respiratoires. Celles-ci constituent de ce fait une cause majeure de consultations, d’hospitalisations et de décès dans les pays en développement, mais aussi industrialisés. Elles sont la première cause de mortalité chez les jeunes enfants (plus de 2 millions de morts par an) et leur coût direct annuel pour nos sociétés est estimé à 2.5 milliards d'euros. Malgré cela, l’arsenal thérapeutique et / ou prophylactique est très peu fourni ou inexistant, excepté en ce qui concerne les virus influenza, dont les solutions restent malgré tout, limitées. L’émergence de souches influenza résistantes aux quelques antiviraux sur le marché est en effet une source de préoccupation importante, au même titre que la protection conférée par les vaccins annuels qui est parfois sous-optimale, du fait de la grande variabilité des souches virales saisonnières. Dans ce contexte de besoins médicaux non pourvus et d’enjeux sanitaires et économiques majeurs, mon travail de thèse s’est inscrit dans un programme de recherche (RESPIROMIX) visant à proposer une stratégie innovante de développement d’antiviraux, basée sur le repositionnement de médicaments déjà sur le marché pour de nouvelles indications thérapeutiques anti-infectieuses. Mon travail s’est focalisé sur la caractérisation et l’exploitation de signatures transcriptomiques d’infections in vivo (banque d'échantillons biologiques de patients infectés) et in vitro (modèle d’épithéliums respiratoires humains cultivés en interface air/liquide), obtenues par hybridation sur puces Affymetrix et par séquençage à haut débit (NGS), respectivement. Nos choix éclairés d’outils et la mise en place d’un pipeline adapté et optimisé ont permis l’analyse différentielle et fonctionnelle de ces signatures virogénomiques, ainsi que leur comparaison avec un ensemble de signatures chemogénomiques, issues de la base de données Connectivity Map (CMap). Cette base de données est une collection de données d’expression issues de cellules humaines en culture, traitées ou non avec de petites molécules bioactives (plus de 7 000 profils d'expression génique correspondant à 1 309 composés). Mes résultats ont notamment contribué (i) à l’identification et au repositionnement comme inhibiteur des virus influenza du diltiazem, un médicament usuellement utilisé comme antihypertenseur, et (ii) à la caractérisation en modèles in vitro et murin de son mode d’action (MoA), qui se traduit par l’activation endogène des gènes codant pour les interférons de type III et des voies de biosynthèse métabolique. Ces résultats ont ainsi permis la mise en place d’un essai clinique multicentrique de phase II (FLUNEXT TRIAL PHRC n°15-0442), visant à évaluer le Diltiazem dans la prise en charge de patients admis en réanimation pour des grippes sévères. Dans la même dynamique, la méthodologie et le pipeline développés au cours de mon travail doctoral ont également conduit, sur la base de signatures de patients infectés, à la sélection de plusieurs autres médicaments pour leur repositionnement thérapeutique contre les infections à pneumovirus (HRSV et HMPV). Certains de ces candidats, sont actuellement en cours de validation dans nos modèles in vitro et murin d’infection

Analysis and exploitation of transcriptomic signatures of patients infected with respiratory viruses for repositioning drugs for new antiviral therapeutic indications

Les infections respiratoires aigües (IRAs) sont causées par de nombreux pathogènes et parmi eux, les virus respiratoires tiennent une place très privilégiée. Les études épidémiologiques mettent en lumière de nombreux cas d’infections causées par des rhinovirus, des adénovirus, des pneumovirus (Virus Respiratoire Syncytial et Métapneumovirus humain) ou encore des coronavirus, mais également des virus influenza et parainfluenza. Les jeunes enfants et les personnes immunodéprimées ou âgées sont considérés comme des populations à risque, mais aucune tranche d’âge n’est épargnée par ces infections virales respiratoires. Celles-ci constituent de ce fait une cause majeure de consultations, d’hospitalisations et de décès dans les pays en développement, mais aussi industrialisés. Elles sont la première cause de mortalité chez les jeunes enfants (plus de 2 millions de morts par an) et leur coût direct annuel pour nos sociétés est estimé à 2.5 milliards d'euros. Malgré cela, l’arsenal thérapeutique et / ou prophylactique est très peu fourni ou inexistant, excepté en ce qui concerne les virus influenza, dont les solutions restent malgré tout, limitées. L’émergence de souches influenza résistantes aux quelques antiviraux sur le marché est en effet une source de préoccupation importante, au même titre que la protection conférée par les vaccins annuels qui est parfois sous-optimale, du fait de la grande variabilité des souches virales saisonnières. Dans ce contexte de besoins médicaux non pourvus et d’enjeux sanitaires et économiques majeurs, mon travail de thèse s’est inscrit dans un programme de recherche (RESPIROMIX) visant à proposer une stratégie innovante de développement d’antiviraux, basée sur le repositionnement de médicaments déjà sur le marché pour de nouvelles indications thérapeutiques anti-infectieuses. Mon travail s’est focalisé sur la caractérisation et l’exploitation de signatures transcriptomiques d’infections in vivo (banque d'échantillons biologiques de patients infectés) et in vitro (modèle d’épithéliums respiratoires humains cultivés en interface air/liquide), obtenues par hybridation sur puces Affymetrix et par séquençage à haut débit (NGS), respectivement. Nos choix éclairés d’outils et la mise en place d’un pipeline adapté et optimisé ont permis l’analyse différentielle et fonctionnelle de ces signatures virogénomiques, ainsi que leur comparaison avec un ensemble de signatures chemogénomiques, issues de la base de données Connectivity Map (CMap). Cette base de données est une collection de données d’expression issues de cellules humaines en culture, traitées ou non avec de petites molécules bioactives (plus de 7 000 profils d'expression génique correspondant à 1 309 composés). Mes résultats ont notamment contribué (i) à l’identification et au repositionnement comme inhibiteur des virus influenza du diltiazem, un médicament usuellement utilisé comme antihypertenseur, et (ii) à la caractérisation en modèles in vitro et murin de son mode d’action (MoA), qui se traduit par l’activation endogène des gènes codant pour les interférons de type III et des voies de biosynthèse métabolique. Ces résultats ont ainsi permis la mise en place d’un essai clinique multicentrique de phase II (FLUNEXT TRIAL PHRC n°15-0442), visant à évaluer le Diltiazem dans la prise en charge de patients admis en réanimation pour des grippes sévères. Dans la même dynamique, la méthodologie et le pipeline développés au cours de mon travail doctoral ont également conduit, sur la base de signatures de patients infectés, à la sélection de plusieurs autres médicaments pour leur repositionnement thérapeutique contre les infections à pneumovirus (HRSV et HMPV). Certains de ces candidats, sont actuellement en cours de validation dans nos modèles in vitro et murin d’infectionsAcute respiratory infections (ARIs) are caused by many pathogens and among them, respiratory viruses hold a very privileged place. Epidemiological studies have highlighted numerous cases of infections caused by rhinoviruses, adenoviruses, pneumoviruses (Respiratory Syncytial Virus and Human Metapneumovirus) or even coronaviruses, but also influenza and parainfluenza viruses. Young children and immunocompromised or elderly people are considered at risk populations, but no age group is spared from these viral respiratory infections. These remain a major cause of consultations, hospitalizations and deaths in both developing and industrialized countries. They are the leading cause of death in young children (more than 2 million deaths per year) and their annual direct cost to our societies is estimated at 2.5 billion euros. Despite this, the therapeutic and / or prophylactic arsenal is very scarce except for influenza viruses, but remains nevertheless limited. The emergence of influenza strains resistant to the few antivirals on the market is indeed a source of major concern, as is the protection conferred by annual vaccines which is sometimes suboptimal, due to the variability of seasonal viral strains. In this context of unmet medical needs and major health and economic issues, my thesis work was part of a research program (RESPIROMIX) which proposes an innovative strategy for the development of antivirals, based on the repositioning of drugs already on the market for new anti- infectious therapeutic indications. My work focused on the characterization and exploitation of transcriptomic signatures of in vivo (bank of biological samples from infected patients) and in vitro (model of human respiratory epithelium cultivated at the air / liquid interface) infections, obtained by hybridization on Affymetrix chips and by high throughput sequencing (NGS), respectively. Our informed choice of tools and the implementation of an adapted and optimized pipeline enabled the differential and functional analyses of these virogenomic signatures, as well as their comparison with a set of chemogenomic signatures, from the Connectivity Map database (CMap). This database is a collection of expression data from human cells in culture treated or not with small bioactive molecules (more than 7 000 gene expression profiles corresponding to 1 309 compounds). My results have notably contributed to (i) the identification and repositioning of Diltiazem, a drug usually used as an antihypertensive, as an inhibitor of influenza viruses, and (ii) the characterization in experimental in vitro and murine models of diltiazem mode of action (MoA), which leads to the endogenous activation of type III interferon genes and metabolic biosynthesis pathways. These results enabled the setup of a multicentric phase II clinical trial (FLUNEXT TRIAL PHRC n°15-0442) aiming to evaluate the use of Diltiazem in the management of patients admitted to intensive care for severe influenza. In the same dynamic, the methodology and pipeline developed during my doctoral work have also led, based on signatures of infected patients, to the selection of several drugs for their therapeutic repositioning against pneumovirus infections (HRSV and HMPV). Some of these candidates are currently being validated in our in vitro and murine models of infection

Characterization of cellular transcriptomic signatures induced by different respiratory viruses in human reconstituted airway epithelia

International audienceAcute respiratory infections, a large part being of viral origin, constitute a major public health issue. To propose alternative and/or new therapeutic approaches, it is necessary to increase our knowledge about the interactions between respiratory viruses and their primary cellular targets using the most biologically relevant experimental models. In this study, we used RNAseq to characterize and compare the transcriptomic signature of infection induced by different major respiratory viruses (Influenza viruses, hRSV and hMPV) in a model of reconstituted human airway epithelia. Our results confirm the importance of several cellular pathways commonly or specifically induced by these respiratory viruses, such as the innate immune response or antiviral defense. A very interesting common feature revealed by the global virogenomic signature shared between hRSV, hMPV and influenza viruses is the global downregulation of cilium-related gene expression, in good agreement with experimental evaluation of mucociliary clearance. Beyond providing new information about respiratory virus/host interactions, our study also underlines the interest of using biologically relevant experimental models to study human respiratory viruses. Acute respiratory infections (ARI) constitute a leading cause of acute illness worldwide and a major cause of death among young children, with nearly 2 million deaths per year 1-3. Among a panoply of different viral and bacterial pathogens, respiratory viruses, such as influenza A and B viruses (IAV and IBV), respiratory syncytial virus (hRSV-A and hRSV-B), human metapneumovirus (hMPV-A and hMPV-B) or rhinoviruses (RV), represent the main etiologic agents of these infections 2,4,5. In that regard, the limited or non-existent prophylactic and therapeutic arsenal available, coupled with the emergence of antiviral resistance, highlight the public health burden imposed by these respiratory pathogens. It appears therefore urgent to develop alternative and/or new therapeutic approaches, for which it is a necessary condition to increase our knowledge of the interactions between respiratory viruses and their primary cellular targets, namely respiratory epithelial cells. The last decade has witnessed the development of high-throughput "omics" approaches that have contributed to deepen our understanding of the multiple levels of interplay between respiratory viruses and the host cell. Numerous studies have described the impact of infection on host gene expression in vitro or in vivo, mainly in the context of influenza viruses or hRSV and to a lesser extent for other respiratory viruses such hMPV 6-10. For example , several mRNA profiling studies, including ours, have highlighted the role of NF-kB, p53, or MAPK cellula

Transcriptional Profiling of Immune and Inflammatory Responses in the Context of SARS-CoV-2 Fungal Superinfection in a Human Airway Epithelial Model

An increasing amount of evidence indicates a relatively high prevalence of superinfections associated with coronavirus disease 2019 (COVID-19), including invasive aspergillosis, but the underlying mechanisms remain to be characterized. In the present study, to better understand the biological impact of superinfection, we determine and compare the host transcriptional response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) versus Aspergillus superinfection, using a model of reconstituted human airway epithelium. Our analyses reveal that both simple infection and superinfection induce strong deregulation of core components of innate immune and inflammatory responses, with a stronger response to superinfection in the bronchial epithelial model compared to its nasal counterpart. Our results also highlight unique transcriptional footprints of SARS-CoV-2 Aspergillus superinfection, such as an imbalanced type I/type III IFN, and an induction of several monocyte and neutrophil associated chemokines, that could be useful for the understanding of Aspergillus-associated COVID-19 and also the management of severe forms of aspergillosis in this specific context

Transcriptional profiling of immune and inflammatory responses in the context of SARS-CoV-2 fungal superinfection in a human airway epithelial model

International audienceSuperinfections of bacterial/fungal origin are known to affect the course and severity of respiratory viral infections. An increasing number of evidence indicate a relatively high prevalence of superinfections associated with COVID-19, including invasive aspergillosis, but the underlying mechanisms remain to be characterized. In the present study, to better understand the biological impact of superinfection we sought to determine and compare the host transcriptional response to SARS-CoV-2 versus Aspergillus superinfection, using a model of reconstituted humain airway epithelium. Our analyses reveal that both simple infection and superinfection induce a strong deregulation of core components of innate immune and inflammatory responses, with a stronger response to superinfection in the bronchial epithelial model compared to its nasal counterpart. Our results also highlight unique transcriptional footprints of SARS-CoV-2 Aspergillus superinfection, such as an imbalanced type I/type III IFN, and an induction of several monocyte- and neutrophil associated chemokines, that could be useful for the understanding of Aspergillus -associated COVID-19 and but also management of severe forms of aspergillosis in this specific context

Arboviral Risk Associated with Solid Organ and Hematopoietic Stem Cell Grafts: The Prophylactic Answers Proposed by the French High Council of Public Health in a National Context

Diseases caused by arboviruses are on the increase worldwide. In addition to arthropod bites, most arboviruses can be transmitted via accessory routes. Products of human origin (labile blood products, solid organs, hematopoietic stem cells, tissues) present a risk of contamination for the recipient if the donation is made when the donor is viremic. Mainland France and its overseas territories are exposed to a complex array of imported and endemic arboviruses, which differ according to their respective location. This narrative review describes the risks of acquiring certain arboviral diseases from human products, mainly solid organs and hematopoietic stem cells, in the French context. The main risks considered in this study are infections by West Nile virus, dengue virus, and tick-borne encephalitis virus. The ancillary risks represented by Usutu virus infection, chikungunya, and Zika are also addressed more briefly. For each disease, the guidelines issued by the French High Council of Public Health, which is responsible for mitigating the risks associated with products of human origin and for supporting public health policy decisions, are briefly outlined. This review highlights the need for a “One Health” approach and to standardize recommendations at the international level in areas with the same viral epidemiology

Human Respiratory Syncytial Virus-induced immune signature of infection revealed by transcriptome analysis of clinical pediatric nasopharyngeal swab samples

International audienceHuman Respiratory Syncytial Virus (HRSV) constitutes one the main causes of respiratory infection in neonates and infants worldwide. Transcriptome analysis of clinical samples using high-throughput technologies remains an important tool to better understand virus-host complex interactions in the real-life setting but also to identify new diagnosis/prognosis markers or therapeutics targets. A major challenge when exploiting clinical samples such as nasal swabs, washes or bronchoalveolar lavages is the poor quantity and integrity of nucleic acids. In this study, we applied a tailored transcriptomics workflow to exploit nasal wash samples from children who tested positive for HRSV. Our analysis revealed a characteristic immune signature as a direct reflection of HRSV pathogenesis and highlighted putative biomarkers of interest such as IP-10, TMEM190, MCEMP1 or TIMM23

Repurposing of Drugs as Novel Influenza Inhibitors From Clinical Gene Expression Infection Signatures

Influenza virus infections remain a major and recurrent public health burden. The intrinsic ever-evolving nature of this virus, the suboptimal efficacy of current influenza inactivated vaccines, as well as the emergence of resistance against a limited antiviral arsenal, highlight the critical need for novel therapeutic approaches. In this context, the aim of this study was to develop and validate an innovative strategy for drug repurposing as host-targeted inhibitors of influenza viruses and the rapid evaluation of the most promising candidates in Phase II clinical trials. We exploited in vivo global transcriptomic signatures of infection directly obtained from a patient cohort to determine a shortlist of already marketed drugs with newly identified, host-targeted inhibitory properties against influenza virus. The antiviral potential of selected repurposing candidates was further evaluated in vitro, in vivo, and ex vivo. Our strategy allowed the selection of a shortlist of 35 high potential candidates out of a rationalized computational screening of 1,309 FDA-approved bioactive molecules, 31 of which were validated for their significant in vitro antiviral activity. Our in vivo and ex vivo results highlight diltiazem, a calcium channel blocker currently used in the treatment of hypertension, as a promising option for the treatment of influenza infections. Additionally, transcriptomic signature analysis further revealed the so far undescribed capacity of diltiazem to modulate the expression of specific genes related to the host antiviral response and cholesterol metabolism. Finally, combination treatment with diltiazem and virus-targeted oseltamivir neuraminidase inhibitor further increased antiviral efficacy, prompting rapid authorization for the initiation of a Phase II clinical trial. This original, host-targeted, drug repurposing strategy constitutes an effective and highly reactive process for the rapid identificatio