Thesis
Analysis and exploitation of transcriptomic signatures of patients infected with respiratory viruses for repositioning drugs for new antiviral therapeutic indications
Claire NICOLAS DE LAMBALLERIE – JULY 2020
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 infections.
Keywords: Influenza Virus, Human Metapneumovirus (HMPV), Respiratory Syncytial Virus (HRSV), human respiratory epithelium model, Transcriptome, Next-Generation Sequencing (NGS), antivirals, drug repositioning.
Study of human MetaPneumoVirus (hMPV) infection and their virulence factors. Development of live-attenuated hMPV vaccine candidate
Julia DUBOIS – January 2018
Human metapneumovirus (hMPV) is a major pathogen responsible of acute respiratory tract infections, such as bronchiolitis or pneumonia, affecting especially infants, under five years old, elderly individuals and immunocompromised adults. Identified since 2001, this virus and its pathogenesis still remain largely unknown and no licensed vaccines or specific antivirals against hMPV are currently available. In this context, my research project was built over two main subjects:
(i) The study of the fusion F glycoprotein that is the major antigenic protein of hMPV and is responsible of viral entry into host cell. By its crucial role for the virus, the F protein has already been characterized in several structural and/or functional studies. Thus, it has been described that the hMPV F protein induces membrane fusion autonomously, resulting in variable cytopathic effects in vitro, in a strain-dependent manner. However, as the determinants of the hMPV fusogenic activity are not well characterized yet, we focused on identification of some of these, located in heptad repeats domains of the protein.
(ii) The modifications of hMPV genome for viral attenuation. Live-attenuated hMPV vaccine candidates for immunization of infants could be very promising. Several engineered hMPV candidate vaccine were constructed by reverse genetic and evaluated in vitro, ex vivo and in vivo for their replication and property to protect against viral challenge.
Keywords: Human metapneumovirus, Pneumovirus, viral pneumonia, fusion protein, attenuated virus, vaccine, reverse genetics
Host-pathogens interactions during RSV / S. pneumoniae infection : immune response and p53 pathway
Daniela Bandeira – December 2017
Respiratory viruses play a leading role in the etiology of respiratory infections. Currently, respiratory syncytial virus (RSV) is generally considered to be the etiologic agent of respiratory disease in pediatric importance, as children can develop bronchiolitis and pneumonia when infected with the virus. The firt RSV infection occurs in the first two years of life in about 95% of children, with the peak incidence occurring in the first few months of life. An important aspect of the prognosis of viral infections is the role of bacterial co-infection. The combination of viral and bacterial agents has been reported between RSV and Streptococcus pneumoniae bacteria. Because of the clinical importance of this co-infection and the high rate of RSV circulation, it is important to understand how the immune system is affected by the infection of both pathogens. Our study was designed to evaluate the immune response in macrophages, in addition to interactions between RSV and p53 transcription factor. The results show a particular profile of this mixed co-infection in macrophages and p53 regulation that implies several modifications in the innate immune response and that allowed us to better understand the mechanisms of pathogenesis of RSV in pulmonary epithelial cells. In the last part, we evaluated the direct impact of mixed co-infection in non-human primates and this model showed us the difficulties and complexities of establishing severe pneumonia.
Keywords: Respiratory Syncytial Virus (RSV), Streptococcus pneumoniae (Sp), co-infection, p53
[Etude des mécanismes moléculaires gouvernant le réassortiment génétique et la modulation des glycoprotéines de surface des virus influenza de type A].
Matthieu YVER - December 2013.
The genome of the influenza A virus (IAV) comprises eight single-stranded negativesense RNA segments (vRNAs). All eight vRNAs are selectively packaged into each progeny virion via packaging signal sequences that are located at both ends of the vRNAs. How these signals ensure packaging of all eight vRNAs remains unclear. It was hypothesized that selective packaging might be driven by direct interactions between vRNAs. Combination of biochemical and reverse genetic approaches allowed us to identify short nucleotide regions on vRNAs interacting with each other in vitro. Here, we demonstrated the importance of these interactions in the packaging process of the human H3N2 and avian H5N2 viral genomes. Furthermore, our results suggest that the packaging process could regulate genetic reassortment. Indeed, we observed that the genetic reassortment between H3N2 and H5N2 viruses is restricted as the avian vRNA HA cannot be incorporated into the human genetic background. Our investigations indicated that (i) the packaging signals are crucial for genetic reassortment and (ii) the modulation of the vRNAs interaction network may be required for the incorporation of the avian HA gene into the human genetic background. Characterization of seed viruses showed that the genetic composition is important for both high growth ability and antigen production. Indeed, cryo-electronic microscopy observations of reassortant virus indicated that the PB1 gene can strongly influence the antigen glycoprotein spike density.
[La glycoprotéine de fusion F des paramyxovirus. Etude structure-fonction et ingénierie de F en vue du développement d'applications thérapeutiques].
Jean-Christophe LE BAYON - September 2013.
Human respiratory paramyxoviruses are responsible of infectious diseases and hospitalisations among infants, children, elderly and immune-compromised. These viruses harbour two glycoproteins implicated in the virus entry into the cell. The attachment glycoprotein (HN, G or H) is implicated the virus attachment on a targeted cell receptor, and HN is also suspected to activate the second glycoprotein, the fusion protein (F). This latter can perform the fusion between the cellular membrane and the viral envelope. This mechanism of activation of the F protein, is actually not well-defined, even with the structural characterisation for some viruses studied in this thesis. This thesis work is focalised onto the viral glycoprotein of parainfluenza virus type 2 (hPIV-2), parainfluenza virus type 5 (PIV-5), and the fusion glycoprotein of human Metapneumovirus (hMPV). The first part of this project was the characterization of a mutation observed in the F protein natural variants of hPIV-2. This work highlights the importance of the F2 subunit of F in the fusion regulation. A second part of the project consisted on the study of the hMPV entry into the cell mechanism, induced by F glycoprotein. This work showed that it was possible to dissociate the characteristics of the F glycoprotein, in order to allow a better understanding of those. This engineering work on the F protein was also targeted onto applied science and could be used in the development of therapeutic tools. This therapeutic use of F PIV-5 was also evaluated in an oncolytic vector based on adenovirus type 5 (AdV-5). Its expression among tumours showed a highly cytotoxic activity for the targeted cells in vivo, but also in vitro on immune-competent rodents.
[Etude transcriptomique des réponses cellulaires à l'infection par différents virus influenza de type A : caractérisation des signatures spécifiques et communes pour la recherche d'antiviraux]
Laurence JOSSET - December 2010
The current treatment of flu relies on antiviral drug targeting viral proteins that can induce the appearance of resistant virus. To limit this risk, alternative therapies are developed that target essential cellular partners of the virus. This thesis is part of this search for new therapeutic and of the study of host-pathogen interactions essential to their development. We proposed an innovative method for the identification of antiviral drugs based on the cell gene- expression profile associated with infection with five different influenza A virus strains belonging to different subtypes (H1N1, H3N2, H5N1, H5N2 and H7N1). Eight molecules inversing the infection signature were selected from the Connectivity Map database and 7 molecules inhibited viral growth on at least one of the viruses tested in vitro. This is the first study showing that gene expression- based screening can be used to identify antivirals. This transcriptomic study provides further characterization of strain specific cellular responses. While HlN1 virus changes slightly cellular transcription, H3N2, H5N1, H5N2 and H7N1 viruses modulate the expression of genes involved in MAPK, NF-kB, IRF3 and p53 pathways. Analysis of the functional involvement of specific modifications of cellular transduction pathways could help to better understand the pathogenesis of some particular viral strains.
[Etude des mécanismes moléculaires gouvernant le réassortiment génétique des virus influenza de type A].
Boris ESSERE - June 2010.
The Flu is a frequent viral infectious disease caused by the Influenza viruses. Their genomes are composed by eight negative single-stranded RNA organised as vRNPs. During the viral cycle, the terminal non-coding and coding regions of viral genome have been shown to be crucial for the selective incorporation of a complete set of the eight vRNPs into influenza viral particles. Band shift assay and electron tomography allowed us to show that all gene segments interact together by RNA/RNA interactions involving their packaging region. Our results suggest that the eight genomic vRNAs are selected and packaged as an organized supramolecular complex held together between identified packaging regions into neosynthesized virions. Due to genome segmented nature, genetic reassortment can occur during co-infection. In order to identify molecular mechanisms responsible for the observed restriction during the genetic reassortment, we have developed a new competitive reverse genetic strategy allowing us to evaluate the genetic reassortment between H3N2 and H5N2 viruses. Our results suggest that mechanism controlling the packaging should regulate genetic reassortment. We have shown that the modulation of RNA/RNA interaction between HA and M gene segment have allowed us to increase HA H5 gene segment incorporation rate into a viral human genetic background, prerequisite for pandemic virus emergency.
[Construction et évaluation fonctionnelle de vecteurs adénovirauxoncolytiques armés exprimant des protéines hyperfusogènes du virusparainfluenza de type 5].
François DURUPT - June 2010.
Oncolytic adenoviral vectors are emerging as promising anti-cancer therapies. These infectious agents derived from human Adenovirus serotype 5 are genetically engineered to infect and kill tumor cells. Several modifications are brought to transduce specifically target cells, restrict the viral replication to tumor cells and insert transgenes in the viral genome in order to enhance anti-tumor efficiency. Among these transgenes, viral fusogenic proteins are of particular interest. These glycoproteins are harbored at the surface of enveloped viruses. They are able to induce strong cytotoxicity (including to non transduced cells), to modify tumor tissue architecture through cell fusion, and to induce beneficial anti-tumor immune response. Replication competent or incompetent adenoviral vectors expressing such fusogenic genes have been constructed by other groups and seem to be promising. The first part of this thesis focused on the engineering of hyperfusogenic mutants of the F protein of the Parainfluenza virus type 5 (F-PIV5). A library of mutants was created, combining different mutations described in the literature together with original mutations determined through the structural study of F-PIV5. This work analyzed the critical mutations involved in the fusion process of F-PIV5 and allowed the creation of hyperfusogenic and autonomous mutants.In the second part of this work, 3 hyperfusogenic genes were inserted into replication-competent adenoviral vectors. These vectors were produced at high titers, induced fusion in tumor cells and showed enhanced cytotoxicity on human melanoma cell lines. Finally, a three-dimensional model of vascularized tumors was implemented on fertilized chicken eggs. This model was easy to handle and allowed high fidelity to the histology of human metastases. The fusogenic replication-competent vectors constructed in this thesis were tested on this model, and showed cytotoxicity and fusogenicity on solid melanoma tumor masses.
[Les glycoprotéines d'enveloppe des virus Parainfluenza : étude structure versus fonctions et développement d'applications diagnostiques et thérapeutiques].
Olivier TERRIER - January 2009
Human parainfluenza viruses (hPIV, family Paramyxoviridae) are respiratory viruses, often responsible ofinfections at the young children, the elderly and also the immuno-deficient patients. These viruses possess twoglycoproteins at the surface of their envelope, that both play a role in the viral entry into the target cell. Thehemagglutinin-neuraminidase glycoprotein (HN) allows the virus to become attached to the cellular receptor.Then, HN "activates" the second glycoprotein, the protein of fusion (F). This last achieves the fusion betweenthe envelope of the virus and the cellular membrane. The mechanism by which the HN protein "activates" the Fprotein remains unknowns, despite the recent determination of the glycoprotein structures. Several models arecurrently proposed in the literature. This thesis work was mainly focused on the human parainfluenza virus type 2 (hPIV-2) and the animalparainfluenza virus type 5 (PIV-5) envelope glycoproteins.A first step of this project has consisted to characterize several circulating hPIV-2 strains at the level of theirglycoproteins and to analyze the differences observed in their functional domains. This work has underlined theimportance of the diagnostic tools update especially in the surveillance of these respiratory viruses.A second part of the project has consisted to study one of viral entry steps of parainfluenza viruses, themembrane fusion led by the F glycoprotein, in a PIV-5 viral model This work has shown that it was to a certainextent possible, by a combinative mutagenesis approach, to dissociate the features of F PIV-5. The engineeringof a viral glycoprotein like PIV-5 F could contribute to the development of tools in therapeutic.The first description of PIV-5 in cryo-electron microscopy has been achieved during this work. This work hasrevealed that the morphology of PIV-5, in a state close to its fully hydrated natural state, was less heterogeneousthan what was generally described in the literature. It was possible to calculate the surface density of theglycoproteins, a new and interesting data, regarding to the actual knowledge on parainfluenza viruses viral entrymechanisms. Works done during this thesis were also accompanied by the development of diagnostic and therapeuticapplications.
