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All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.

In dieser Arbeit wurden Vektorimpfstoffe auf der Basis rekombinanter Vacciniaviren hinsichtlich ihrer Eignung zur Immunisierung gegen Infektionen mit Respiratorischen Synzytialviren (RSV) untersucht. Hierfür standen genetisches Material und Viruspräparationen des Respiratorischen Synzytialvirus des Rindes (BRSV, Stamm Odijk) sowie des Respiratorischen Synzytialvirus des Menschen (HRSV, Subtyp A2) sowie rekombinante Vacciniaviren MVA-HRSV-F bzw. MVA-HRSV-G zur Verfügung. Rekombinante MVA-Viren, welche die Gene der BRSV-Oberflächenproteine G und F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu), sowie Viren in welchen die Fremdgensequenzen durch Deletion wieder entfernt sind (Revertante Viren MVA-∆BRSV-F und MVA-∆BRSV-G), wurden gentechnologisch hergestellt. Alle rekombinanten MVA-Viren wurden molekular-virologisch charakterisiert und dienten zur Gewinnung und Prüfung von Testimpfstoffen im Tiermodell. Die Untersuchungen zeigen: 1. Alle neu konstruierten rekombinanten MVA-BRSV-Viren produzierten nach Infektion von Zellkulturen die erwünschten Zielantigene, die BRSV-Glykoproteine F und G. Für das durch MVA-Expression hergestellte BRSV-F-Glykoprotein konnte außerdem die biologische Funktionalität in einem Fusionstest in infizierten HeLa-Zellen nachgewiesen werden. 2. Die Charakterisierung der Genome aller MVA-BRSV- sowie MVA-HRSV-Vektorviren bestätigte die exakte Insertion der Fremdgensequenzen im anvisierten Genombereich und zeigte die genetische Stabilität der Virusisolate nach Passagierung. 3. Bei der Untersuchung des Wachstumsverhaltens von MVA-BRSV-F und MVA-BRSV-G zeigte sich die eingeschränkte Vermehrungsfähigkeit des Virus MVA-BRSV-G. Die Konstruktion und Untersuchung der revertanten Viren MVA-∆BRSV-F und MVA-∆BRSV-G belegte die Koproduktion des G-Proteins als Ursache des verminderten Replikationsvermögens. Dieser für ein mögliches Impfvirus erhebliche Nachteil konnte durch die Verwendung eines moderateren Vacciniavirus-Promotors zur Fremdgenexpression (rekombinantes Virus MVA-BRSV-Gneu) behoben werden. 4. Die Prüfung von Testimpfstoffen auf der Grundlage der rekombinanten MVA-HRSV-Viren in einem Maus-HRSV-Infektionsmodell zeigte, dass MVA-HRSV-Impfstoffe, im Gegensatz zu Impfstoffen aus mit Formalin-inaktiviertem HRSV, Immunantworten mit einem ausgewogenen TH1/TH2-assoziierten Zytokinprofil induzierten. Eine infolge von Immunisierung verstärkte Einwanderung eosinophiler Zellen (Marker für Immunpathogenese) in die Lungen HRSV-infizierter Tiere, konnte nach MVA-Impfung nicht beziehungsweise in nur sehr geringem Ausmaß festgestellt werden (OLSZEWSKA et al. 2004). 5. Wichtige erste Daten hinsichtlich der Verträglichkeit, Immunogenität und Schutzwirkung rekombinanter Impfstoffe auf der Basis von MVA-BRSV-F und MVA-BRSV-G konnten in einem Kälber BRSV-Infektionsmodell erhoben werden. Die zweimalige Immunisierung mit MVA-Impfstoff verlief bei allen Tieren ohne feststellbare Nebenwirkungen und die Anregung Vaccinia- bzw. BRSV-F-spezifischer Antikörper bestätigte die Immunogenität der Vektorvakzinen. Schließlich belegten klinische Daten, insbesondere die fehlende Fieberreaktion bei Impflingen nach BRSV-Belastungsinfektion, die Schutzwirkung der MVA-BRSV-Impfstoffe. Insgesamt unterstützen die erzielten Ergebnisse dieser Arbeiten die weitere präklinische und klinische Untersuchung von MVA-Vektorimpfstoffen zur wirksameren und sichereren Bekämpfung von Infektionen mit Respiratorischen Synzytialviren. / This study investigated vector vaccines based on recombinant vaccinia virus MVA for their suitability to immunize against infections with respiratory syncytial viruses. Genetic material and virus stocks of bovine respiratory syncytial virus (BRSV, Strain Odijk) and human respiratory syncytial virus (HRSV, Strain A2) and recombinant vaccinia viruses MVA-HRSV-F and MVA-HRSV-G were provided and used in this study. The project work included the genetical engineering of recombinant MVA expressing gene sequences encoding the BRSV surface proteins G and F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu) and the secondary generation of mutant viruses in which recombinant gene sequences have been removed (revertant viruses MVA-∆BRSV-F, MVA-∆BRSV-G). All recombinant MVA were carefully characterized in in vitro experiments and served for generation of vaccine preparations being tested in animal model systems. The investigations demonstrate: 1. All recombinant MVA-BRSV viruses produced the target antigens (BRSV-F and -G proteins) upon tissue culture infections. Functional activity of BRSV-F protein was demonstrated in a cell fusion assay using virus-infected HeLa cells. 2. The characterization of the genomes of all MVA recombinant viruses confirmed the correct insertion of foreign gene sequences into the target site of the MVA genome and demonstrated the genetic stability of the vector viruses upon tissue culture passage. 3. In vitro studies on virus growth revealed a reduced replicative capacity of the recombinant virus MVA-BRSV-G. Construction and growth analysis of revertant viruses MVA-∆BRSV-F and MVA-∆BRSV-G demonstrated that over expression of BRSV-G protein caused this replication deficiency which could be avoided by using a more moderate vaccinia virus promoter for transcriptional control of recombinant gene expression (recombinant virus MVA-BRSV-Gneu). 4. Upon characterization in a mouse-HRSV challenge model candidate vaccines based on recombinant MVA-HRSV viruses, in contrast to formalin inactivated HRSV, and induced a well balanced TH1 and TH2 cytokine profile. In addition, none of the MVA-HRSV-F vaccinated animals and only two of the MVA-HRSV-G immunized mice showed low-level eosinophilia in the lungs after HRSV challenge infection (OLSZEWSKA et al. 2004). 5. Vaccination experiments in the calf-BRSV challenge model generated first relevant data on safety, immunogenicity and protective capacity of MVA-BRSV recombinant vaccines. The repeated application of MVA vaccine was well tolerated by all vaccinated animals and the induction of vaccinia- and BRSV-F-specific antibody responses confirmed the immunogenicity of the MVA vector vaccines. Moreover, clinical data (lack of fever response in vaccines) suggested the protective capacity of MVA-BRSV immunization upon BRSV challenge. The obtained results from these studies clearly support further preclinical and clinical evaluation of recombinant MVA candidate vaccines to immunize against disease caused by RSV infections in cattle and humans.

Le virus respiratoire syncytial (RSV) est un virus à ARN de polarité négative. Les études démontrent que toute la population sera infectée par ce virus au moins deux fois avant l’âge de 3 ans. Le RSV peut provoquer plusieurs pathologies respiratoires telles que la bronchiolite aiguë et la pneumonie. Les infections sévères corrèlent avec le développement de l’asthme. Lors d’une infection virale, les particules du RSV sont détectées par le senseur RIG-I qui induit l’activation des facteurs de transcription NF-κB et IRF-3. Respectivement, les facteurs de transcription activeront les réponses inflammatoire et antivirale. Au coeur des pathologies induites par le RSV se trouve une réponse immunitaire mal adaptée. Plus précisément, par l’entremise de NF-κB, le RSV provoque une production exagérée de cytokines et chimiokines qui induisent une réponse inflammatoire démesurée provoquant du dommage tissulaire. Paradoxalement, le RSV est capable d’échapper à la réponse antivirale. Ces deux phénomènes sont contrôlés par l’entremise des protéines non structurales NS1 et NS2. Le mécanisme délimitant le mode d’action de NS1 et NS2 sur la réponse antivirale reste à être déterminé. Avec pour objectif d’élucider comment NS1 et NS2 inhibent la réponse antivirale, nous avons investigué le mécanisme de reconnaissance de l’hôte vis-à-vis de RSV. Nous démontrerons, pour la première fois, que le senseur cytosolique MDA5 est impliqué dans la réponse antivirale contre le RSV. Nous présenterons des résultats préliminaires qui suggèrent que le rôle de MDA5 est non redondant à RIG-I. À l’aide d’ARN interférant dirigé contre RIG-I et de transfection de MDA5, nous démontrerons que MDA5 ne contribue pas à la phosphorylation d’IRF-3, mais plutôt qu’elle régit la stabilité du facteur de transcription. Nous démontrerons aussi que, contrairement à l’hypothèse actuelle sur le fonctionnement de NS1 et NS2, l’inhibition de ces derniers ne provoque pas une augmentation de la cytokine antivirale IFN−β. Cependant, l’expression ectopique de NS1 et NS2 réduit l’activité du promoteur de l’IFN-β et de la protéine cytoplasmic antivirale ISG56 lorsqu’elle est mesurée par essai luciférase. / Respiratory Syncytial Virus (RSV) is a RNA virus with negative polarity. RSV infections are the most common cause of hospitalization among infants. Among populations at risk, infection of RSV can be quite severe. RSV infections can cause bronchiolitis, pneumonia, while severe infections are linked to the development of asthma. Early in the infectious cycle of RSV, the cytosolic sensor RIG-I captures viral particles, and activates the immune response by engaging the transcription factors IRF-3 and NF-κB. At the heart of RSV mediated pathologies is a skewed immune response. More precisely, RSV over stimulates the release of proinflammatory chemokines and cytokines. Intriguingly, while RSV is able to stimulate the production of proinflammatory cytokines and chemokines, RSV under stimulates the antiviral response. The ability of RSV to evade the antiviral response is thought to be mediated by its non-structural proteins: NS1 and NS2. However, the mechanism by which NS1 and NS2 enable RSV to evade the antiviral response remains to be determined. In this memoir we investigated, how RSV is recognized by the innate immune response in airway epithelial cells. With this information we hope to improve our understanding of how NS1 and NS2 allow RSV to circumvent the antiviral response. We show for the first time that cytosolic sensor MDA5 plays a role in the recognition of RSV particles. Using a combination of interfering RNA directed against RIG-I, and transfection of MDA5, we show that MDA5 does not contribute to the phosphorylation of IRF-3. According to the data presented, we suggest that MDA5’s role in the immune response is to prevent the degradation of IRF-3. Contrary to previous research, we show that the inhibition of the nonstructural protein does not increase the production of the antiviral cytokine IFN-β. However, the ectopic expression of NS1 and NS2 does lead to a reduction of the promoter activity of IFN-β and the antiviral protein ISG56 when measured by luciferase assay. This research highlights the importance of MDA5 as a potential therapeutic target in the development of a cure for RSV.

Afin de se répliquer, les rétrovirus doivent disposer à la fois d’ARN épissés et non épissés. Chez le virus du Sarcome de Rous (RSV), l’accumulation de l’ARN non épissé dépend de l’élément NRS (Negative Regulator of Splicing). L’élément NRS est un élément bipartite. Sa région 5’ est assimilée à une séquence ESE (Exon Splicing Enhancer) à laquelle se fixent de nombreuses protéines SR tandis que sa région 3’ contient un pseudo-site 5’ non fonctionnel qui constitue un leurre qui est responsable de l’inhibition de l’épissage à l’unique site 5’ fonctionnel du virus. Seule la structure 3D de la partie 3’ du NRS qui contient le pseudo-site a été expérimentalement établie. Dans ce travail, nous avons déterminé la structure 2D de la totalité de l’élément NRS à l’aide de sondes chimiques et enzymatiques. La comparaison de cette structure expérimentale à celles que nous avons établies pour d’autres éléments NRS mutants fonctionnels et non fonctionnel ainsi qu’à celles théoriques de la totalité des virus aviaires séquencés argumente en faveur de la forte signification biologique de notre modèle. Des expériences d’épissage in vitro réalisées sur l’élément NRS sauvage ainsi que ses formes tronquées ont permis de mettre en évidence le rôle crucial de deux structures tige-boucles dans la fonction du NRS. Les expériences de purification de complexes formés avec un extrait nucléaire de cellules HeLa sur ces différents éléments NRS par des techniques chromatographie d’affinité ont permis de démontrer l’importance de l’association de ces deux structures tige-boucles avec les protéines SR et la snRNP U1. Nous avons défini un nouvel élément NRS minimal fonctionnel capable d’inhiber l’épissage et nous avons démontré l’activation de l’inhibition de l’épissage de l’élément NRS par la protéine 9G8 in vitro et in cellulo / Retroviruses require both spliced and unspliced RNAs for productive replication. Accumulation of unspliced RNA in Rous Sarcoma Virus (RSV) depends on the NRS element, (Negative Regulator of Splicing). The NRS element is bipartite. Its 5’ terminal part is considered as an ESE that binds SR proteins and its 3’ part contains a decoy 5’-splice site (ss), which inhibits splicing at the bona fide 5’ ss. Only the 3D structure of a small NRS fragment including the decoy 5’ ss had been experimentally studied. Here, by chemical and enzymatic probing of entire RSV NRS, we determine its 2D structure. By comparative analysis of 2D structures of functional and non-functional avian NRS variants and of all sequenced avian NRSs, we bring strong arguments for a biological significance of the established structure. By in vitro splicing assays, we show a crucial role of two of the established stem-loop structures and by affinity purification of complexes formed by WT and truncated NRSs in HeLa cell nuclear extract, we demonstrate their importance for SR protein and U1 snRNP association. We define a new small NRS element retaining splicing inhibitory properties and finally demonstrate the capability of the SR protein 9G8 to increase NRS activity in vitro and in cellulo

RSV is the most common cause of infant bronchiolitis, leading to morbidity and mortality in both infants and the elderly. The relationship between RSV and asthma development further highlights the need to fully understand the immune responses involved in order to develop effective vaccines and therapeutics to aid prevention and treatment of RSV infection respectively. Helminths have long been studied both as a major pathogen of humans, infecting approximately 3 billion people worldwide, and also their ability to modulate the host immune response to allow survival and chronic infection to ensue. Specifically, helminth infections are thought to modulate the host immune response through regulatory mechanisms which are not fully understood. This not only confers protection and survival of the parasites themselves, but also modulates the immune response to unrelated antigens and pathogens. In this thesis, the potential role of a strictly enteric helminth infection, with Heligmosomoides polygyrus, in the modulation of respiratory syncytial virus (RSV) infection was investigated and the associated immune mechanisms were investigated. Firstly, the effects of prior H. polygyrus infection on RSV infection and immune responses in the lung were analysed. H. polygyrus significantly reduced the number of natural killer cells, CD8+ T cells, B cells and conventional dendritic cells in the lung following RSV infection. Co-infection also reduced the production of pro-inflammatory cytokines IL-6 and TNF-α in the lungs. All of these reductions were associated with significantly lower viral titres on day 4 of RSV infection. Interestingly, this attenuation of immune responses and viral titres, correlated with reduced severity of clinical disease, as assessed by weight loss and lung function. H. polygyrus excretory secretory product (HES) was not found to be the immune-modulatory factor in this system, as HES failed to suppress viral titres and reduce immune cell responses to RSV infection. However, irradiated larvae with stunted maturation to adult worms, revealed that larval stages were sufficient to suppress viral titres. Next, the role of type 2 signalling for H. polygyrus effects on RSV infection were examined, using IL-4Rα-/- mice. H. polygyrus infection maintained the ability to attenuate RSV infection and subsequent immune responses in IL-4Rα-/- mice. Furthermore, the presence of the adaptive immune response was not required for H. polygyrus-induced attenuation of RSV infection, as demonstrated in recombinase-activating gene (RAG-/-) deficient mice. H. polygyrus induces innate type 2 immune responses indicating the release of the innate alarmin, IL-33, in the lung and consequently an accumulation of group 2 innate lymphoid cells (ILC2). Their contribution to H. polygyrus effects remain to be fully elucidated. Finally, the role of antiviral responses was explored in H. polygyrus and RSV co-infection. H. polygyrus infection alone induced expression of antiviral genes, IFN-β, OAS1A, Viperin and the antimicrobial peptide CRAMP, in both the duodenum and the lung. Expression of these genes was still higher in the lung 1 hour after RSV in H. polygyrus co-infected mice compared to controls without co-infection. The importance of type I IFN signalling pathway was demonstrated using mice deficient in the type I IFN receptor in H. polygyrus co-infection, which failed to suppress RSV titres and subsequent lung immune cell infiltration. These data highlight the ability of the strictly enteric helminth H. polygyrus to attenuate RSV infection and subsequent immune responses in the lung through the potentiation of type I IFN signalling and consequent upregulation of antiviral immune responses in the lung.

Human respiratory syncytial virus (HRSV) is a negative sense, single strand RNA virus that causes respiratory tract infection with common cold-like symptoms, which can be severe in children, immunocompromised, and the elderly. Even with 60 years of research, the need for vaccine and effective treatment has not been met. In this work, recombinant viruses have been generated which will be valuable in gaining a better understanding of HRSV subtypes, glycoprotein evolution, and the polymerase localization, which would contribute to HRSV vaccine and therapeutics development. The differences in the fitness of A and B antigenic subtypes of HRSV and how it affects the regional circulation pattern is not well understood. To study and compare the two subtypes, it is important to use clinically relevant recombinant viruses and to use animal models that best represent human infection. Using a wild-type virus strain (A11 and B05) from each HRSV subtype, a wild-type like recombinant (r) virus, rHRSVA11, and recombinant viruses expressing fluorescent proteins, rHRSVA11EGFP(5) and rHRSVB05dTom(5), were generated. Characterization of rB05 viruses demonstrated that the differences in the fluorescent protein expressed did not affect virus growth kinetics. To prepare for an experiment in cotton rats, recombinant HRSVs generated were used to infect cotton rat lung cells in vitro. With confirmation of infection of cotton rat lung cells by rHRSV, cotton rat co-infection experiment was planned for the recombinant A11 and B05 viruses and a microneutralization assay was developed for post-infection processing of the in vivo samples. The BA genotype of HRSV B subtype is a strain of HRSV B subtype containing a 60 nucleotide duplication in the glycoprotein (G) gene. HRSV BA genotype was first isolated in 1998 and has quickly become the predominant genotype circulating globally. Although a role of immune evasion by the strains of BA genotype has been suggested to explain this phenomenon, few studies have supported this hypothesis. To compare the HRSV B subtype virus with and without the duplication, rB05 virus lacking the duplication, rHRSVB05EGFP(5)GΔ60b, and containing an epitope tag within the duplication, rHRSVB05EGFP(5)Gmycb, were generated. A serial passage experiment was set up using rHRSVB05EGFP(5) and rHRSVB05EGFP(5)GΔ60b to understand the mutations that accumulate in the G protein gene of each virus. This will be valuable in setting up a similar experiment in the presence of immune pressure to understand the advantage that is conferred to the virus containing the duplication. Expression of Gmyc was confirmed in rHRSVB05EGFP(5)Gmyc infection, which validated that this virus can be used to study the HRSVB05 G protein and modifications in the duplicated region. The HRSV large (L) protein is essential in HRSV transcription and replication, but is difficult to study due to lack of immunologic reagents and challenges with purification. Recombinant viruses expressing reporter and polymerase fusion proteins have been generated and used for studying various other viral polymerases. Expression plasmids for HRSV L protein containing a reporter protein in its variable region 2 have been published. However, the modification resulted in downregulation in the function of the protein and rHRSV expressing modified L protein have not yet been published. In this study, rHRSVB05LVenus was generated to study the effects of modification of HRSV L protein variable region and the localization of HRSV L protein. LVenus protein in rHRSVB05LVenus infected cells was visualized by confocal laser scanning microscopy and the expression levels were examined by immunoblotting. rHRSVB05LVenus was compared to rHRSVB05EGFP(5) with unmodified L protein to show that modification of HRSV L protein had no effect on virus replication. Viruses had equivalent growth kinetics and were equally sensitive to ribavirin, a known HRSV inhibitor. The recombinant viruses generated in this study are valuable tools in answering questions that are difficult to pursue without clinically relevant recombinant viruses. Characterization of the rHRSVs demonstrated that these viruses will have many applications. In this study, viruses were characterized for the basic growth kinetics, expression of proteins of interest, and assay development. With these validated tools, questions such as the cause of the epidemiological pattern observed for HRSV A and B subtypes, the role of host immune response in advantage conferred to HRSV BA genotype, and the effects of inhibitors to formation of HRSV polymerase complex can be addressed. / 2018-10-31T00:00:00Z

De nombreuses études ont impliqué des TLR dans le développement et la progression tumorale. Précédemment, il a été démontré que les cellules tumorales expriment TLR7, un récepteur à ARNsb, et qu’une forte expression de TLR7 par les cellules tumorales de patients atteints de cancer du poumon est associée à un mauvais pronostic. Dans un modèle murin de cancer du poumon, cet effet pro-tumoral a été reproduit lors de l’injection d’agoniste de TLR7. Mes travaux de thèse ont eu pour objectif de déterminer les mécanismes impliqués dans les effets pro-tumoraux de TLR7. La stimulation de ce récepteur au niveau des cellules tumorales induit une production plus importante de CCL2 et GM-CSF, ainsi qu’un fort recrutement de MDSC au site de la tumeur. Ces MDSC, de par leurs propriétés immunosuppressives sont responsables de l’effet pro-tumoral à la suite de la stimulation de TLR7. Nous avons également mis évidence que la stimulation de TLR7 était pro-métastatique dans un modèle murin de cancer du poumon et que les MDSC étaient également impliquées dans cet effet. Ces effets pro-métastatiques associés au TLR7 ont été confirmés chez l’homme, grâce à l’étude de transcrits de molécules associées à l’invasion, l’angiogénèse, la transition épithélio-mésenchymateuse et les métastases. Enfin nous avons démontré la présence de ligand de TLR7 chez les patients atteints de cancer du poumon et démontré que l’injection intratumorale de virus respiratoires, IAV et RSV, a un effet pro-tumoral dans notre modèle de cancer du poumon. Ces virus respiratoires pourraient donc être à l’origine de la surexpression de TLR7 et du mauvais pronostic associé à ce récepteur chez les patients atteints de cancer du poumon. Ces recherches ont donc permis de mettre en évidence de nouveaux facteurs aggravants dans le cancer du poumon, dont les virus respiratoires, et de découvrir les mécanismes impliqués. / Numerous studies have implicated some TLR in tumor development. Previously, we have demonstrated that lung tumor cells express TLR7, a receptor for ssRNA, and that high TLR7 expression confers to NSCLC patients bad clinical outcome. In mice models of lung cancer, we further demonstrated that the injection of TLR7 agonists led to a pro-tumoral effect.My thesis work has firstly demonstrated the mechanisms involved in the pro-tumoral effects of TLR7 in lung cancer: TLR7 stimulation on tumor cells induces a high production of CCL2 and GM -CSF, as well as a sharp MDSC recruitment within the tumor. These MDSC, by their immunosuppressive properties, are implicated in the pro-tumoral effect upon TLR7 stimulation. We also demonstrated that TLR7 stimulation was pro-metastatic in a mice model of lung cancer and that MDSC were also involved in this effect. These pro-metastatic effects associated with TLR7 have been confirmed in humans through the studies of transcripts and proteins involved in invasion, angiogenesis, Epithelial–mesenchymal transition and metastasis. Finally, we demonstrated that TLR7 ligands are present in tumor microenvironment of lung cancer patients and that intratumoral injection of respiratory viral infections such as IAV and RSV, have a pro-tumoral effect in lung cancer mice model. These respiratory viruses could therefore be at the origin of the overexpression of TLR7 and the poor clinical outcome associated with this receptor in lung cancer patients. This research has thus made possible to highlight new aggravating factors in lung cancer, including respiratory viruses, and to discover the mechanisms involved.

Respiratory virus infections are associated with asthma pathogenesis and exacerbations in children and adults. Unfortunately, it remains largely unknown whether innate and adaptive T cell anti-viral immunity differs in allergic disease versus health. Here, we established a short-term primary cell culture system using human peripheral blood mononuclear cells (PBMC) optimized for measuring immune responses to reovirus, respiratory syncytial virus (RSV) and metapneumovirus (MPV) based on virus-specific cytokine and chemokine production. The prevalence and intensity of innate and adaptive responses in children and adult populations was addressed. Using this in vitro model of human anti-viral immunity, we tested our global hypothesis that asthmatics mount anti-viral cytokine responses to respiratory viruses that differ from those of healthy individuals. MPV and RSV, although both ubiquitous and leading to very high levels of infection, seroconversion and clinically similar presentation in the population, evoke distinct innate and adaptive T cell-dependent cytokine responses. Reovirus induced exceptionally strong IFN recall responses concomitant with intense IL-10 production, which were independent of viral replication in PBMC. Surprisingly, despite Type 1 cytokine production dominated adaptive immune responses in both asthmatic and non-asthmatic individuals, asthmatics exhibited significantly stronger pro-inflammatory IFNγ and IL-10 production towards virus stimulation than non-asthmatic children and adults. Moreover, children with current AHR, regardless of asthmatic status, exhibit a greater frequency and intensity of IFNγ responses towards pneumoviruses than do non-AHR counterparts. Conversely, expression of chemokine CCL5 was substantially weaker in asthmatics, and was further decreased in children with AHR and familial history of asthma. This pattern of enhanced pro-inflammatory and deficient anti-viral CCL5 responses towards pneumoviruses in children with markers of symptomatic asthma or AHR may underlie the enhanced sensitivity of these children to experience breathing difficulties following infection with respiratory viruses. Furthermore, we have clearly demonstrated a gene by environment interaction, whereby ETS exposure in children with familial asthma results in suppressed anti-viral IFNγ and IL-10 production. Therefore, we have attempted to determine whether genetic variation affects the intermediate phenotype of anti-viral immunity, in the population and dependent on clinical status. In summary, we have demonstrated that asthma, childhood exposures and genetics shape anti-viral cytokine responses in human. These findings have a substantial impact for physicians deciding the contextually appropriate treatment for asthma symptoms in their patients and could have implications for experimentation relating to mechanisms of disease, clinical practice and development of appropriate therapeutics. / October 2007

Gold nanoparticles are excellent candidates for all the biomedical applications due to their size and shape dependent optical and physiological properties. In this study, gold nanoparticles were synthesized chemically for bio-application. It was observed that the size and shape of gold nanoparticles depend strongly on the concentration of chemical solution, type of reducing agent used in the reaction, temperature of the solution and stabilizing agent for reaction. Transmission electron microscopy (TEM) has been used extensively to determine the size and shape of the gold nanoparticles. Optical properties of the size and shape selected nanoparticles were studied using UV-vis spectrophotometer in absorption mode. The chemically synthesized gold nanoparticles were observed to show excellent absorption property which is reflected by the presence of the characteristic surface plasmon resonance (SPR) band peak. The SPR peak was found to be predominantly dependent on the size of nanoparticles. We have observed a strong red shift with increasing the size of gold nanoparticles. The position of the SPR peak was also observed to change with shape of gold nanostructures. Synthesis and characterization of the composites of gold nanoparticles and Poly (Oanisidine) (POAS) have been carried out in this thesis. Gold-POAS materials system was characterized using UV-vis spectroscopy, TEM, Fourier Transform Infrared Spectroscopy. The chemically synthesized gold nanoparticles were successfully utilized for the study of Respiratory Syncycial Virus (RSV) interaction. Gold nanoparticles were found to inhibit the RSV infection. The electrochemical behavior of gold nanoparticles was studied and their potentials for biosensing applications were tested. Cyclic voltaammetry was performed for the detection of dopamine and ascorbic acid using gold nanoparticles of different sizes. Interaction of gold nanoparticles with Bovine Serum Albumin (BSA) has been studied via absorption spectroscopy and TEM measurements. The absorption spectra of the GNP-BSA show remarkable shift in SPR band peak towards high wavelength. Catalytic properties of the gold nanoparticles were studied by using them as a catalytic activator for the gas sensing applications.