Keywords: RSV, S. pneumoniae, mixed-infection, immune response, p53 pathway

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 first 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.

LIST OF TABLES

En raison de l'importance clinique de cette co-infection et le taux élevé de circulation du VRS, il est important de comprendre comment le système immunitaire est affecté à l'infection de ces deux agents pathogènes. Notre étude identifie la réponse immunitaire dans les macrophages, ainsi comme les interactions entre le VRS et le facteur de transcription p53. Les résultats montrent un profil particulier a cette coinfection mixte dans le macrophages et des modifications dans la réponse immune innée que nous a permis de mieux comprendre les mécanismes de pathogenèse du VRS dans les cellules épithélial pulmonaires en regardant la régulation de p53. Dans la dernière partie, nous avons évalué l'impact direct de l'infection mixte chez les primates non-humain et ce modèle nous a montré les difficultés et complexités des établir une pneumonie sévère.

Mots-clés : VRS ; S. pneumoniae ; co-infection ; réponse immune ; p53 pathway.

INTRODUCTION 12

Tous les résultats présentés ici ont été développés dans le cadre du projet de thèse visant à mieux comprendre les infections mixtes au cours de la pneumonie. Ce travail a eu la collaboration ferme et efficace entre les Laboratoire de Pathogènes Emergents et Virpath, permettant l'approfondissement et menant à des conclusions importantes dans la pathogenèse des virus respiratoires lors de la présence ou l'absence de bactéries dans des modèles in vitro et in vivo.

ACUTE LOWER RESPIRATORY TRACT INFECTIONS

The respiratory tract constitutes a wide and critical frontier at the interface between the body and the environment. This complex organ system is divided into the upper airways and lower airways. The upper airways or upper respiratory tract includes the nose and nasal passages, paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (cords). The lower airways or lower respiratory tract includes the portion of the larynx below the vocal folds, trachea, bronchi, and bronchioles. The lungs can be included in the lower respiratory tract or as a separate entity and include the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli [1]. Upper respiratory tract infections are less severe whereas lower infections are often associated with high mortality rates [START_REF] Van Den Bergh | Associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria[END_REF]. Acute lower respiratory tract infections constitute the third leading cause of human death worldwide with 3.2 million deaths in 2015 (Figure 1), and the first cause of mortality in children under five years of age, according to the World Health Organization (WHO) [START_REF] Who | The top 10 causes of death[END_REF][START_REF] Who | Children: reducing mortality[END_REF]. Lower respiratory infections constitute the third cause of death in the world population (red bar) being responsible for 3.2 million of deaths in 2015. Heart diseases (Ischemic heart disease -blue bar -and strokes -pink bar) were the most cause of deaths. Among the 5 main causes of global death described, lower respiratory infections are the only transmissible infectious disease [START_REF] Who | The top 10 causes of death[END_REF].

The diversity of pathological agents makes it difficult to prevent, diagnose and treat these diseases, contributing to high mortality rates [START_REF] Shaughnessy | Community-acquired pneumonia in the post 13-valent pneumococcal conjugate vaccine era[END_REF][START_REF] Tramper-Stranders | Childhood community-acquired pneumonia: A review of etiology-and antimicrobial treatment studies[END_REF][START_REF] Lee | The role of respiratory viruses in the etiology of bacterial pneumonia: An ecological perspective[END_REF][START_REF] Figueiredo-Mello | Prospective etiological investigation of communityacquired pulmonary infections in hospitalized people living with HIV[END_REF]. These diseases can affect the general population, but severe cases and high mortality rates are found among children up to age 5, immunosuppressed adults and elderly [START_REF] Nair | Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis[END_REF][START_REF] Benito | Pulmonary infections in HIV-infected patients: an update in the 21st century[END_REF][START_REF] Stupka | Community-acquired pneumonia in elderly patients[END_REF].

Acute lower respiratory infections constitute a major global health burden due to the emergence of resistance to antimicrobial treatments, the presence of multiple pathogens and the recurrence of infections throughout life [START_REF] Feldman | Recent advances in our understanding of Streptococcus pneumoniae infection[END_REF][START_REF] Fukutani | Pathogen transcriptional profile in nasopharyngeal aspirates of children with acute respiratory tract infection[END_REF][START_REF] Groothuis | Prevention of serious respiratory syncytial virus-related illness. I: Disease pathogenesis and early attempts at prevention[END_REF]. In this context, more knowledge about respiratory diseases and their etiological agents are very important to improve or propose novel prophylactic and therapeutic approaches.

Definition and clinical symptoms

Respiratory tract infections are responsible for a variety of clinical features that range from milder manifestations, such as the common cold to acute lower respiratory tract infections, represented by bronchiolitis and pneumonia [START_REF] Hancock | The heterogeneity of viral bronchiolitis: A lack of universal consensus definitions[END_REF][START_REF] Ralston | Clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis[END_REF] Pneumonia is an inflammatory process that takes place in the alveolar spaces, whereas, in bronchiolitis, inflammation rather occurs in bronchi (Figure 2). The symptoms are relatively similar between pneumonia and bronchiolitis; they begin as a common cold (nasal congestion, high fever, and decreased appetite) and after 2 to 3 days it is possible to observe several complications of the disease. Pneumonia usually accompanies a dry cough, thoracic pain, and extreme tiredness while bronchiolitis accompanies a characteristic wheezing. At this stage of both diseases, cough is persistent and the difficulty of feeding is marked, besides the accelerated breathing. Apnea is a very common symptom in infants less than 2 months during bronchiolitis while confusion occurs in the elderly with pneumonia. Complications such as hypoxia (low oxygen level) and cyanosis (blue-tinged skin) are indicative of both severe diseases [START_REF] Ralston | Clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis[END_REF][START_REF]Bronchiolitis: diagnosis and management of bronchiolitis in children[END_REF]. Bronchiolitis affects children younger than 2 years old with a high mortality rate among preterm infants, due to the characteristics of this age group in which the lung and immune system are functionally immature, forming ideal spots for viral infection [START_REF] Holt | Contemporaneous maturation of immunologic and respiratory functions during early childhood: implications for development of asthma prevention strategies[END_REF]. Pneumonia affects all ages but is extremely severe in children up to 5 years of age, immunosuppressed adults and the elderly [START_REF] Nair | Global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis[END_REF][START_REF] Pilie | Adult patients with respiratory syncytial virus infection: impact of solid organ and hematopoietic stem cell transplantation on outcomes[END_REF][START_REF] Falsey | Respiratory syncytial virus infection in elderly and high-risk adults[END_REF]. The most common pneumonia is community-acquired pneumonia, which is acquired in an extra-hospital environmental [START_REF] Tramper-Stranders | Childhood community-acquired pneumonia: A review of etiology-and antimicrobial treatment studies[END_REF][START_REF] Musher | Community-acquired pneumonia[END_REF][START_REF] Kim | Pneumonia and Streptococcus pneumoniae vaccine[END_REF].

The pathogenesis of respiratory infections involves a complex interplay between virulence factors of a number of different pathogens -including bacteria, virus and/or fungi -and host response [START_REF] Troy | Respiratory viral infections and host responses; insights from genomics[END_REF]. An overview of the etiological agents of pneumonia and bronchiolitis is presented in the following chapter and the host/pathogens interactions involved in these respiratory infections will be approached in the second part of this manuscript.

Etiology

The upper respiratory tract, mainly the nasopharynx, constitutes a rich and diverse niche in microbes. It is believed that most respiratory infections of the lower respiratory tract must have originated from this microbial niche. Thus, commensal microorganisms are also found in cases of severe disease, making it difficult to determine precisely etiological pathogens [START_REF] Van Den Bergh | Associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria[END_REF][START_REF] Bogaert | Streptococcus pneumoniae colonisation: the key to pneumococcal disease[END_REF].

Respiratory viral infections

The main viruses associated with lower respiratory tract infections are Influenza Viruses, human respiratory syncytial viruses (RSV), human parainfluenza viruses (hPIV), human metapneumoviruses (hMPV) and human Coronaviruses (CoV) [START_REF] Nair | Global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis[END_REF][START_REF] Jain | Community-acquired pneumonia requiring hospitalization among U.S. children[END_REF][START_REF] Self | Respiratory Viral Detection in Children and Adults: Comparing Asymptomatic Controls and Patients With Community-Acquired Pneumonia[END_REF].

a) Influenza Viruses

Influenza viruses cause an acute infection popularly known as Flu that has a high rate of recurrence and the ability to infect individuals in all age groups. Influenza viruses epidemics are estimated to result in about 3 to 5 million cases of severe illness, and about 250000 to 500000 deaths [29].

Influenza A, B and C viruses belong to the family Orthomyxoviridae [30].

Among these three types, Influenza A viruses (IAV) is responsible for the major pandemic and seasonal epidemics events being considered more virulent and genetically more variable [31]. IAV have the ability to achieve a large spectrum of animal reservoirs, among mammals and birds. Instead, Influenza B types are composed of two circulating lineages (Victoria and Yamagata) in the human population causing seasonal epidemics but without the capacity to promote pandemic events. Influenza C type is the less common and often only cause a mild infection in children [START_REF] Hardelid | Effectiveness of pandemic and seasonal influenza vaccine in preventing pandemic influenza A(H1N1)2009 infection in England and Scotland 2009-2010[END_REF][START_REF] Salez | Influenza C virus high seroprevalence rates observed in 3 different population groups[END_REF][START_REF] Who | WHO recommendations on the composition of influenza virus vaccines[END_REF].

IAV is divided into subtypes according to antigenic properties of the two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Historically, three subtypes of HA (H1, H2, and H3) have acquired the ability to be transmitted efficiently among humans and seasonal subtypes H1 and H3 are most co-circulate detected [START_REF] Schrauwen | Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments[END_REF]. However, other subtypes, such as H5, H6, H7, and H9 occasionally affect humans and are considered possible threats to a future pandemic [START_REF] Wang | Epidemiology of avian influenza A H7N9 virus in human beings across five epidemics in mainland China, 2013-17: an epidemiological study of laboratory-confirmed case series[END_REF]. IAV can cause pandemics when a strain which was not previously circulating among humans emerges and transmits among humans. As the majority of the population has no immunity against these viruses, the proportion of persons in a population getting infected may be quite large [START_REF] Reperant | The immune response and within-host emergence of pandemic influenza virus[END_REF]. Also, zoonotic Influenza viruses can cause sporadic severe infection in humans through direct contact with infected animals, such as swine or birds but rarely spread very far among humans [START_REF] Cardona | Avian influenza in birds and mammals[END_REF].

IAV is spherical and elongated virions with a diameter varying between 95 to 120 nanometers (nm) and morphological virions are shown in Figure 3A [START_REF] Moules | Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles[END_REF]. In the viral envelope are inserted the surface glycoproteins HA and NA, and also the M2 channel, which is a protons channel. The viral envelope is constituted from host cell membrane and M1 proteins delimit the inner portion of the viral particle. The negative single strand RNA viral genome is composed of 8 segments and each single RNA strand is coated by nucleoprotein (NP) and associated to an RNA-polymerase-RNAdependent complex composed by basic polymerase 1 (PB1), basic polymerase 2 (PB2) and acid polymerase (PA) [START_REF] Eisfeld | At the centre: influenza A virus ribonucleoproteins[END_REF]. IAV genome encodes up to 17 structural and nonstructural proteins that play different roles in the architecture and dynamics of viral replication [START_REF] Schrauwen | Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments[END_REF][START_REF] Eisfeld | At the centre: influenza A virus ribonucleoproteins[END_REF][START_REF] Medina | Influenza A viruses: new research developments[END_REF][START_REF] Wise | A complicated message: Identification of a novel PB1-related protein translated from influenza A virus segment 2 mRNA[END_REF][START_REF] Dubois | Influenza viruses and mRNA splicing: doing more with less[END_REF]. A schematic viral particle is represented in Figure 3B. andM2). Each segment of the viral genome is also represented and RNP complex is highlighted to show each protein that forms this complex. Also, nonstructural proteins are listed. Adapted from Moulès et al [START_REF] Moules | Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles[END_REF].

The HA glycoprotein possesses two primordial functions for the replicative cycle. First, the HA recognizes and binds to sialic acid receptors on the cell surface and then allows the fusion of the viral envelope with the internal membrane of the endosome, allowing the release of vRNPs in the cytoplasm [START_REF] Gamblin | Influenza Hemagglutinin and Neuraminidase Membrane Glycoproteins[END_REF]. The other glycoprotein, NA is involved in the liberation of new virions from the surface of the host cell due to its sialidase activity, thus allowing viral dissemination [START_REF] Nayak | Influenza virus morphogenesis and budding[END_REF]. A schematic representation of the different steps of the replicative cycle Influenza A viruses is presented in Figure 4. Two important evolutionary mechanisms are involved in the replicative cycle of influenza viruses, the antigenic drift and shift, which allow it to evade the immune system and adapt to new hosts [31]. The antigenic drift mechanism results from point mutations inserted during the replicative process, caused by the low fidelity of the viral RNA-dependent RNA polymerase enzyme, facilitating rapid viral evolution [START_REF] Medina | Influenza A viruses: new research developments[END_REF].

This process allows viral antigenic evolution by the selection of new variants containing amino acid alterations in HA and NA proteins [START_REF] Medina | Influenza A viruses: new research developments[END_REF]. More drastic changes in the viral genome may occur, such as the rearrangement of gene segments (antigenic shift), resulting in the emergence of a new variant with pandemic potential, as occurred with the A (H1N1) virus pdm09 [START_REF] Smith | Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic[END_REF]. The mechanism of antigenic shift occurs due to the rearrangement between the gene segments of viruses of distinct origins during the simultaneous infection of two viral particles in the same cell.

The seasonality of IAV is complex and influenced by a set of socio-economic (social, behavioral and cultural interactions), viral (the continuous process of generation and selection of new strains) and ecological/environmental factors [START_REF] Lofgren | Influenza seasonality: underlying causes and modeling theories[END_REF].

Influenza viruses have their peak epidemic from May to September in the temperate regions of the Southern Hemisphere, between December and March in the temperate regions of the Northern Hemisphere, and throughout the year (with a higher incidence in the rainy season) in Tropical and subtropical regions [START_REF] Tamerius | Global influenza seasonality: reconciling patterns across temperate and tropical regions[END_REF].

Due to the evolutionary characteristics of influenza viruses, the annual impact on morbidity and mortality of their epidemics and their pandemic potential, the constant surveillance of these viruses as well as the rapid identification of new emergent strains are extremely important.

b) Respiratory Syncytial Virus

The human Respiratory Syncytial Virus (RSV) is considered as an etiological agent of major pediatric importance in respiratory infectious disease such as pneumonia and bronchiolitis [START_REF] Karron | Determining the burden of respiratory syncytial virus disease: the known and the unknown[END_REF][START_REF] Heikkinen | Clinical and Socioeconomic Burden of Respiratory Syncytial Virus Infection in Children[END_REF].The first RSV infection occurs in the first two years of age and the peak incidence usually occurs in the first months of life [START_REF] Scheltema | Global respiratory syncytial virus-associated mortality in young children (RSV GOLD): a retrospective case series[END_REF].

Also, RSV infection during the first year is associated with the development of recurrent wheezing, asthma, and others chronics lung diseases later in life [START_REF] Perez-Yarza | The association between respiratory syncytial virus infection and the development of childhood asthma: a systematic review of the literature[END_REF][START_REF] Holtzman | Asthma as a chronic disease of the innate and adaptive immune systems responding to viruses and allergens[END_REF].

Cases of reinfection by RSV are common throughout life, but the clinical symptoms in children with older age and adults are milder nature [START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF]. Some studies associate RSV infection with a relevant morbidity and mortality in children with prematurity, bronchopulmonary dysplasia, and congenital heart disease, in the elderly [START_REF] Falsey | Respiratory syncytial virus infection in elderly and high-risk adults[END_REF] and in immunocompromised individuals [START_REF] Falsey | Respiratory syncytial virus infection in adults[END_REF][START_REF] Hall | Respiratory syncytial viral infection in children with compromised immune function[END_REF].

Human RSV is a member of the new family Pneumoviridae [START_REF] Afonso | Taxonomy of the order Mononegavirales: update 2016[END_REF] with Bovine

Respiratory Syncytial Virus and murine pneumonia virus. RSV particles have irregular spherical morphology, with a diameter around 100-350 nm (Figure 5A).

Long filamentous particles having a diameter from 60 to 200 nanometers and more than 10µm in length have also been described in the literature [START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF].

RSV possesses a negative, non-segmented single-stranded RNA genome [START_REF] Huang | The genome of respiratory syncytial virus is a negativestranded RNA that codes for at least seven mRNA species[END_REF]. Three surface glycoproteins are inserted at the surface of the viral envelope: the fusion protein (F), the attachment glycoprotein (G) and the small hydrophobic protein (SH). Two proteins compose the viral matrix and are known as M and M2-1. The nucleocapsid -a protein complex associated with the vRNA -is formed by the phosphoprotein (P), the nucleoprotein (N), the largest subunit of the polymerase (L)

and the M2-2 transcription factor. RSV genome encodes 11 proteins, with two nonstructural proteins, NS1 and NS2 which are expressed only during cell infection and are not packaged into the viral particle [START_REF] Levine | Polypeptides of respiratory syncytial virus[END_REF][START_REF] Huang | Respiratory syncytial virus mRNA coding assignments[END_REF][START_REF] Collins | Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes[END_REF][START_REF] Bernstein | Respiratory syncytial virus proteins: identification by immunoprecipitation[END_REF][START_REF] Dubovi | Analysis of proteins synthesized in respiratory syncytial virus-infected cells[END_REF][START_REF] Fernie | Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion[END_REF][START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF]. A schematic representation of RSV particle is represented in Figure 5B and RSV genome in Figure 5C. There are two major groups of the virus, RSV A, and B, based on differences in reaction with monoclonal antibodies against the major structural glycoproteins G and F, and also by genetic comparison analysis [START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF]. Each group was further subdivided into genotypes based on nucleotide sequence variability [START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF][START_REF] Sullender | Respiratory syncytial virus genetic and antigenic diversity[END_REF].

Overall, the role of surface glycoproteins F and G in mediating receptor binding are not completely understood. Some studies have shown that heparin sulfate present at the cell surface is essential for RSV entry into continuous cell lines and interactions with nucleolin, annexin II, ICAMs and Toll-like receptors may also be associated with the process of binding the RSV to cells in vitro [START_REF] Crim | Identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity[END_REF][START_REF] Tayyari | Identification of nucleolin as a cellular receptor for human respiratory syncytial virus[END_REF][START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF][START_REF] Langedijk | Proposed three-dimensional model for the attachment protein G of respiratory syncytial virus[END_REF][START_REF] Lentz | The recognition event between virus and host cell receptor: a target for antiviral agents[END_REF][START_REF] Mettenleiter | Interaction of glycoprotein gIII with a cellular heparinlike substance mediates adsorption of pseudorabies virus[END_REF][START_REF] Hallak | Interaction between respiratory syncytial virus and glycosaminoglycans, including heparan sulfate[END_REF][START_REF] Krusat | Heparin-dependent attachment of respiratory syncytial virus (RSV) to host cells[END_REF][START_REF] Teng | Contribution of the respiratory syncytial virus G glycoprotein and its secreted and membrane-bound forms to virus replication in vitro and in vivo[END_REF]. The F protein also mediates the fusion of infected cells with adjacent cells that are not infected, contributing to the formation of large multinucleated cells called syncytia [START_REF] Fearns | Mapping the transcription and replication promoters of respiratory syncytial virus[END_REF]. A schematic of RSV replication showing principle steps is represented in Figure 6. Replication cycle begins when viral surface proteins interact with a cell receptor. After viral attachment fusion between viral and cell membrane occurs in a pH-independent process. All RSV replication steps occur in the cell cytoplasm and viral maturation occurs with the assembly of the nucleocapsid by combining genomic vRNA with N protein, which is accompanied by the addition of P and L proteins for envelope assembly. Matrix proteins aggregate with the viral surface proteins in the cellular membrane and the complete viral particle is released by budding, taking a portion of the plasma membrane in a reverse process to penetration by fusion. In addition to transcription and translation of proteins, viral genome produces a positively stranded RNA intermediate, which serves as a template to generate copies of the viral genome. Adapted from Collins et al. [START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF] Patterns of seasonality and duration of RSV outbreaks vary considerably between geographical regions. In temperate climates, epidemics have been described in the winter months [START_REF] Salter | Molecular epidemiology of human respiratory syncytial virus subgroups A and B identified in adults with hematological malignancy attending an Irish hospital between 2004 and 2009[END_REF][START_REF] Zhang | Genetic variability of respiratory syncytial viruses (RSV) prevalent in Southwestern China from 2006 to 2009: emergence of subgroup B and A RSV as dominant strains[END_REF][START_REF] Fujitsuka | A molecular epidemiological study of respiratory viruses detected in Japanese children with acute wheezing illness[END_REF] while in tropical regions, epidemics appear to occur in rainy seasons [START_REF] Law | An update on respiratory syncytial virus epidemiology: a developed country perspective[END_REF]. However, it is possible that the seasonality of the virus is not only related to climatic factors but also to socioeconomic factors increasing the risk of RSV contamination [START_REF] Hillis | Respiratory syncytial virus infection in children in West Bengal[END_REF][START_REF] Weber | Respiratory syncytial virus infection in tropical and developing countries[END_REF][START_REF] Waris | Seasonality of respiratory syncytial virus infection[END_REF]. In most RSV epidemics reported, the cocirculation of different genotypes of groups A and B were detected [START_REF] Salter | Molecular epidemiology of human respiratory syncytial virus subgroups A and B identified in adults with hematological malignancy attending an Irish hospital between 2004 and 2009[END_REF][START_REF] Zhang | Genetic variability of respiratory syncytial viruses (RSV) prevalent in Southwestern China from 2006 to 2009: emergence of subgroup B and A RSV as dominant strains[END_REF][START_REF] Ramaekers | Prevalence and seasonality of six respiratory viruses during five consecutive epidemic seasons in Belgium[END_REF].

c) Others Human Viruses

Others respiratory viruses are capable to cause acute lower respiratory tract infections in humans such as:

Human Parainfluenza viruses (hPIVs) are common respiratory pathogens that induce acute respiratory tract diseases in infants and immunocompromised adults [START_REF] Shorr | Viruses are prevalent in non-ventilated hospital-acquired pneumonia[END_REF][START_REF] Abedi | Estimates of Parainfluenza Virus-Associated Hospitalizations and Cost Among Children Aged Less Than 5 Years in the United States, 1998-2010[END_REF]. Serological surveys have indicated that 80% of children are infected with hPIV-3 by 4 years of age and hPIV infections re-occur throughout life. The hPIV belong to a diverse group of enveloped single-stranded RNA viruses within the family Paramyxoviridae and based on genetic and antigenic analyses, hPIVs have been divided into four major subtypes (hPIV-1 to hPIV-4), with subtypes 1 and 3 being most frequently found in severe cases [START_REF] Pawelczyk | The Role of Human Parainfluenza Virus Infections in the Immunopathology of the Respiratory Tract[END_REF].

Human metapneumoviruses (hMPV) were first identified in 2001 and constitute a common cause of acute respiratory infection in individuals of all ages worldwide [START_REF] Vogel | What We Have Learned from the Influenza A pH1N1 2009/10 Pandemic: High Clinical Impact of Human Metapneumovirus and Respiratory Syncytial Virus in Hospitalized Pediatric Patients[END_REF][START_REF] Principi | Paediatric human metapneumovirus infection: epidemiology, prevention and therapy[END_REF]. hMPV is a member of the family Pneumoviridae, that also includes RSV, and two distinct hMPV genotypes, A and B, which can be divided into two subgroups: A1, A2, B1, and B2 circulate worldwide. hMPV and RSV share similar clinic features causing severe disease in the same range of age between children with an incidence around 15% of all respiratory tract infections [START_REF] Kahn | Epidemiology of human metapneumovirus[END_REF][START_REF] Williams | Human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children[END_REF][START_REF] Guerrero-Plata | Human metapneumovirus induces a profile of lung cytokines distinct from that of respiratory syncytial virus[END_REF][START_REF] Bao | Airway epithelial cell response to human metapneumovirus infection[END_REF][START_REF] Edwards | Burden of human metapneumovirus infection in young children[END_REF].

Human Coronaviruses (HCoV) infections display a wide range of symptoms and their role in pediatric lower respiratory infections is still not clear [START_REF] Kuypers | Clinical disease in children associated with newly described coronavirus subtypes[END_REF][START_REF] Talbot | The pediatric burden of human coronaviruses evaluated for twenty years[END_REF]. There are currently five coronaviruses (family Coronaviridae) known to infect humans and they are associated with both upper and lower respiratory tract infections in all age groups [START_REF] Kuypers | Clinical disease in children associated with newly described coronavirus subtypes[END_REF][START_REF] Gaunt | Epidemiology and clinical presentations of the four human coronaviruses 229E, HKU1, NL63, and OC43 detected over 3 years using a novel multiplex real-time PCR method[END_REF][START_REF] Van Der Hoek | Burden of disease due to human coronavirus NL63 infections and periodicity of infection[END_REF][100]. Thus, the role of coronaviruses in pneumonia has not been

a) Streptococcus pneumoniae

Streptococcus pneumoniae (S. pneumoniae) can colonize the nasopharynx asymptomatically but is one of the leading causes of high mortality and morbidity in infants, the elderly and immunocompromised people [111][112][113][114]. Prior to the use of antibiotics, more than 75% of pneumonia cases were caused by S. pneumoniae [START_REF] Musher | Community-acquired pneumonia[END_REF].

However, nowadays, studies show that only 5 to 15% of pneumonia cases are caused by S. pneumoniae in developed countries and a higher proportion of cases described in low and income countries [START_REF] Musher | Community-acquired pneumonia[END_REF][START_REF] Jain | Community-acquired pneumonia requiring hospitalization among U.S. children[END_REF]115,116] Colonization state (the first step to infection) occurs when bacteria promotes adhesion on the mucosal surface of the nasopharynx. The surface of the bacterium consists of 3 structures with several virulence factors that could contribute to colonization and development of pneumococcal diseases as described in Figure 7 [START_REF] Bogaert | Streptococcus pneumoniae colonisation: the key to pneumococcal disease[END_REF]123,124]. remains a particular problem in the HIV-infected population [START_REF] Figueiredo-Mello | Prospective etiological investigation of communityacquired pulmonary infections in hospitalized people living with HIV[END_REF]130]. C. pneumoniae is an obligate intracellular pathogen that induces an inflammatory reaction which contributes to damages in epithelial respiratory tract [136,137].

Mixed Respiratory infections

The upper respiratory tract constitutes a dynamic and equilibrated microbiological niche, notably composed of commensal viruses and bacteria.

Perturbation of this equilibrium, by the emergence of a pathogen and/or imbalance of the host immunity, can constitute the starting point of respiratory diseases [START_REF] Van Den Bergh | Associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria[END_REF][START_REF] Bogaert | Streptococcus pneumoniae colonisation: the key to pneumococcal disease[END_REF]138]. Usually, the opportunistic bacteria, such as S. pneumoniae, S. aureus, and H.

influenza are co-detected with respiratory viruses during lower respiratory tract infections [127,[139][140][141][142]. However, determining the contribution of viral/bacterial coinfection to disease severity is highly complex. There is an abundance of distinct Pathogen isolation is a method where samples are incubated in different conditions and the most abundant pathogen is detected and identified. During a long time, this type of identification was considered the preferred method in diagnostics.

For bacterial culture, the problems of this method are the false-negative detection due antibiotic pretreatment and nonculturable bacteria [174]. For viral isolation in cell culture, the support and conditions can vary a lot. Influenza virus can be isolated in embryonated chicken eggs or mammalian cell lineages (MDCK) while for RSV isolation, other mammalian cell lineages (HEp-2) are most common. The main disadvantage of these procedures is the relatively long period of time required between 7 to 10 days, depending on the pathogen [173].

Molecular detection is based on nucleic acid amplification and nowadays polymerase chain reaction (PCR) assay is considered a primordial technique for pathogen characterization. This method can be used directly on clinical samples and the rapid nature of the results can greatly facilitate investigation of outbreaks of respiratory illness. In addition, this method allows detecting multiples pathogens together being capable to identify different respiratory pathogens and its subtypes [173]. Also, it is possible to make quantitative analyses correlating pathogen load to the severity of disease [175,176]. However, although PCR is highly specific, sensitivity has been shown to vary depending on the patient sample tested [177,178].

Indirect immunofluorescence assay is the most common test in the diagnosis of various respiratory viruses. This technique is based on antibody staining of virusinfected cells in original clinical specimens and is a rapid and sensitive method for diagnosing viral infections [173].

Serological diagnoses are important approaches when clinical specimens are unobtainable or when a laboratory does not have the resources required for pathogen isolation. Serological methods such as the haemagglutination inhibition test are essential for many epidemiological and immunological studies and for evaluation of the antibody response following vaccination for Influenza virus, for example [173].

Biomarkers approaches can help to predict or to recognize potential cases of severity. Biomarkers are biological markers that function as indicators of a pathogenrelated disease, or of disease severity [179]. The first biomarker proposed during an infection were white cell count and erythrocyte sedimentary rate but nowadays they have been replaced by C-reactive protein (CRP) and procalcitonin (PCT) which have higher sensitivity and specificity for severity prognostic [180][181][182][183][184][185][186][187][188][189][190][191][192][193][194][195][196][197][198][199]. They seem to have suboptimal sensitivity and specificity for differentiated bacterial to viral pneumonia [187,200,201].

Several others biomarkers capable of identifying the etiology and predict complications, outcomes, and mortality of pneumonia have been studied. Tumor necrosis factor (TNF) receptor 2 and interleukin (IL)-10 characterization studies don't

show success but tissue inhibitor of metalloproteinases has shown promise for the identification of bacterial pneumonia in children [195,[202][203][204][205]. Also, an association of different biomarkers like CRP, TRAIL, and IP-10 were described and might offer advantages in the differentiation of viral or bacterial pneumonia.

In addition, innovative technologies, including microarray-based whole genome expression arrays, proteomics, and metabolomics, can be a basis for biomarker discovery. For example, specific host responses induced show a microRNA bio-signatures that can be identified using microRNA analyses [199].

However, further studies are necessary before routine use of biomarker assays [199,206].

Treatments for lower respiratory tract infections

Treatments for lower respiratory tract infections depend on the nature of the etiological agent, resulting in antibiotic or antiviral treatment, in the case of bacterial or viral infections, respectively.

Antiviral treatments

Viral replication is linked to metabolic processes of the host cells and safe antivirals offering benefits by reducing mortality as well as the duration of disease Viral NA protein is an attractive target for drug action as it is essential for infectivity and has a highly conserved active site across influenza A and B viruses [31] Despite the importance of this viral pathogen, there are not adequate treatment options available. Thus, it is important to continue to identify and characterize possible targets for antiviral drugs.

Antibiotics

Against bacterial infections, antibiotics are widely used worldwide. There are cytotoxic or cytostatic towards the microorganism and often act by inhibiting the synthesis of a bacterial cell wall [229,230]. For mild to moderate pneumonia suspected to be of bacterial origin, amoxicillin is recommended first-line therapy.

Amoxicillin appropriately covers the most prominent invasive bacterial pathogen, S.

pneumoniae [231]. During an atypical bacterial suspicion, a macrolide (azithromycin)

is recommended [232]. In addition, a third-generation cephalosporin is recommended for a specific group of patients [START_REF] Shaughnessy | Community-acquired pneumonia in the post 13-valent pneumococcal conjugate vaccine era[END_REF].

Due to the indiscriminate use of antibiotics, the emergence of antibioticresistant strains is considered a serious problem [230]. Thus, preventing lower respiratory tract infections through vaccination and prophylaxis is important.

Prevention of lower respiratory tract infection

Prophylaxis is considered one of the best alternatives for combating respiratory tract infections. The prophylaxis method most common are vaccines, but passive immunization has also been overspread worldwide.

Viral prophylaxis

The Inactivated vaccines are composed of purified virus chemically inactivated and are capable to generate humoral and cellular immunity. The annual Influenza vaccine around the world is an inactivated vaccine [31,233,234]. This vaccine, during decades, was composed of 2 IAV strains and one strain of Influenza B virus according to circulated subtypes detected by surveillance. In 2016, WHO decided to add another Influenza B virus subtype to try to increase vaccine coverture [START_REF] Who | WHO recommendations on the composition of influenza virus vaccines[END_REF]233].

Even if this Influenza vaccine has a great coverture and seroconversion, the

Pneumococcal vaccine

The polysaccharide capsule from encapsulated bacteria is a major virulence factor and can be used as an antigen for vaccine development [256,257]. However, that does not induce a complete response and cannot provide adequate protection against pneumococcal infection in children [START_REF] Kim | Pneumonia and Streptococcus pneumoniae vaccine[END_REF]. Thus, the polysaccharide was chemically conjugated to different bacterial protein [258] Therefore, new potential vaccines that effectively protect against pneumonia have been investigated and are undergoing clinical trials [START_REF] Kim | Pneumonia and Streptococcus pneumoniae vaccine[END_REF][272][273][274].

Host-pathogen interactions

The human body is constantly exposed to microbes and prevention of

Immune response

In

Innate immune response

The innate immune response begins as soon as the pathogen enters the target cell and is implicated in recognition and protection of infections. The innate system consists of different cells as shown in Viral attachment and fusion proteins, as well as the components of bacterial cell wall, are able to be recognized by these receptors. The summary of TLR receptors and pathogens ligands are described in Table 2.

TLR Microbial ligands Reference TLR1

Recognize lipoproteins; minority role in pneumococcus infection.

[ Once the ligand is recognized, TLRs dimerize and initiate a signaling cascade leading to the activation of the proinflammatory response [296]. Signaling cascade is divided into two major families of transcription factors -Nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kB) and interferon regulatory factor (IRF) [START_REF] Troy | Respiratory viral infections and host responses; insights from genomics[END_REF] which will be detailed in the next part of this manuscript.

Adaptive immunity

The adaptive immunity is a durable response to pathogens and has been extensively studied for many years, contributing to vaccine development. Also, an exacerbated activation of this response contributes to the severity of the disease

Inflammatory response

The severity of respiratory infections is closely linked to inflammation and tissue damage in the airways. Inflammation is induced by multiple signaling pathways such as NF-kB and interferon regulatory factor (IRF) -the two major transcription factors -and others more recently described like p53 pathway [285,315,316].

During mixed infection, both S. pneumoniae and RSV have been shown to stimulate inflammasome activation. The culmination of the multiple signaling pathways triggered by S. pneumoniae primarily results in an NF-kB-mediated inflammatory response, while RSV infection results in a strong interferon response.

There is, however, the potential for considerable overlap between the signaling pathways triggered by each pathogen, which could lead to synergistic stimulation of inflammation during co-infection [304,317,318].

NF-kB pathway

The NF-κB family of transcription factors consists of five protein subunits that share an affinity for the κB DNA sequence motif and are divided into two classes: the NF-κB proteins (p105/p50 or NFkB1 and p100/p52 or NFkB2) and the Rel proteins There are a number of different mechanisms to regulate the transcriptional response downstream of NF-κB pathway activation and some viral proteins were described to interact with NF-kb pathway such as the NS2 protein of Influenza virus [318].

Antiinflammatory drugs such as aspirin, dexamethasone, and prednisone can indeed inhibit pathway activation, placing NF-κB as a prime target for therapies against inflammatory disease [319].

Interferon pathway

Interferon

b) Interferon-stimulated genes (ISGs)

The interferon signaling cascade induces the expression of many genes known as Interferon stimulated genes (ISGs). The transcription induction of nearly 1000 genes is regulated by IFNs and some are capable to encode direct antiviral effectors or molecules [337]. Some ISG already described to be modulated during virus infection are described in Table 3.

Further research on their expression and function is needed to better elucidate their contribution of ISG to the immune response during respiratory infection [283,337,338].

p53 pathway

The p53 protein, discovered in 1979, was described as "guardian of the genome" due to its major function as a tumor suppressor. The new classification "guardian of homeostasis" is maybe more appropriate, covering all known p53 biological activities [341][342][343][344]. The transcription factor p53 can activate several genes that regulate large quantities of cellular mechanisms in response to different types and intensity of stresses [345][346][347].

The p53 transcriptional activity binds to the promoter of the target to regulate gene expression [348][349][350]. However, even if this protein acts as a nuclear transcription factor, p53 can also play a role outside the nucleus through protein- In conclusion, there are different types of interactions with p53 for example, DNA damage promotes p53 activation driving cell-cycle arrest, senescence, or apoptosis [361,362] while hypoxia, thermal shock, and oncogenes promote p53 stabilization [363,364]. Also, pathogens can modulate p53 to optimize their infection as shown in Figure 14.

Mechanisms of p53 regulation

The regulation of p53 is complex and controlled by many factors including

b) Phosphorylation

Phosphorylation of p53 or Mdm2 can occurs on many different sites of these proteins. These modifications can be important in the activation of the p53 network [381]. The regulation and consequences of this phosphorylation are defined by different levels of stimuli for example, during a low DNA-damage, phosphorylation results in partial activation of p53 but in too severe damage, p53 is fully activated and lead to cell death [356,366,[382][383][384][385].

c) Other modifications

Several other post-translational modifications play an important role in the p53 regulation. Acetylation of p53 occurs on several lysine residues at the C-terminal domain and mainly results in increased DNA binding of p53, promoting the activation of its target gene [386]. Methylation in a specific region of p53 can suppress transcriptional activity of p53 while if it occurs in another region, can activate p53.

Also, some lysine residues of p53 that are modified by acetylation can also be methylated [386][387][388][389][390]. Others modifications of p53, such as prolyl isomerization and glycosylation, may also contribute to the regulation of p53 stability and activity [356,366,391].

p53 isoforms

TP53 gene encodes several isoforms of p53 due to the usage of multiple promoters, alternative translation initiation sites, and alternative splicing, as shown in

Figure 16 [392-396].

P53α is the canonical full-length p53 protein and inclusion of alternative exons gives rise to p53β and p53γ protein isoforms. Some studies reported that these isoforms retain characteristics of a tumor suppressor and both expressions are lost in cancer tumors [392,397]. Also, changes in the relative abundance of p53 isoforms have been implicated in senescence and aging as p53β was described to modulates p53α transcriptional activity in response to stress and promotes apoptosis and senescence [392,[398][399][400][401].

These multiple p53 isoforms might contribute significantly to the generation of distinct p53 pathways [402]. Altogether, modulate p53 isoform expression and thus cellular response can be a therapeutic target to control p53 pathway.

Biological responses induced by p53 target genes

In the past two decades, a huge number of p53 target genes have been

Functional interplay between pathogens and p53

Modulation of p53 is an essential step for pathogens to extend the survival of the host cell, to access nutrients during intracellular replication and to modulate the immune response. However, targeting p53 may compromise the natural safety system of a cell promoting mutations and tumorigenesis, correlating to a number of pathogens that are considered oncogenic [419][420][421].

The p53 protein was discovered during a study with the oncogenic virus SV-40

which has a protein capable to physically interact with p53 and inhibit p53-mediated transcription [342]. Since then, a large number of other oncogenic and nononcogenic pathogens were described to interact in different ways with p53.

The strategy of impairing the transcriptional activity of p53 is also used by others viruses, including hepatitis B virus, human T lymphotropic virus, hepatitis C virus and HIV, and some bacteria, like Neisseria gonorrhoeae [422,423].

Another strategy of p53 inhibition was described by different pathogens, for example, human papillomavirus, adenovirus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus and some bacteria such as Shigella flexneri, Helicobacter pylori, and Chlamydia trachomati promoting proteasomal degradation [424][425][426][427]. In contrast to these pathogens, Salmonella enterica and P.aeruginosa induces acetylation of p53, which is a stabilizing modification that is associated with cell cycle arrest in infected cells [419,428]. Some virus and their interactions with p53 pathway are shown in Table 4. The functional interactions between influenza viruses or RSV and p53 have been reported in a limited number of studies, and infection of influenza viruses significantly alters the gene expression of several host factors belonging to the p53 pathway [438,[440][441][442][443]. This thesis work has mainly based on the interactions RSV / host in the context of simple infections or co-infections, due to the clinical importance of mixed infection, the high rate of circulation of these pathogens and lack of existing therapeutic arsenal.

Indeed, it is important to understand how the immune system is modulated at different levels, in vivo and in vitro, which helps to better understand the pathogenesis mechanisms, identify biomarkers in the control of inflammation or to find potential targets for new antiviral strategies.

58

RESULTS

59

Les résultats de la thèse sont divisés en trois chapitres pour faciliter la compréhension de chaque groupe de résultats. La connexion entre les chapitres est représentée dans la figure ci-dessous.

Les résultats du chapitre 1 obtenus au Laboratoire des pathogènes émergents (Fondation Mérieux) ont permis de consolider les résultats en cours du groupe et de renforcer le modèle de co-infection des macrophages en plus de favoriser l'identification des biomarqueurs.

Le chapitre 2 était le résultat d'un partenariat avec le laboratoire Virpath, et en profitant de l'expertise de ce groupe, nous visions à approfondir la connaissance de la co-infection dans des mécanismes importants pour la cellule, visant à corréler avec la réponse immunitaire trouvée dans le étude des macrophages (chapitre 1).

Le chapitre 3 a été développé en collaboration avec la société Cynbiose (incluant LPE et Virpath), pour examiner les différents niveaux d'interaction proposés et aussi pour appliquer les conclusions obtenues dans les études in vitro (chapitres 1 et 2).

The results of the thesis are divided into three chapters to facilitate comprehension of each group of results. The connection between the chapters is represented in the figure below.

Chapter 1 results obtained at Laboratoire des Pathogenes Emergents (Fondation Mérieux) provided the consolidation of the results that were in progress by the group and strengthened the model of co-infection in macrophages in addition to promoting the identification of biomarkers.

Chapter 2 was the result of a partnership with the Virpath laboratory, and taking advantage of the expertise of this group, we aimed to deepen the knowledge of co-infection in important mechanisms for the cell, aiming to correlate with the immune response found in the macrophages study (chapter 1).

Chapter 3 was developed in collaboration with the company Cynbiose (including LPE and Virpath), to look the different levels of interaction proposed and also to apply the conclusions obtained in the in vitro studies (Chapter 1 and 2). to control the pathogens and contributes to severity disease [156,309,[463][464][465][466][467][468].

Clinical evidence suggests that infection with particular combinations of pathogens results in the more severe clinical outcome [140,168]. The most detailed mixed infection described in the literature is Influenza virus and S. pneumoniae.

Bacterial superinfection after IAV pandemics was described and highlights the importance of Influenza infection in disease severity [147,323,[469][470][471][472]. Thus, in our first study published in Scientific Reports, we set-up an optimization in IAV / S. pneumoniae macrophages infection and observed a potential biomarker and the mechanism involved in its expression after the double infection on these cells.

Another mixed infection that appears to be very important in disease severity in pediatrics community is RSV and S. pneumoniae. Some studies show bacterial superinfection as an important risk factor for hospitalization for RSV infection [128,129,473,474]. Co-infections RSV / S. pneumoniae are frequently detected in respiratory diseases and pneumococcal bacterial load and carried serotypes can indicate clinical severity when co-infected with 473,[475][476][477][478][479]. Thus, in the second manuscript, we look mixed infection of RSV and pneumococcus in macrophage infection.

The main objective of these studies is to better understand how pathogens interactions and cell infection can lead to disease severity. For this purpose, we establish a macrophages model for each co-infection. Macrophages are strategically situated in the primary lung defense and play a central role in innate host defense.

They are capable of initiating innate and adaptive immune responses. In addition, they are a primary source of inflammatory and immunomodulatory cytokines in lungs against invading pathogens. Some studies show that macrophages are able to limit RSV or Influenza virus replication participating in the clearance viral. In vivo studies which macrophages are depleted show an enhanced disease in both viral infections [459,480,481].

Several studies have also identified an important role for macrophages during the initial phase of host defense against S. pneumoniae even if this bacterium has a polysaccharide capsule which limits bacterial phagocytosis by macrophages [322,323,482].

The majority of mixed co-infection models described in the literature is realized using respiratory tract cells and focuses on interactions between the pathogens. It is well established that Influenza viruses enhance susceptibility to secondary bacterial infection promoting bacterial superinfection [309,483]. Also, direct interaction of RSV and S. pneumoniae was shown to increases bacterial virulence [159]. In macrophages cells infected with Influenza virus decreased of binds and phagocytosis of S. pneumoniae was shown [309,[484][485][486]. In the literature, some mechanisms of RSV and other bacterial co-infection were already described, but the information is incomplete and scarce mainly when restricting information about RSV and S.

pneumoniae co-infections is searched.

In addition to better thoughtful the role of macrophages during simple and mixed infection, we also tried to better understand cellular mechanisms and we described a set of potential biomarkers capable to indicate the severity of the disease or the etiology of infection. The relevance of new biomarkers to help diagnostics methods was highlighted in the diagnostic topic of introduction chapter of this manuscript. In our two articles, we confirmed the importance of biomarkers and we contribute to the literature with a description of new biomarkers. Also, to show the close interactions between mixed infections and macrophages we described the cellular mechanisms and activated-pathway in the expression of these biomarkers.

In the last part of the first article, we performed a translational study to correlates in vitro experiments with clinical specimens of children with pneumonia.

The correlations between severity of disease and pathogens were shown and confirmed the selected cytokine as a potential biomarker.

The results suggest potential biomarkers to generate an assay that could discriminate between single or mixed infection during pneumonia. Further research is needed to clarify how mixed infection modulates the cytokines/chemokines expression in macrophages. A better understanding of the host immune responses to mixed infections is important for designing vaccines and other preventative agents in the future. This group of results enriches the literature on respiratory infections and the role of macrophages in the response to mixed infection and promotes greater knowledge about the role of IP-10 as a potential biomarker.

Article 1. Viral and bacterial co-infection in severe pneumonia

Respiratory viruses have been shown to modulate the host response in a variety of mechanism while host cells have numerous strategies to resist infection, replication and virus spread using different cellular mechanisms to the elimination of pathogens. RSV infections are characterized by an extensive inflammatory response through the activation of cell networks and immunoregulatory genes. However, the mechanisms involved in the inducible expression, regulation of these immune modulators and the dynamics of the virus-host interface are not well-understood [START_REF] Troy | Respiratory viral infections and host responses; insights from genomics[END_REF]487].

Several different signaling pathways were described during RSV infection Due the epidemiological importance and urgency to develop an effective vaccine or RSV treatment, we investigated the role of p53 during this infection. To study the role of p53 in RSV infection we used several methodologies already described in the literature to identify and measure the intensity of p53 activity as well as using molecules and RNA silencing techniques to manipulate p53 activity over the course of infection. Also in this article, we used a cell lineage that does not have the TP53 gene, to better identify virus replication under these conditions.

In addition to the RSV results presented in the first article, we performed some experiments in parallel with parainfluenza 3 virus, because although it has recently been separated from the family and is no longer part of the RSV family, this virus has several structural similarities as well as a cycle replicative. Also, for this viruses, we started to look p53 isoforms expression.

After characterizing the interaction of RSV with the p53 pathway, we attempted to show the role of co-infection in the modulation of this pathway. Only

Does viral-bacterial coinfection influence the severity of disease?

A large number of clinical studies have described an enhanced severity of disease in the context of mixed infections, highlighting the importance of performing studies to better understand the role of each pathogen as well as host/pathogen interactions [156,[492][493][494][495][496][497][498][499]. In light of the major public health burden, pneumonia caused by S. pneumoniae following IAV infection has been extensively studied in children and adults with a correlation of disease severity [155,500,501]. In contrast, the clinical significance of bacterial co-infections during RSV remains relatively unclear. Children with RSV/bacterial co-infection have been found to be hospitalized for longer periods and required ventilator support for longer than sole RSV infections but more studies are needed to confirming this correlation [140,141,476].

In our translational study (article #1, chapter 1), the patient cohort studied was restricted (n=74) but the classification of non-severe and severe pneumonia was accurately defined, according to an exhaustive list of criteria. Thus, we managed to demonstrate an increase in disease severity in mixed infection cases. However, A better understanding of pathogens involvement and their contribution to disease severity is required for the development of future strategies for the prevention and treatment of severe respiratory tract infection. In vivo studies constituted a good methodology for an attempt this goal confirming the necessity of in vivo models. Also, another important point to better understand the contribution of each pathogen for disease severity is to better understand interactions and strategies that the pathogen can utilize in cells during infection. For this attempt, I focused my interest on host/pathogen interactions at the level of p53, a key transcription factor at the crossroads between large numbers of cellular pathways -including innate immune responses.

determining

Are macrophages activated cells during acute lower respiratory infections?

Macrophages are a population of phagocytic cells composed of different phenotypes according to the local environment and differentiate stimuli [503,504].

They are known to constitute long-lived reservoirs for some persistent viruses (e.g.,

HIV and Chikungunya virus) [505,506] already published in different macrophages subtypes [322,480,481,484,519,525,526]. An in vivo model could also contribute to this aspect since on obtaining bronchoalveolar aspirate the quantification and identification of active macrophages during co-infection could be performed.

To substantiate the important role of macrophages in co-infection, the expression of cytokines and chemokines by these cells was measured. In a model in vitro developed in article 1 and 2 (Chapter 1), IAV and RSV infection altered biological functions in the monocytes-derived macrophages, inducing an antiviral state and bacterial co-infection show an exacerbated inflammatory response, mediated at least by IP-10 expression.

Importance of cytokines and chemokines expression during respiratory

infections: a good strategy for the development of biomarkers of interest?

The disease severity is not only determined by the causal agent, but also by interactions between the immune response and pathogen. The balance of this interaction is regulated through complex interactions between immune cells and pro- The interplay between influenza viruses and p53 has been reported in different studies [438,440,452,514] and influenza viruses modulate p53 transcriptional activity is modulated during time-course of infection, with a significant increase in early infection [455]. In contrast, this activity is significantly inhibited during the later stages of infection, correlating to elevated NS1 protein levels, which has been described to inhibit p53-mediated transcriptional activity [455]. Results in article #1 (Chapter 2) show that RSV infection modulates p53 transcriptional activity and an important role for non-structural protein NS1, contributing to inhibition of p53 activity by proteasomal degradation.

Further studies are required to understand the impact of RSV infection on p53 biological functions. To explore others possible p53 regulation by RSV and PIV-3

preliminary results shows the possible role of p53 isoforms in their self-regulation.

Expression of p53 isoforms during viral infections: insights of p53 regulation

Since some interplays between p53 and respiratory viruses have been described, Terrier and colleagues investigated the role of ∆133p53α and p53β in the context of an IAV infection [396,402]. In the literature, it was reported that p53β can promote p53 transcriptional activity while ∆133p53α inhibits in different cell lineages [397,401,402]. In addition, several studies reported a deregulation of p53 isoforms expression in human cancers [393,395,[397][398][399][400].

For Influenza virus, distinct roles of the ∆133p53α and p53β isoforms were described in the literature, showing a modulation of the expressions of ∆133p53α and p53β at the transcriptional and posttranscriptional levels affecting viral production.

Preliminary results can't show the impact of RSV in the expression of mRNA of these two isoforms, however, hPIV-3 show an impact on ∆133p53α and p53β expression. During early stages of infections, no differences in p53 isoforms expression were detected during RSV infection. Controversially, hPIV-3 promotes a diminution of p53 isoform expression.

In order to evaluate the role of p53 isoforms mRNA expression during RSV (light grey) or hPIV-3 (dark grey) infection, A549 cells were infected and results from 6 and 12 hours post-infection are shown. No significant differences in the isoforms expression were detected during RSV infection while hPIV-3 is probably capable to impaired p53 isoforms expression with a transcriptional strategy.

This preliminary results according to with the other preliminary result comparing hPIV-3 to RSV, showing that these viruses don't have the same strategies to regulate p53 activity. Further studies like inhibition of both p53 isoforms during infection to look the impact on viral replication and western blot analyses to conclude if these differences in mRNA level are representative of protein level are necessary to understand the impact of different p53 isoforms during viral infection and confirming the antiviral role of the p53 pathway.

Development of severe pneumonia model: application and difficulties

A severe pneumonia model could be important to help to understand the development of severity and also to evaluate antiviral approaches and vaccine candidates due to proximity respiratory tract among non-human primates and humans.

Animal models of viral or bacterial infection that have been studied include the ferret and rodents, particularly cotton rats and mice [160,273,[535][536][537]. Bacterial and host factors contributing to colonization have been defined in animal models and in a mouse acute pneumonia model [START_REF] Kim | Pneumonia and Streptococcus pneumoniae vaccine[END_REF]123]. For RSV infection, they have shown viral replication and antibody response mimicking infection in humans but these models not develop the measurable clinical disease and cannot be utilized to model cellular immunity [START_REF] Groothuis | Prevention of serious respiratory syncytial virus-related illness. I: Disease pathogenesis and early attempts at prevention[END_REF][START_REF] Schrauwen | Reassortment between Avian H5N1 and human influenza viruses is mainly restricted to the matrix and neuraminidase gene segments[END_REF][START_REF] Cardona | Avian influenza in birds and mammals[END_REF]160,535,538]. Also, utilization of some non-human primates as a model of viral or bacterial respiratory infection was already described.

For example, RSV-infected chimpanzees were used to evaluate live-attenuated RSV vaccine candidates, while African green monkeys and rhesus macaques have been used to model the FI-RSV vaccine-enhanced efficacity [538]. Therefore, currently available NHP models of RSV are not sufficiently permissive to use them as a gatekeeper for either efficacy or safety [539] and clinical symptoms are not well detailed and severe pneumonia was not described during RSV infection agreeing with results on article #1 (Chapter 3).

Differences in the route of inoculation were described to contribute to viral replication and disease severity [169,243,540]. In article 1 (chapter 3) results showed that even with an invasive inoculation of pathogens (intratracheal route) the development of pneumonia was not severe. Thus, may be indicated that the quantities of the pathogen are the most important criteria for disease severity.

Improvement in the development of severe pneumonia in NHP model: particularities of RSV infection

Different strains of RSV have already been described to infect primates, and reference strains and isolated clinical sample are usually adapted to the animal to be studied [540][541][542]The objective of this work was to develop a model of severe pneumonia caused by mixed infection, thus, a standard strain, well described and without passage in the animal was chosen. Its known that may be adapted strains can induce a more effective immune response and maybe help to explain why the severe disease was not detected. Also, more studies of efficient entry of RSV are needed to help to identify fully permissive animal models. Initial attachment processes of RSV infection involve binding to glycosaminoglycans, or to C-type lectins which are abundantly expressed in many cell types across many species [START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF]. Thus, maybe are others necessary receptors that promote viral entry and viral tropism.

Severe pneumonia caused by RSV occurs mainly in newborns, so younger primates may potentiate the impact of infections, as described in humans. In summary, a possible strategy to optimize the severe pneumonia model may be, in addition to increasing the number of bacteria, use in vivo adapted virus and/or younger animals.

The literature describes the use of several species of monkeys as a model of respiratory infection, associated with good permissiveness and spontaneous production of the disease after a few days of infection.

Model of severe pneumonia, benefits, and challenges

To conclude, a model of severe pneumonia can help the development of safe vaccines and treatment. NHP model can be used to evaluate therapeutics and pathogenesis for each pathogen and also in combination. Also, it can be used to study various aspects of pathogenesis, particularly the immune response patterns due to similarities in respiratory tract infection.

With the development of a model of severe pneumonia due to mixed infection, many innate immune response mechanisms can be the answer and drug targets can be explored. In view of all works presented in this thesis, in vivo model could be a strategy of research to clarify an innate immune response, cellular mechanisms, and complexities in disease severity development.

Lower respiratory tract infections are caused by many pathogens and the impact of co-infections is an important factor of the severe disease. The impact of coinfections on the modulation of the innate immune response was investigated. To this end, a model for the in vitro study of mixed infection in macrophages by RSV and S. pneumoniae was developed and the modulation of the inflammatory response by cytokines expression was studied. The results demonstrate that co-infection of immune cells deregulates the inflammatory response with IP-10 expression, identified in our study, as a potential biomarker prognostic of severe pneumonia.

In addition, the role of p53 pathway during RSV infection was investigated to better characterize viral pathogeneses, strategies to hijack cell mechanisms and modulation of immune response. An important aspect of this modulation was described highlighting the participation of two important viral proteins.

To conclude, the pilot study to the establishment of an in vivo model of severe pneumonia was developed showing difficulties and challenges of this type of model.

Particularities of pathogens that can contribute to the development of an in vivo model were also highlighted.

Article 1. Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase

Mdm2 during the time-course of infection

In parallel to the thesis, I contributed to another study that leads to a submission article. This work shows detailed interactions between Influenza virus and Mdm2 protein, highlighting the importance of p53 pathway during viral infection.

FIGURE 1

 1 FIGURE 1 CAUSES OF DEATH WORLDWIDE. ...............................................................................................

FIGURE 2 DIFFERENCES

 2 FIGURE 2 DIFFERENCES BETWEEN PNEUMONIA AND BRONCHIOLITIS. .........................................................

FIGURE 3 INFLUENZA

 3 FIGURE 3 INFLUENZA VIRUS PARTICLE. .....................................................................................................

FIGURE 4 THE

 4 FIGURE 4 THE REPLICATIVE CYCLE OF IAV. ..............................................................................................

FIGURE 5 RESPIRATORY

 5 FIGURE 5 RESPIRATORY SYNCYTIAL VIRUS PARTICLE AND GENOME. ...........................................................

FIGURE 6 REPLICATIVE

 6 FIGURE 6 REPLICATIVE CYCLE OF RSV. ...................................................................................................

FIGURE 7 PNEUMOCOCCUSFIGURE 8

 78 FIGURE 7 PNEUMOCOCCUS SURFACE STRUCTURE AND MAJOR VIRULENCE FACTORS. ................................. FIGURE 8 HOST-PATHOGEN INTERACTIONS. ..............................................................................................

FIGURE 9 CHRONOLOGICAL

 9 FIGURE 9 CHRONOLOGICAL COURSE OF INNATE AND ADAPTIVE IMMUNITIES. ...............................................

FIGURE 10 INNATE

 10 FIGURE 10 INNATE AND ADAPTIVE CELLS. ................................................................................................. FIGURE 11 PRRS SIGNALING PATHWAY. ................................................................................................... FIGURE 12 INFLAMMATORY RESPONSES. .................................................................................................. FIGURE 13 THE P53 NETWORK. ................................................................................................................

FIGURE 14 SCHEME

 14 FIGURE 14 SCHEME OF THE P53 PATHWAY. ..............................................................................................

FIGURE 15 P53

 15 FIGURE 15 P53/MDM2 COMPLEX. .............................................................................................................

FIGURE 16 SCHEMATIC

 16 FIGURE 16 SCHEMATIC OF THE P53 ISOFORMS. .........................................................................................

FIGURE 17 P53

 17 FIGURE 17 P53 PATHWAY ROLE DURING THE ANTIVIRAL RESPONSE. ...........................................................

FIGURE 18 P53

 18 FIGURE 18 P53 AND NF-KB MODULATION. ................................................................................................

FIGURE 19 P53

 19 FIGURE 19 P53 PATHWAY MODULATION BY IAV AND RSV. ........................................................................

FIGURE 20 POST

 20 FIGURE 20 POST-TRANSCRIPTIONALLY P53 MODULATION DURING HPIV-3 INFECTION. ..............................

FIGURE 21 A

 21 FIGURE 21 A DIFFERENT PATTERN OF P53 EXPRESSION DURING VIRAL AND MIXED INFECTIONS. .................

FIGURE 22 EXPRESSION

 22 FIGURE 22 EXPRESSION OF P53 ISOFORMS DURING RSV OR HPIV-3 INFECTION. ......................................

  ......................................................................................................... TABLE 2 TLR AND MICROBIAL LIGANDS. ................................................................................................... TABLE 3 ISG MODULATED DURING DIFFERENT VIRUS INFECTION. ............................................................... TABLE 4 VIRUS AND THEIR INTERPLAY WITH P53 PATHWAY. ....................................................................... des voies respiratoires inférieures constituent la troisième cause de décès dans la population mondiale, avec 3,2 millions de décès. Parmi cette mortalité, au tour de 1 million c'était des enfants de moins de 5 ans, représentant la première cause de mortalité dans ce groupe d'âge, selon l'OMS en 2015. Le virus respiratoire syncytial (VRS) est considéré comme un agent étiologique important dans le millieux pediatric et les estimations sont que ce virus cause 3 million d'hospitalization par an. Un aspect important du pronostic des infections virales est le rôle de co-infection bactérienne. La combinaison d'agents viraux et bactériens a été signalée entre le VRS et les bactéries Streptococcus pneumoniae.

Figure 1

 1 Figure 1 Causes of death worldwide.Lower respiratory infections constitute the third cause of death in the world population (red bar) being responsible for 3.2 million of deaths in 2015. Heart diseases (Ischemic heart disease -blue bar -and strokes -pink bar) were the most cause of deaths. Among the 5 main causes of global death described, lower respiratory infections are the only transmissible infectious disease[START_REF] Who | The top 10 causes of death[END_REF].

Figure 2

 2 Figure 2 Differences between Pneumonia and Bronchiolitis. Lower respiratory tract diagram showing affected lung regions during respiratory tract infections. A)Pneumonia is characterized by infection in the alveoli airways and normally occurs in the terminal part of the lobular lung. B) Bronchiolitis is restricted in the bronchial region and remains near to principal bronchi. Adapted from Eugenia et al[START_REF] Eugenia | Pathogenesis of Viral Respiratory Infection[END_REF].

Figure 3

 3 Figure 3 Influenza virus particle. A) Cryo-electron micrographs (Cryo-EM) presenting different strains of Influenza A subtypes. B) Schematic structure of Influenza viral particle showing surface glycoproteins (HA, NA,and M2). Each segment of the viral genome is also represented and RNP complex is highlighted to show each protein that forms this complex. Also, nonstructural proteins are listed. Adapted from Moulès et al[START_REF] Moules | Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles[END_REF].

Figure 4 The

 4 Figure 4 The Replicative cycle of IAV.IAV binds to the respiratory epithelial cells by residues of sialic acid on the cell surface. Then, the internalization occurs by endocytosis of the virus. The low pH in the endosome triggers the fusion of the viral and endosomal membranes, releasing the genome into the cytoplasm. The genome is imported into the nucleus where they serve as a template for translation and transcription of the viral genome. New proteins are synthesized from messenger RNA (mRNA) viral and the viral genome is replicated by means of a complementary RNA (cRNA) of positive sense. The newly produced vRNA assembles with the viral polymerase and the nucleocapsid protein to form the biologically active ribonucleoprotein complexes (RNPs). After packaging of the RNPs into new virions the virus is released from the cell surface by the action of the NA glycoprotein. Adapted from Dubois et al.[START_REF] Dubois | Influenza viruses and mRNA splicing: doing more with less[END_REF]

Figure 5

 5 Figure 5 Respiratory syncytial virus particle and genome. A) Electron transmission micrograph of different stages of the budding process of RSV particles in the cytoplasm membrane. B) A schematic figure representing RSV viral particle with glycoproteins of the surface (F, G, and SH), matrix protein and viral RNA bound to N, P and L proteins. C) RSV genome showing representative proteins of each genome region. Adapted from Norrby et al. [66].

Figure 6

 6 Figure 6 Replicative cycle of RSV.Replication cycle begins when viral surface proteins interact with a cell receptor. After viral attachment fusion between viral and cell membrane occurs in a pH-independent process. All RSV replication steps occur in the cell cytoplasm and viral maturation occurs with the assembly of the nucleocapsid by combining genomic vRNA with N protein, which is accompanied by the addition of P and L proteins for envelope assembly. Matrix proteins aggregate with the viral surface proteins in the cellular membrane and the complete viral particle is released by budding, taking a portion of the plasma membrane in a reverse process to penetration by fusion. In addition to transcription and translation of proteins, viral genome produces a positively stranded RNA intermediate, which serves as a template to generate copies of the viral genome. Adapted from Collins et al.[START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF]

Figure 7

 7 Figure 7 Pneumococcus surface structure and major virulence factors. A) Scheme of S. pneumoniae surface structure showing capsule, cell wall, and cell membrane. The principal's virulence factor PSpA, PspC, LytA, Pneumolysis and ABC complex are identified. B) The virulence factors showed in part A with each main role in respiratory tract infection. Adapted from Kadioglu et al. [123].

1. 3 .

 3 viruses and bacterial species carried commensally in the nasopharynx and samples for laboratory diagnosis are generally contaminated with components of upper respiratory tract[143]. The use of the same pathways, cofactors, and the overlap in the inflammatory mediators produced by different pathogens create an opportunity for augmentation of the immune response during dual or sequential infection. The complexity of microbiome interactions in the airways possibly contributes to the susceptibility to exacerbations and the natural course of airway diseases[144, 145].Thus, many aspects of the relationship between co-infection detection and disease severity remain unclear. However, in the literature, some studies have classified viruses/bacteria interactions in two distinct scenarios: Bacterial superinfection is described when viral infection promotes favorable conditions to commensal bacteria causes an infection in the lower respiratory tract.The association of viruses and bacteria is described by epidemiological studies that show a high prevalence of bacteria in severe disease during seasonal epidemics of the respiratory virus[146]. During IAV pandemics, bacterial superinfection was observed in adults and children associated with increased morbidity and mortality[147][148][149]. Bacteria superinfection is also demonstrated by quantitative studies that show an increasing of commensal bacteria during a viral infection. For example, IAV and RSV infection increase colonization rates of S. pneumoniae and H. influenzae which can lead to secondary complications contributing to the disease severity[128, 142, 146,[150][151][152][153][154][155].Mechanisms associated with viruses predisposing the respiratory tract to bacterial superinfection[156] are poorly understood with two potential explanations:(I) Viral infection can increase bacterial adherence into epithelial cells, as described for IAV infection which is capable to exposes bacterial receptors on the surface of host cells by cleaving sialic acids residues in the upper respiratory tract[128,[157][158][159][160][161]. RSV, on the other hand, is thought to bind directly to H. influenzae and S. pneumoniae[129, 159, 162], increasing bacterial proximity to the epithelial monolayer and augmenting attachment to host cell receptors. Also, viral infection can induce disruption of epithelial cell tract enabling bacteria to access into deep epithelial cells [147]. (II) Viral infection can also predispose bacterial superinfection via the alteration of host's innate immune response. Viral replication may increase recruitment and activation of pro-inflammatory immune cells and may also directly affect the immune system [163-166]. Additionally, viral presence also affects the production and biological activity of cytokines [167] impairing bacterial clearance in its initial phase. Bidirectional synergism or bacterial predisposition to viral disease is represented by an increased viral susceptibility to bacterial infection. It might be possible that microbial interactions may disturb the equilibrium of the microbiota, creating an opportunity for viral invasion and transmission in the lower respiratory tract.Epidemiological studies show that pneumococcal conjugate vaccine not only reduced the incidence of pneumonia due to S. pneumoniae but also prevented approximately 33% of pneumonia associated with respiratory viruses[168]. Also, several studies have shown that the presence of a specific bacterial species may promote viral infection in the respiratory tract such as S. pneumoniae was shown to enhanced RSV infection in vitro and in vivo[144, 169, 170]. In addition, pre-exposure of epithelial cells to bacteria alters the response to subsequent viral infection, suggesting that bacterial presence could facilitate viral attachment to host cells[171].Diagnostic of lower respiratory tract infectionsThe diagnostic of pneumonia or bronchiolitis is performed through clinical examinations taking into account the history and the age of the patient. This clinical diagnosis should be performed according toWHO criteria [120]. In addition, to confirm the diagnosis of pneumonia, a chest X-ray can be performed, being able to show the extent of the disease and to identify the presence of complications that increase the severity of the disease. Usually, clinicians start the treatment without an etiologic detection due to the low sensitivity and/or lack of specificity of current diagnostic tools. In severe cases, laboratory tests capable to identify the pathogen are usually requested and a great effort has been made to improve etiological diagnosis methods[172, 173]. The quality of the collection, packaging, and transport of clinical samples are essential for an optimal diagnosis. In general, nasopharyngeal secretion samples are used for detection of viruses while blood samples are analyzed for bacterial detection[172, 173]. The most common laboratory detection methods are pathogen isolation, molecular detection, immunofluorescence and antibodies detection.

  symptoms and complications are described. The available antivirals include classical approaches focused on targeting the viral cycle and new antiviral strategies targeting the host instead of the virus [207]. a) Anti-influenza drugs According to their targets in the viral replicative site, anti-influenza drugs are classified into three groups: targeting M2 channel, neuraminidase inhibitors, or polymerase inhibitors. Inhibition of viral envelope fusion to cell membranes by M2 proton channel blockers is represented by amantadine and rimantadine. These inhibitors were approved by health authorities but transmissible resistance variants rapidly emerged from patients after treatment and since 2009, they are not recommended for clinical use anymore [208].

  Some anti-RSV treatment targets viral replication cycle by intervention in membrane fusion and RNA synthesis. Membrane fusion inhibition, such as GS-5806, prevents virus-cell fusion and cell-cell syncytium formation and has shown promise results in early-phase clinical trials with efficacy and safety in hospitalized adults [223, 224]. Inhibitors of RNA synthesis during RSV replication are divided into two groups. Nucleoside analogs represented by ALS-8112 and ALS-8176 target the active site of the polymerization domain, and non-nucleoside inhibitors (BI-compoundD) bind to other regions of the polymerase. These molecules are in ongoing studies and show promising results but further evaluation is necessary to determine effectivity and safety [225-228].

  constantly evolving of influenza viruses requires continuous global monitoring and annually reformulation of influenza vaccines [250, 251]. For RSV, during 60's, a clinical trial of a formalin-inactivated RSV vaccine not only failed to prevent RSV infection but caused an increase in severe disease [252]. This experience had a profound negative impact on subsequent RSV vaccine development and the immune mechanisms that led to enhanced disease in this clinical study are not yet fully elucidated, making difficult to advances into clinical evaluation of inactivated RSV vaccines [253].For RSV vaccine, lability and heterogeneity in particle size represent obstacles for the formulation of a stable vaccine[254] and despite the many approaches developed and tested, there is still no vaccine defined for use in humans.Thus, the prophylactic palivizumab is indicated in months prior to the seasonality of RSV for premature babies; children with congenital pathology or with chronic lung disease by WHO. Palivizumab is a humanized mouse IgG1 monoclonal antibody directed against a conserved epitope on the surface fusion protein of RSV.This passive prophylaxis shows a potent RSV neutralizing activity and has been clearly demonstrated to protect against RSV. The administration of palivizumab in specific risk groups is limited by its expensive cost in many low and income countries [221]. Consequently, prevention of RSV infection is a public health priority, and global initiatives have advanced numerous efforts to expand the field [255]. Continued research into the pathogenesis of RSV disease and immune responses are important to contribute to the development of RSV vaccines.

A

  reduction in pneumococcal disease among vaccinated children have been observed since the introduction of the first PCV vaccine in 2000 [151, 260-264]. In addition, vaccination in children has been shown to reduce pneumococcal disease among the elderly by preventing the transmission due the diminished of the carriage in general population [265-267].The challenge of the pneumococcal vaccine is the existence of 92 serotypes since vaccinated individuals remain susceptible to serotypes not included in the vaccine[268][269][270]. Also, changes of serotypes carried out in the nasopharynx of children[271] were detected and the potential risk of other serotypes infections make the pneumococcal disease an important problem of global health, especially in children and must be a priority.

  opportunistic infections is made by physical or anatomical mechanisms (skin, mucosa), mechanical (cilia in respiratory tract cells, tight junctions) and biochemical (tears, saliva) barriers as well as cellular mechanisms including innate and immune responses [1].The pathogenesis of respiratory infections involves the complex interplay between virulence factors, environmental conditions, the magnitude and temporal dynamics of the host response, and host susceptibility factors. The severity of disease is associated with an enhanced host immune response which is essential for pathogen control but can cause collateral damage to the tissues, leading to mortality in some cases. In addition, several pathogens evolved strategies to counteract or hijack host responses constitute by multiple elements including diverse cell types (epithelial cells, dendritic cells, macrophages, monocytes, and granulocytes), various pattern recognition receptors, a large array of cytokines and chemokines, cellular stress, and different pathways[START_REF] Troy | Respiratory viral infections and host responses; insights from genomics[END_REF] 275]. Thus, in this chapter, immune response and others cellular mechanisms important during Influenza virus, RSV and S. pneumoniae infection was highlighted as resumed in Figure8.

Figure 8

 8 Figure 8 Host-pathogen interactions. Important complex cell physiological processes in the control of pathogen infection showing an immune and inflammatory response, in addition to p53 pathway. all these cellular mechanisms are often targeted or modulated by pathogens during infection. Each pathway will be explained in the next part. Adapted from Sun et al. [275].

  Figure 9.

Figure 9

 9 Figure 9 Chronological course of innate and adaptive immunities. Innate mechanisms confer rapid protection, keeping the invading microorganisms under control until the development of adaptive immunity. It may take several days and even weeks for adaptive immunity to become effective.

Figure 10

 10 Figure 10 Innate and adaptive cells.Innate immune cells (macrophages, dendritic cells, natural killers, basophil, neutrophil and mast cells) exists before the invading microorganism, they are effectors cells capable of recognized several molecules of various pathogens. Adaptive cells are developed following exposure to a particular invading agent. It is able to react more quickly and more effectively to subsequent contacts, they specifically identify a molecule with high specificity. Adaptive immunity is divided into a cellular and humoral response. During the cellular response, CD8+ T cells are capable to identify and kill infected cells while CD4+ T cells increase macrophages quantity and/or increases antibodies expression by B cells (humoral response).

Figure 10 .

 10 The recognition of the pathogen by receptors is the first step in the host cell membrane during infection. This recognition is based on a limited repertoire of receptors called pattern recognition receptors (PRRs) that detect conserved microbial components known as pathogen-associated molecular patterns (PAMPs)[276]. This initial response triggered by infection is mediated by three major receptor families -PRRs: Toll-like receptors, RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs) that will be detailed above.Toll-Like Receptors (TLRs) are type 1 transmembrane proteins that are able to recognize PAMPs from bacteria, parasites, fungi, and viruses[277]. TLRs are one of the largest classes of PRRs with 10 receptors (TLRs 1-10). TLRs family is well conserved among organisms and homologous receptors are found in plants, insects and other vertebrates[276, 278]. Normally TLR 1, 2, 4, 5, 6 and 10 are expressed on the cell surface whileTLR 3,[START_REF] Lee | The role of respiratory viruses in the etiology of bacterial pneumonia: An ecological perspective[END_REF] 279]. The expression profile of TLRs in different cell types may be tissue-specific and highlight the different role between cells of the immune system (macrophages, dendritic cells, neutrophils, B and T cells) and epithelial cells[277].

  TLR signaling is finely regulated to differentiate resident flora and to avoid excessive inflammation, which can cause damage to the tissue contributing to the severity of disease[288]. Thus, the activated cascade may be negatively regulated at different levels by different mechanisms such as protein phosphorylation and degradation[282, 297]. Another family of PRRs, RIG-I-Like Receptors (retinoic acid-inducible gene-I; RLRs) is intracellular helicases capable to detect [207] most respiratory viruses, including influenza viruses, RSV and hPIV, and others RNA viruses. Activated-RIG-I leads to the regulation of transcription factor IRF and NF-kB, inducing a very important antiviral response [280, 281, 298, 299]. NOD-Like Receptors (nucleotide oligomerization domain; NLRs) is another cytosolic PRR [207]. This family of more than 20 proteins is involved in various cellular processes of the immune response [300]. Some NLRs, including NLRP3, are critical for the formation of inflammasome complexes which are involved in the proinflammatory responses [301, 302]. NLRP3 is expressed by monocytes, dendritic cells, neutrophils, macrophages, and human bronchial epithelial cell and is essential for the recruitment of innate immune cells during RSV and Influenza infection [303-307]. Another attendant of this family is the NOD2 receptor that should mediate the recognition of viral single-strand RNA. NLR NOD2 binds RSV genome and triggers the innate response, also described for Influenza virus and S. pneumoniae, leading to the production of type I IFN [276, 280, 281, 301]. All PRRs are represented in Figure 11.

Figure 11

 11 Figure 11 PRRs signaling pathway. Different classes of host PRRs (TLRs, RLRs, and NLRs) that trigger distinct signaling pathways culminating in the induction of IFNs and/or proinflammatory cytokines. Adapted from Kumar et al 2009 [277].

[

  308]. Overall, this response is highly specific being capable to recognize various antigens and to produces a specific immune response to each one. Once produced, memory cells have a long life and are able to recognize antigen for years. Thus, subsequent responses to the same antigen are usually faster, greater, and qualitatively different from the first. Adaptive immune cells are shown in Figure10and are divided into two types: humoral and cellular response. The adaptive humoral immune response is mediated by antibodies expression produced by B lymphocytes. Antibodies recognize bacterial antigens, neutralize the infection and eliminate these antigens by various effector mechanisms. During viral infection, the virus specifics antibodies can block viral entry and subsequent establishment of infection. In Influenza virus infection, HA protein is neutralized by host antibodies while for RSV, F protein is probably the viral protein neutralized [275, 281].The adaptive cellular immune is characterized by a T-lymphocyte-mediated response against intracellular microorganisms, such as viruses and some bacteria. T lymphocytes occur in two main classes when activated. The first, CD8 + or cytotoxic, kill infected cells while the second class, the CD4 + or helper, coordinate the immune response promoting the activation of B lymphocytes and macrophages.CD8 + cytotoxic T cells play an important role performing direct elimination of infected cells. They are recruited to the site of infection, where they recognize and eliminate the infected cell preventing the production of progeny. In the other hand, CD4 + T cells produce various cytokines profile. The classic profile is helper T cells type 1 (Th1), which promote the activation of macrophages while Th2 cells, regulates antibodies production by B lymphocytes[309]. More recently, other profiles have been described, such as cells of Th17 and regulatory T cells, which control the inflammatory process[280, 281].A particularity of the immune system in neonates is their reduced ability to produce Th1-responsive cytokines with normal or increased cytokine production to the response of type Th2 and Th17[221] During RSV infection, T and B cells are downregulated[310] leading to a poor induction of long-lasting immunity resulting in difficulties to the development of an effective vaccine[275,[311][312][313][314].

Figure 12

 12 Figure 12 Inflammatory responses.NF-kb pathway and Interferon pathway activation promote transcriptional activity of a number of genes responsible for inflammatory response. Adapted fromLester et al. [297]

  (c-Rel, RelB, and RelA/p65)[319].The NF-κB, composed of a p50 and RelA/p65 subunit, is considered a key transcription factor in antiviral cytokines production. Upon a variety of stimuli, such as viruses or bacteria, NF-κB migrates into the nucleus whereby it binds and activates target gene promoters of numerous cytokines [320] like ICAM, IL-1, IL-6, IL-8, and TNF-α, which are crucial for the response to inflammation[321].

  (IFN) is a family of cytokine capable to produce an antiviral response, mediated by type I and type III, and to promotes macrophages activation by type II interferon[298].Type I IFNs, including IFN-α and IFN-β, has an important role in restricts virus replication and spread. In addition, there is evidence that they are also produced in response to pneumococcal colonization resulting in a synergistic stimulation during mixed infection[298].More recently, type III IFN (IFN-λ1, 2 and 3) have been proposed to have a crucial role in virus control in the respiratory tract, as they are expressed at higher levels than type I IFNs in human airway[322][323][324][325].Due to IFN response importance in preventing viral infection, viruses have different strategies to evade to this response. For influenza viruses, the principal IFN antagonist is NS1 and NS2 proteins that target the IFN system at multiple stages (Talon J, Horvath CM 2000)[283].RSV also encodes two proteins, NS1 and NS2, which prevents the synthesis and signaling of type I and III IFNs. NS1 and NS2 work either individually or synergistically to block almost every essential step of the IFN pathways. These viral proteins promote the degradation of a number of elements of these pathways, including interferon regulatory factor and proteins coded by Interferon stimulate genes through proteasomal degradation[326][327][328][329][330][331][332][333].a) Interferon regulatory factors (IRFs)There are two major interferon regulatory factors: IRF 3 and IRF 7 in the antiviral response. IRF 3 is constitutively expressed while IRF 7 expression is enhanced significantly upon stimulation. Viral activation of the IRFs results in homoor heterodimerization of IRF3 and/or IRF7 and subsequent nuclear translocation to bind to type I and III IFN promoters. In general, IFN-β and IFN-λ1 are predominantly activated by IRF 3, while IRF 7 promotes the transcription of 335]. During RSV infection, IRF3 degradation is promoted, reducing drastically.

  protein interactions[351]. The p53 protein, formed by 393 amino acids, is expressed at low concentrations in various cell types and tissues and has a short half-life being continuously degraded[352][353][354][355].Alterations in the structure and function of p53 are detected in a large number of cancers types and TP53 gene mutation is linked to the poor patient prognosis of cancer[356, 357]. Emerging role in various physiological processes and coordination of diverse cellular responses to different stress are shown in Figure13 [358]. In recent years, p53 regulation in several cell programs including cell metabolism, fertility, and immune regulation was described[345, 359, 360].

Figure 13

 13 Figure 13 The p53 network. Genes are represented by node and interactions by lines. P53 is activated by a large range of regulators, shows on blue line, which controls many distinct biological processes showed in the red line. Interactions are annotated as positive (arrow), negative (T-bar), or modifying (solid circle). Adapted from Kastenhuber et al. [348].

Figure 14

 14 Figure 14 Scheme of the p53 pathway. Different types and intensities of stresses activate p53 by mediators like Mdm2. Activated p53 initiates transcription of hundreds of target genes (transducers) that subsequently mediate cellular responses such as cell cycle arrest, apoptosis or senescence. Adapted from Vousden et al. [361].

  protein stabilization, activation, and posttranslational modifications. The most important posttranslational modifications of p53 include ubiquitinylation, phosphorylation, acetylation, and methylation [365].a) UbiquitinylationUbiquitinylation is a highly regulated, flexible and reversible process that mark proteins for degradation, changes in activity and re-localization[366]. Ubiquitinylation of p53 is mainly mediated by mouse double minute 2 (Mdm2)[366, 367] an E3ubiquitin ligase protein which binds to p53, forming p53 / Mdm2 complex. This complex allows p53 degradation by the proteasome[368][369][370]. Controversially, the Mdm2 expression is transcriptionally regulated by p53 defining a negative-feedback loop, where p53 increases the expression of Mdm2, and this, promotes p53 degradation as showed in Figure15 [367, 371, 372]. Several others E3-ubiquitin ligases have been identified that could play a role of substitute/alternative of Mdm2 such as Cop1, Pirh-1, Pirh-2, Trim24, Arf-BP1, Topors, Chip, Carp1, Carp2, p300, and CBP. However, their role is not already understood[373][374][375].Due to this major role of Mdm2 in p53 activation and the critical need for p53 stabilization during stress or cancer development, some studies look for Mdm2 inhibitors[376]. A small molecule Nutlin-3a, which can disrupt the p53 / MDM2 complex, has proven to be highly specific in inducing p53 stabilization in phase I clinical trials with promising results[377][378][379].

Figure 15

 15 Figure 15 p53/Mdm2 complex. A) P53 and MDM2 form A negative-feedback loop, where p53 induces transcription of MDM2, which repress the transcriptional activity and proteasomal degradation of p53, in unstressed cells. B) Stress or MDM2 antagonists lead to MDM2 degradation promoting p53 accumulation, resulting in cell cycle arrest and other p53 responses. If the stress can be resolved, the cells may return to the pre-stress state. If the p53 activating signal cannot be alleviated, p53 initiates specific responses, such as apoptosis or senescence [380].

Figure 16

 16 Figure 16 Schematic of the p53 isoforms. p53a is composed of two transactivation domains (TA-1 and TA-2), a proline-rich domain (PRD), the DNA-binding domain (DBD), the hinge domain (HD), the oligomerization domain (OD), and a regulatory domain. The theoretical molecular weight of each protein isoform is indicated and the color of the protein domains represents different exon. Adapted from Joruiz et. al. [395].

  identified in various cell types. The classical role of proteins encoded by p53 target genes is apoptosis modulation [403-407] and cell cycle regulation [408]. Apoptosis is a type of programmed cell death activated by a variety of intracellular or extracellular death signals and is a common cellular response to infection. Multiple strategies to modulate, positively or negatively, the apoptotic responses of the host cell have been described for different pathogens [409, 410]. In addition to apoptosis, p53 promotes DNA repair by cell cycle and by modulation of the activity of various DNA repair systems [362]. Nowadays, the non-canonical p53 pathway show modulation in numerous aspects of cellular metabolism [411], inflammation pathway and immune response [348]. p53 changes the activity of multiple metabolic pathways, including glycolysis, mitochondrial oxidative phosphorylation and fatty acid synthesis via transcriptional and non-transcriptional regulation, promoting the preservation of cellular energy under conditions of nutrient restriction [412]. Another non-canonical role of p53 recently described is during inflammation and immune response [345, 413-415]. p53 inactivation in immune cells augments inflammation via multiple pathways, such as enhancing the production of inflammatory cytokines and chemokines in macrophages and promoting the differentiation and function of T cells. p53 also regulates many target genes involved in the immune response creating a loop of amplification of the immune response triggered by infection and mediated by p53. The antiviral role played by p53 limits virus replication, enhances antiviral activity modulated by IFN type I [416], and regulates TLR3 expression and function [417] as represented in Figure 17.

Figure 17 p53

 17 Figure 17 p53 pathway role during the antiviral response. Viral activation of TLR 4 promotes IFN expression that induces p53 transcriptional activation. Target genes important for the antiviral response, such as TLR3, IRF5, ISG15, and IRF9, are up-regulated promoting important implications in the immune response. Adapted from Munoz-Fontela [345]

Figure 18 p53

 18 Figure 18 p53 and NF-kB modulation.During extrinsic or intrinsic stress, p53 and NF-kB pathway is activated and they can reciprocally regulate each other by different interactions. Adapted fromAk et al.[418]

  Influenza viruses and RSV induce apoptosis in numerous cell types and the role of viral proteins which participates in the induction of cell death and cell cycle arrest was described in the literature [444-451]. Several levels of regulation of p53 transcriptional activity are affected during the time course of IAV infection. The global down-regulation of p53 target genes during initial phases of influenza infection is in accordance with the inhibition of p53 pathway during this transient context [452]. After, IAV induces posttranslational modifications that stabilize the p53 protein throughout the course of infection [410, 452-454]. The enhance of p53 transcriptional activity limited viral replication and has an important role in the induction of the IFN response [410]. The viral protein NS1 participates in the stabilization of p53 [454, 455] while NP protein induces apoptosis and cell death [440]. p53 isoforms are involved in the regulation of these p53dependent antiviral properties [452, 455]. Infection differentially modulates the expression of ∆133p53α and p53β at both transcriptional and posttranscriptional levels. The modulation of ∆133p53α and p53β isoforms play distinct roles in the viral cycle by acting as regulators of the p53-dependent antiviral activity [396]. Further investigations are needed to better understand p53 pathway during the time-course of infection. Groskreutz and colleagues have shown that RSV induces the down-regulation of p53 during the time-course of infection, with a consecutive impact on apoptosis and survival of airway epithelial cells [441]. However, the underlying mechanisms of p53 modulation, possibly involving viral proteins, remain to be determined. The NS1 and NS2 RSV proteins play an essential role in suppressing apoptosis and facilitating virus growth through the inhibition of interferon responses [456], NS1 also interacted with components involved in cell cycle regulation and DNA damage repair, promoting a G1-phase arrest in the cell cycle [457]. The F protein triggers p53-dependent apoptosis in the late phase of acute infection [442] while P protein may inhibit apoptosis [458]. This discordance in the literature may reflect cell-specific responses to RSV infection or differences in the strain and infectivity of the virus [433]. The effect of viral modulation in p53 protein may be important targets for therapy in RSV infection. P53 modulation by IAV and RSV are represented in Figure 19.

Figure 19 p53 56 OBJECTIVE 57 Ce

 195657 Figure 19 p53 pathway modulation by IAV and RSV.NS1 protein of IAV is capable to interacts with p53 protein and inhibit transcriptional activity during infection. Also, an NS1 protein of RSV is capable to inhibit p53 by Mdm2 interaction.

  , two works will be presented, in which we observe an innate immune response during viral or mixed infection. The importance of each pathogen (IAV, RSV and S. pneumoniae) has already been highlighted by the introduction of this manuscript. These two works highlighted certain aspects of the in vitro host innate immune response during exposition by single or mixed infections. The majority of respiratory viruses, including IAV and RSV, and bacteria are recognized in human airway respiratory cells by TLRs and RIG-I as reviewed in general introduction. This recognition leads to the initial production of antiviral inflammatory response and activation of immune cells like macrophages with an expression of cytokines and chemokines [459-461]. Individually, each pathogen interacts with immune cells and has different strategies to evade host immunity response. Influenza virus and RSV are capable to make modifications in cellular mechanisms during virus replication while S. pneumoniae possesses a number of virulence factors which may confound immune responses [462]. Together, viralbacterial co-infection subverts many aspects of immunity, of which result in a failure

  such as protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and NF-kB. MAPK are involved in RSV replication in human airway epithelial cells [488]. The precise trigger of NF-kB activation is uncertain and may involve oxidative stress, virus replication, viral nucleic acid, or expression of individual viral proteins. NF-kB is a family of inducible transcription factors that control or modulate the expression of several hundred genes, many of which are involved in a variety of activities including cell proliferation, differentiation, and death, as well as modulating the host immune response to viral invasion [489]. In addition to signaling pathways well studied in the literature, there are some others important pathway in the interactions between virus and host response responsible for regulating inflammatory cytokine and chemokine production, To go forward into the knowledge between pathogen interactions and cellular mechanisms, we decided to look at the p53 pathway that, as was shown in the Introduction chapter of this thesis, participates in important cellular mechanisms and is a target of the pathogens. For this purpose, we performed a set of experiments on the respiratory tract of epithelial cells as they are known to express wild-type p53. These cells are the first site of virus infection and try to limit viral infection through cellular mechanisms. Thus, we can highlight the role of p53 during viral infection. Studies on influenza virus and the interactions with p53 have been conducted since 2011 by the VirPath team and the results have already been published and are available in the literature. As the interactions are complex and involve several factors of transcription, a lot of work is still underway and in parallel of my thesis, I contributed to the attached article in appendices which described Mdm2 interactions and influenza virus. This manuscript was submitted to Scientific Reports journal.

  preliminary results were presented in discussion due to difficulty in standardizing the co-infection. Adding the bacteria means more manipulation of the cells, with changes in the medium and more external changes, which causes stress in the cell and makes it difficult to identify the role of p53. As the Influenza virus is more easily manipulated in vitro and has mechanisms more accurate than compared to RSV, we also performed the co-infection of IAV and bacteria, to identify the role of the p53 protein. common inflammatory illness of the lungs which was described in the introduction part of this manuscript. The etiology of pneumonia can be either bacterial and/or viral, thus, mixed infection is a key element that promotes severe inflammatory disease and mortality.Currently, the mechanisms involved in the lethal synergism between RSV and S. pneumoniae co-infection still remains misunderstood. We tried to develop a coinfection Nonhuman Primates (NHP) model of severe pneumonia susceptible to RSV and S. pneumoniae infection like the human. NHP lungs closely resemble the human lung anatomically and physiologically. NHP also exhibit immune responses to bacterial infections similar to the human and have some studies in vaccine development, including for S. pneumoniae.RSV infection occurs an early age when maternal antibodies are present and in the failure of natural infection to prevent reinfection. A history of a formalininactivated RSV vaccine that enhanced disease in young children, and the lack of animal models that fully reproduce the pathogenesis of RSV infection in human difficult an efficacious vaccine development. RSV infection has already been observed in cynomolgus[490, 491]. Importantly, no study focus on pneumonia induced by mixed viral and bacterial infection in cynomolgus macaques. Very few new models are in development and no relevant models for respiratory diseases on primate are currently available due to the limited qualified centers in Europe.In this study, we tried to develop a nonhuman primate model of severe pneumonia to evaluate pneumonia onset induced by mixed infection. Given the suboptimal performance of current diagnostic tests, we would like to characterize potential clinically relevant molecular profiles and to investigate the feasibility of identifying biomarker to severe pneumonia.

  the precise contribution of each pathogen to disease severity by the clinical setting remained extremely difficult. Studies focused on the identification of etiological agents are difficult to compare due to a lack of standards for information on severity, In addition, variation in techniques, diagnostic cut-off values, and pathogens targeted between studies may confound the determination of the overall clinical significance of viral/bacterial co-infection.In addition to clinical studies, several reports show the interactions between pathogens, suggesting a correlation with severity disease. For example, It was demonstrated that respiratory viral infection promotes an increasing of S. pneumoniae density in children with pneumonia[502]. Moreover, direct interactions between virus and bacteria are capable of increased virulence of the other pathogens, such as was described for RSV that is capable to interact with a protein of S. pneumoniae increasing bacterial virulence[159]. Bacterial-virus interactions were not studied in this thesis, however, the detection of the two pathogens in the same macrophage showed by immunofluorescence in article #1 (chapter 1) reveals that the model developed in Chapter 1 can be used in viral-bacterial interactions studies. Complementary studies, like immunoprecipitation, will be necessary to identify possible close viral-bacterial interactions.

  and anti-inflammatory cytokines[527]. Also, different pathogens may trigger different inflammatory responses depending on their intrinsic properties[528, 529]. It is well known that the cytokine storm is present in severe pneumonia, so, cytokines appear to be good biomarkers targeting the immune response. In chapter 1 (articles #1 and #2), mixed co-infection show an increase of cytokines expression when compared with RSV or IAV single infection, suggesting an increased inflammatory pathway. In addition, CXCL-10/IP-10 expression pattern found in bacterial and viral pneumonia highlight its importance as severity biomarker. IP-10 is expressed by macrophages in early response to infection and attracts activated T-cells to the site of inflammation IP-10 expression in macrophages infected with RSV was already described[513, 530, 531]. Also, Hayney et al described that an increased IP-10 concentration measured in serum of adult with pneumonia correlates with the severity, duration, and illness symptoms and concluded that IP-10 could serve as a useful marker or predictor of respiratory infections severity in adults[532]. In addition, Principi et al. described IP-10, in addition to CRP and TRAIL expression capable to distinguish bacterial from viral infections[481]. Thus, we propose that IP-10 is an important component during mixed infection and associated with others can be a good set of biomarkers.Macrophages normally express cytokines in response to infection by pathogens. Often it is also activated by the cytokines expressed by the respiratory tract cells that are the primary site of infection. To continue studying the interactions between RSV infection and the immune system, I have chosen to study an important cellular mechanism capable of modulating various pathways in the epithelial cells which play an important role in the immune response.Host-Pathogens interactions: Role of the master cell regulator p53 pathway during viral infection: similarities and differences between IAV, RSV, PIV-3, and mixed-infection. Respiratory tract epithelial cells are the first site of infection and promote the immune response activation. In the immune response, p53 not only promotes the activation of IFN-stimulated genes (ISGs) but also enhances IFN production in virusinfected cells [345].The human parainfluenza viruses (hPIVs) belong to a diverse group of enveloped single-strand RNA viruses within the Paramyxoviridae family, a large group of viruses that was recently reformulated. The hPIV genome is quite the same of RSV genome, and viral replication is almost the same[533]. After RSV, hPIV type 3 is the leading cause of hospitalization for respiratory illness in young children from ages 0 to 2 years, with a marked increase among 1-to 2-year-olds[START_REF] Abedi | Estimates of Parainfluenza Virus-Associated Hospitalizations and Cost Among Children Aged Less Than 5 Years in the United States, 1998-2010[END_REF]. Due to hPIV-3 importance and similarities with RSV, we decided to look for p53 interactions during hPIV-3 infection.As part of complementary results, p53 protein and mRNA expression show that hPIV-3 promotes the downregulation of p53 by transcriptional level while the same group of results shows a posttranscriptional regulation of p53 during RSV infection in article 1 of Chapter 2. The decreased of p53 during these viral infections were already described byEllis et al and Marques et al. [433, 534] with some contrarious results, probably due to differences in virus strain and time-course of infection. However, there are not many data in the literature that describe more details about these interactions.

Figure 20

 20 Figure 20 Post-transcriptionally p53 modulation during hPIV-3 infection. The p53 pathway can be modulated during different stages of infection. A) Results from early stages of infection showing no differences in p53 protein expression by western blot, a decreased in mRNA expression without changes in the p53 transcriptional activity. B) During late stages of infection, western blot revealed a decreased of p53 proteins, while mRNA expression is stabilized but an important decreased of transcriptional activity of p53 is detected, suggesting a post-transcriptional impairment of p53 by hPIV-3. C) The half-life of p53 was measured with cycloheximide technique described by XX. hPIV-3 apparently, don't change half-life of p53, probably they don't promote p53 degradation.

Figure 21 A

 21 Figure 21 A different pattern of p53 expression during viral and mixed infections. RT-PCR was used to quantify mRNA p53 expression in late stages of infection and the modifications apparently are up-regulation of p53 showing a synergism of expression during IAV and Sp infection.

Figure 22

 22 Figure 22 Expression of p53 isoforms during RSV or hPIV-3 infection.During early stages of infections, no differences in p53 isoforms expression were detected during RSV infection. Controversially, hPIV-3 promotes a diminution of p53 isoform expression.

Article 2 .

 2 Phylogenetic analyses of influenza A (H1N1)pdm09 hemagglutinin gene during and after the pandemic event in Brazil Also, in parallel to my thesis, I participated in some evolutionary analyses of Influenza virus. The Brazilian national reference center of Influenza virus work on the surveillance and evolutionary analysis of influenza virus detected in Brazil. The collaboration with this laboratory, where I did my master degree, is still established and I contributed to this article published by the Brazilian team.

TABLE 1 PNEUMOCOCCAL VACCINE.

 1

b) Haemophilus influenzae

	Haemophilus influenza (H. influenzae) is a gram-negative bacterium also
	found in the upper and lower respiratory tract as commensal bacteria. H. influenzae
	can be divided due to differences in the capsular polysaccharide, with six different
	strains (a-f), and nonencapsulated strains (NTHi) [125]. Polysaccharide capsule is a
	major virulence factor in protecting the bacterium from phagocytosis and stimulating
	the inflammatory response [125]. H. influenzae serotype b is considered an important
	agent causing pneumonia in children under five years of age, elderly and
	immunosuppressed [126]. Also, viral-bacterial dynamics has been described
	suggesting that viral infection increases NTHi colonization [127-129].

c) Others respiratory bacteria

	subspecies [132], whereas methicillin-resistant Staphylococcus aureus (MRSA) is the
	principal responsible for hospital infections, among organisms resistant to antibiotics
	[133].
	Mycoplasma pneumoniae (M. pneumoniae) are distributed globally and are
	the smallest prokaryotic microbes present in nature. Mycoplasma is divided into
	seven species that are pathogenic to humans, including M. pneumoniae [134] that
	accounts for approximately 20% of all pneumonia and higher rates correlated with
	the degree of immunosuppression accounts in the HIV-infected population [135].
	Chlamydophila pneumoniae (C. pneumoniae), with the two others species C.
	psittaci and C. trachomatis, are responsible for lung infections and C. pneumoniae
	Some studies have suggested that other bacteria, such as Staphylococcus
	aureus, Mycoplasma pneumoniae, and Clamidophyla pneumoniae, may cause
	pneumonia with elevated mortality among HIV positive population [10, 130].
	Staphylococcus aureus (S. aureus) is another commensal bacteria of the
	human nasopharynx that causes respiratory infections [131]. S. aureus is a gram-
	positive, facultatively anaerobic bacterium, usually without a capsule. This species of
	bacteria is widely distributed, being able to live in a wide variety of environments due
	to its tropism for several tissues. S. aureus is composed of several species and

  Thisapproach was described for influenza virus which HA protein was expressed in insect cells by baculovirus vectors[233]. For RSV, viral antigens expressed in other viruses, such as Sendai virus, vaccinia virus, adenovirus, and parainfluenza or in bacteria was studied. Despite promising results in murine studies, studies in adults showed

	relatively low capacity for inducing neutralizing activity [241-243], so they have not
	been advanced into clinical phases. Another approach of recombinant RSV vaccine
	carrying host cytokines capable to promote immune responses suggests that these
	formulations can modulate the immune response being effective alternatives for
	immunization against RSV [244-249].
	restricted use in USA and Russia [233, 234]. Using this strategy, attenuated RSV
	strains have been developed [235, 236] but failed in some clinical trials [237, 238].
	The disadvantage of this strategy is the that, in rare cases, the live attenuated
	vaccine strain can revert to its virulent wild-type, causing severe disease [239].
	Alternatively, recombinant vaccines consisting viral protein expressed in other

development of viral vaccines are based on attenuated, recombinant, inactivated and subunit composition strategies. A traditional strategy that has worked for several pathogens involves the development of attenuated viral strains. Attenuation can be accomplished by serial passage or cold-adaptation and has the advantage of expressing most of the pathogen's antigens to improve immune response. Attenuated vaccines for Influenza viruses have been produced for more than 50 years, however, this vaccine has a in vivo support have been increasingly used for vaccine development [240].

Table 1 Pneumococcal vaccine. Serotypes

 1

included in each pneumococcal vaccines approved to use worldwide

[259]

.

Table 2 TLR and microbial ligands

 2

. TLR family represented by 10 receptors capable to recognize different microbial ligand.

Table 3 ISG modulated during different virus infection

 3

. ISG already described in the literature to have an important role in viral infection. Adapted from

Schoggins et al [337]

Table 4 Virus and their interplay with p53 pathway. Functional

 4

effects of virus interactions with p53 pathway showing different levels of interaction.

133

  Au cours de mon travail de thèse, l'objectif général était de mieux comprendre les interactions multiples et complexes entre les pathogènes respiratoires et l'hôte, à différents niveaux, in vitro et in vivo. En effet, mon travail était centré sur différents pathogènes ou combinaisons d'entre eux (IAV, RSV, S. pneumoniae), sur différents

	modèles cellulaires in vitro (macrophages, cellules épithéliales pulmonaires
	humaines), et in vivo (primate non humain), et sur différents types des voies
	cellulaires (réponse immunitaire, voie p53 / NF-kB). Ces différents projets ont mis en
	évidence quelques points intéressants de discussion générale que je voulais aborder
	dans ce chapitre.
	During my thesis work, the general objective was to better understand the
	multiple and complex interactions between respiratory pathogens and the host, at
	different levels, in vitro, and in vivo. Indeed, my work was focused on different
	pathogens or combination of them (IAV, RSV, S. pneumoniae), on different in vitro
	cellular models (macrophages, human lung epithelial cells), and in vivo (Nonhuman
	primate), and on different types of cellular pathways (immune response, p53/NF-kB
	pathway). These different projects have highlighted some interesting points of
	general discussion I wanted to address in this chapter.

  special emphasis on Mycoplasma pneumoniae. Jpn J InfectDis, 2007. 60(6): p. 337-41. 136. Burillo, A. and E. Bouza, Chlamydophila pneumoniae. Infect Dis Clin North Am, 2010. 24(1): p.[START_REF] Collins | Identification of a tenth mRNA of respiratory syncytial virus and assignment of polypeptides to the 10 viral genes[END_REF][START_REF] Bernstein | Respiratory syncytial virus proteins: identification by immunoprecipitation[END_REF][START_REF] Dubovi | Analysis of proteins synthesized in respiratory syncytial virus-infected cells[END_REF][START_REF] Fernie | Immunochemical identification of viral and nonviral proteins of the respiratory syncytial virus virion[END_REF][START_REF] Collins | Respiratory Syncytial Virus and Metapneumovirus[END_REF][START_REF] Norrby | Morphogenesis of respiratory syncytial virus in a green monkey kidney cell line (Vero)[END_REF][START_REF] Sullender | Respiratory syncytial virus genetic and antigenic diversity[END_REF][START_REF] Crim | Identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity[END_REF][START_REF] Tayyari | Identification of nucleolin as a cellular receptor for human respiratory syncytial virus[END_REF][START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF][START_REF] Langedijk | Proposed three-dimensional model for the attachment protein G of respiratory syncytial virus[END_REF] C., et al., pneumonia related to intracellular pathogens. Intensive Care Med, 2016. 42(9): p. 1374-86. 138. Brogden, K.A., J.M. Guthmiller, and C.E. Taylor, Human polymicrobial infections. Lancet, 2005. 365(9455): p. 253-5. 139. Duttweiler, L., Pulmonary and systemic bacterial co-infections in severe RSV bronchiolitis. Archives of Disease in Childhood, 2004. 89(12): p. 1155-1157. 140. Thorburn, K., et al., High incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (RSV) bronchiolitis. Thorax, 2006. 61(7): p. 611-5. 141. Kneyber, M.C., et al., Concurrent bacterial infection and prolonged mechanical ventilation in infants with respiratory syncytial virus lower respiratory tract disease. Intensive Care Med, 2005. 31(5): p. 680-5. 142. Honkinen, M., et al., Viruses and bacteria in sputum samples of children with community-acquired pneumonia. Clin Microbiol Infect, 2012. 18(3): p. 300-7. 143. Bogaert, D., et al., Variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis. PLoS One, 2011. 6(2): p. e17035. 144. Bellinghausen, C., et al., Exposure to common respiratory bacteria alters the airway epithelial response to subsequent viral infection. Respir Res, 2016. 17(1): p. 68. 145. Bosch, A.A.T.M., et al., Viral and Bacterial Interactions in the Upper Respiratory Tract. PLoS Pathogens, 2013. 9: p. e1003057. 146. Jansen, A.G., et al., Invasive pneumococcal and meningococcal disease: association with influenza virus and respiratory syncytial virus activity? Epidemiol Infect, 2008. 136(11): p. 1448-54. 147. Morens, David M., Jeffery K. Taubenberger, and Anthony S. Fauci, Predominant Role of Bacterial Pneumonia as a Cause of Death in Pandemic Influenza: Implications for Pandemic Influenza Preparedness. The Journal of Infectious Diseases, 2008. 198(7): p. 962-970. 148. Reid, A.H. and J.K. Taubenberger, The 1918 flu and other influenza pandemics: "over there" and back again. Lab Invest, 1999. 79(2): p. 95-101. 149. Dhanoa, A., et al., Epidemiology and clinical characteristics of hospitalized patients with pandemic influenza A (H1N1) 2009 infections: the effects of bacterial coinfection. Virol J, 2011. 8: p. 501. 150. Garcia-Rodriguez, J.A. and M.J. Fresnadillo Martinez, Dynamics of nasopharyngeal colonization by potential respiratory pathogens. J Antimicrob Chemother, 2002. 50 Suppl S2: p. 59-73. 151. Ruckinger, S., et al., Reduction in the incidence of invasive pneumococcal disease after general vaccination with 7-valent pneumococcal conjugate vaccine in Germany. Vaccine, 2009. 27(31): p. 4136-41. 152. Watson, M., et al., The association of respiratory viruses, temperature, and other climatic parameters with the incidence of invasive pneumococcal disease in Sydney, Australia. Clin Infect Dis, 2006. 42(2): p. 211-5. 153. Klugman, K.P. and S.A. Madhi, Pneumococcal vaccines and flu preparedness. Science, 2007. 316(5821): p. 49-50. 154. Koon, K., et al., Co-detection of pandemic (H1N1) 2009 virus and other respiratory pathogens. Emerg Infect Dis, 2010. 16(12): p. 1976-8. 155. O'Brien, K.L., et al., Severe pneumococcal pneumonia in previously healthy children: the role of preceding influenza infection. Clin Infect Dis, 2000. 30(5): p. 784-9. 156. Bakaletz, L.O., Viral-bacterial co-infections in the respiratory tract. Curr Opin Microbiol, 2016. 35: p. 30-35.

nombreux pathogènes et l'impact des co-infections est un facteur important de la maladie grave. L'impact des co-infections sur la modulation de la réponse immunitaire innée a été étudié. A cette fin, un modèle pour l'étude in vitro de l'infection mixte dans les macrophages par RSV et S. pneumoniae a été développé et la modulation de la réponse inflammatoire par l'expression des cytokines a été étudiée. Les résultats démontrent que la co-infection des cellules immunitaires dérégule la réponse inflammatoire avec l'expression IP-10, identifiée dans notre étude, comme un pronostic biomarqueur potentiel de la pneumonie sévère.En outre, le rôle de la voie p53 au cours de l'infection par RSV a été étudié pour mieux caractériser les pathogénies virales, les stratégies pour détourner les mécanismes cellulaires et la modulation de la réponse immunitaire. Un aspect important de cette modulation a été décrit soulignant la participation de deux protéines virales importantes.En conclusion, l'étude pilote à l'établissement d'un modèle in vivo de pneumonie sévère a été développée montrant les difficultés et les défis de ce type de modèle. Les particularités des pathogènes pouvant contribuer au développement d'un modèle in vivo ont également été soulignées.

LIST OF FIGURES