Le virus respiratoire syncytial (VRS) est le principal agent responsable desbronchopneumonies du jeune veau et des bronchiolites du nourrisson. Il n’existe pas devaccin ni d’antiviraux spécifiques pour l’homme. La réplication du génome et la transcriptiondes gènes viraux sont assurées par un ensemble de protéines virales constituant le complexeARN polymérase ARN-dépendant : la nucléoprotéine N, la phosphoprotéine P, le facteur detranscription M2-1 et la grosse sous-unité L. L’objectif principal de ma thèse était d’obtenirde nouvelles données structurales et fonctionnelles sur le complexe ARN-polymérase ARNdépendante(RdRp) du VRS, en particulier sur le couple P-L. Pour ceci j’ai tout d’aborddéveloppé un protocole de production et purification de la protéine L sous formerecombinante en cellules d’insecte. Ceci m’a permis ensuite de cartographier le sited’interaction de P avec L. J’ai ainsi mis en évidence que la protéine L interagit avec la partieC-terminale de la protéine P, au-niveau des résidus 216 à 239. Les données obtenuessuggèrent que ce domaine peut former un nouvel élément de reconnaissance moléculaire(« MoRE ») se structurant en hélice alpha lors de l’interaction avec la protéine L. De plus, lacartographie de ce domaine d’interaction m’a permis d’identifier entre les résidus 164 et 205de P une nouvelle région impliquée dans le recrutement de la protéine L aux corpsd’inclusions viraux. Ces nouvelles données ouvrent la voie à de nouvelles études structuralesde l’ARN-polymérase du VRS et nous permettent d’envisager de nouvelles stratégiesantivirales ciblant ce complexe. / Respiratory syncytial virus (RSV) is the leading cause of calves bronchopneumonia andinfants bronchiolitis. Neither vaccine nor antiviral treatments are currently available for use inhumans. Viral genome is replicated and transcribed by a set of viral proteins constituting theviral RNA-dependent RNA polymerase (RdRp) complex: the nucleoprotein (N), thephosphoprotein (P), the transcription factor (M2-1) and the large subunit (L). This workaimed to unveil new structural and functional data regarding the viral RdRp, especially the PLcouple. With this aim in view, I have first conceived a protocol to produce and purifyrecombinant L and P proteins expressed in insect cells. This tool enabled the fine mappingand characterization of the L binding domain of the RSV phosphoprotein. This highlightedthe interaction between the L protein and the C-terminal region of the P protein, especiallyresidues 216 to 239. Further data suggests that this area constitutes an alpha helix formingmolecular recognition element (« MoRE ») during P-L interaction. Furthermore, this studyunveiled a new region of the P protein encompassing residues 164 to 205, involved in therecruitment of L protein to viral inclusion bodies. These new results open the way toupcoming structural studies of RSV RdRp and allow us to define a new target for thedevelopment of antiviral drugs against RSV.

vÃrus sincicial respiratÃrio humano (VSRh) à o agente viral mais freqÃentemente relacionado a infecÃÃes do trato respiratÃrio inferior em crianÃas menores de dois anos de idade. O VSRh à caracterizado antigenicamente em dois grupos: A e B, e cada grupo apresenta vÃrios subgrupos. A glicoproteÃna G à a principal responsÃvel pela a variaÃÃo antigÃnica inter e intragrupos desse vÃrus. Os objetivos desse estudo foram caracterizar os perÃodos epidÃmicos e a diversidade antigÃnica e genÃmica dos VSRh circulantes em Fortaleza, Cearà â Brasil, durante cinco perÃodos epidÃmicos consecutivos (2004-2008). A imunofluorescÃncia indireta (IFI) foi utilizada para a triagem de VSRh e de todos os vÃrus analisados e para a caracterizaÃÃo antigÃnica dos VSRh. A RT-nested-PCR seguida do seqÃenciamento parcial do gene G foi utilizada para a caracterizaÃÃo gÃnomica dos VSRh. O VSRh foi detectado em 456 das 2885 (15.8%) amostras. O pico dos perÃodos epidÃmicos de VSRh ocorreu nos meses de marÃo a maio relacionado à ocorrÃncia de chuvas. Um total de 282 VSRh (62,8%) foram caracterizados antigenicamente por IFI, sendo 170 VSRhA (60,3%) e 112 VSRhB (39,7%). Ambos os grupos circularam durante todo o perÃodo analisado sendo observado o predomÃnio de A em todos os anos. Um total de 250 VSRh (54,8%) foi submetido à RT-nested-PCR com amplificaÃÃo de 133 e seqÃenciamento de 86. A caracterizaÃÃo genÃmica dos VSRh identificou os subgrupos GA2 e GA5 para o VSRhA e os subgrupos GB3 e BA para o VSRhB. Esses quatro subgrupos co-circularam durante o ano de 2006. Nos anos de 2004, 2005 e 2007 verificou-se a presenÃa dos dois subgrupos de VSRhA. Em 2008 somente o GA2 circulou. Em 2004, 2007 e 2008 somente o subgrupo BA esteve presente. Em 2005 somente o GB3 circulou. Os VSRhA apresentaram uma maior variabilidade nas seqÃÃncias nucleotÃdicas, indicando uma possÃvel pressÃo seletiva positiva. Houve variaÃÃes no Ãnicio, fim e duraÃÃo de cada perÃodo epidÃmico de VSRh, assim como na circulaÃÃo de grupos e subgrupos. / The human respiratory syncytial virus (HRSV) is the major agent of lower respiratory tract in children under two years old. HRSV is characterized antigenically into two groups: A and B, and each group has several subgroups. Glycoprotein G is primarily responsible for the antigenic variation between and within groups of viruses. The aims of this study were to characterize the epidemic periods and the antigenic and genomic diversity of circulating HRSV in Fortaleza, Cearà - Brazil, for five consecutive epidemic periods (2004-2008). The screening of positive samples to HRSV and other viruses analyzed, as the antigenic characterization of HRSV was carried out by indirect immunofluorescence. RT-nested-PCR followed by partial sequencing of the gene G was used for genomic characterization of HRSV. The HRSV was detected in 456 of 2885 samples (15.8%). The peak of the epidemic periods of HRSV occurred from March to May related to rainfall. A total of 282 HRSV (62.8%) were characterized antigenically, with 170 HRSVA (60.3%) and 112 HRSVB (39.7%). Both groups circulated throughout the period analyzed with a predominance of HRSVA in all years of study. A total of 250 HRSV (54.8%) were submitted to RT-nested-PCR with amplification of 133 and sequencing of 86. The genomic characterization of HRSV identified subgroups GA2 and GA5 for HRSVA and subgroups GB3 and BA for HRSVB. In the years 2004, 2005 and 2007 both subgroups of HRSVA circulated. In 2008 only GA2 circulated. In 2004, 2007 and 2008 only the subgroup BA was present. In 2005 only the GB3 circulated. The HRSV A showed a higher variability in nucleotide sequences, indicating a possible positive selective pressure. There were variations in the beginning, end and duration of each epidemic period of HRSV, as well as in the occurrence of groups and subgroups.

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

The airway epithelium has many roles in innate immunity including detection of pathogens and transmitting danger signals to other cell types. However, its role as a primary defender against infection is not well recognized. We have investigated methods of augmenting antiviral immunity by application of agents that stimulate viral killing, either in the extracellular space or within the cytoplasm. A recently described property of airway epithelial cells is direct oxidative killing of bacteria through the coordination of Duox and lactoperoxidase enzymes. We have exploited this property by supplementing airway cells with the lactoperoxidase substrate iodide to prevent viral infection. A second method for enhancing antiviral defenses is to supply small interfering RNAs (siRNAs) targeting essential viral genes. We have optimized antiviral siRNAs targeting respiratory syncytial virus by designing them to specifically target positive sense viral RNAs. Finally, we have initiated a project to discover host defense genes that are expressed in either the submucosal glands surface epithelium of human airway. This information will enable a better characterization of the roles for these structures in host defense pathways, and may identify other targets for augmentation of antiviral immunity.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection and hospitalization in infants and young children, but no vaccine is currently available. CD4 and CD8 T cells are critical for mediating viral clearance but also contribute to immunopathology following an acute RSV infection. However, few RSV-derived CD4 and CD8 T cell epitopes in the commonly used C57BL/6 mouse strain have been described. I utilized an overlapping peptide library spanning the entire RSV proteome and intracellular cytokine staining for interferon-gamma (IFN-γ) to identify novel CD4 and CD8 T cell epitopes in C57BL/6 mice. I discovered and characterized two novel CD4 T cell epitopes and three novel CD8 T cell epitopes located within multiple RSV proteins. Overall, the novel RSV-derived CD4 and CD8 T cell epitopes identified in C57BL/6 mice will aid in future studies of RSV-specific T cell responses. While CD8 T cells are important for viral clearance following an acute RSV infection, the contribution of memory CD8 T cells in providing protection against reinfection with RSV remains unclear. I used a prime-boost immunization approach to induce robust, systemic memory CD8 T cell responses in the absence of RSV-specific CD4 T cells and antibodies. I determined that high magnitude, systemic memory CD8 T cell responses efficiently reduced lung viral titers following RSV infection, but unexpectedly did so at the expense of severe and fatal immunopathology. The exacerbated disease was mediated by the rapid and excessive production of IFN-γ by memory CD8 T cells in the lung and airways. In contrast, I found that local immunization generated a large population of tissue-resident memory CD8 T cells in the lung that efficiently reduced lung viral titers in the absence of exacerbated disease. Additionally, I observed that pre-existing RSV-specific neutralizing antibodies prevented the immunopathology induced by high magnitude, systemic memory CD8 T cell responses following RSV infection. Prophylactic treatment with neutralizing antibodies against RSV efficiently restricted early virus replication, which resulted in a significant decrease in lung IFN-γ levels, memory CD8 T cell activation, and the frequency of IFN-γ producing CD8 T cells. Thus, my results demonstrate that high magnitude, systemic memory CD8 T cells induce lethal immunopathology following RSV infection, which can be prevented by pre-existing RSV-specific neutralizing antibodies. Overall, my results have important implications for the development of future RSV vaccines. The development of a live-attenuated vaccine for RSV has been prevented by the inability to properly balance attenuation with immunogenicity and efficacy. Recently, a recombinant RSV strain lacking the gene that encodes the matrix (M) protein (RSV M-null) was developed. As the M protein is required for virion assembly following infection of a host cell, RSV M-null induces a single-cycle infection. I evaluated RSV M-null as a potential live-attenuated vaccine candidate by determining its pathogenicity, immunogenicity, and protective capacity in BALB/c mice compared to its recombinant wild-type control virus (RSV recWT). RSV M-null was sufficiently attenuated, as significantly reduced lung viral titers, weight loss, and pulmonary dysfunction were observed compared to mice infected with RSV recWT. Surprisingly, despite its attenuation, I found that RSV M-null infection induced effector T cell, germinal center B cell, serum antibody, and memory T cell responses of similar magnitude to that elicited by infection with RSV recWT. Importantly, RSV M-null immunization provided protection against secondary viral challenge by reducing lung viral titers as efficiently as immunization with RSV recWT. Overall, my results indicate that RSV M-null combines attenuation with high immunogenicity and efficacy and represents a promising novel live-attenuated RSV vaccine candidate.

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis-induced hospitalization in young children. A natural RSV infection fails to elicit long-lasting immunity, further increasing the need for an effective vaccine. Despite the significant healthcare burden, there is no licensed RSV vaccine currently available. While most RSV vaccine strategies focus on the induction of humoral immunity, high antibody titers do not prevent RSV infection. It remains unclear if protective immunity can be achieved through robust cellular immunity. Previous work has indicated that a relatively low frequency of virus-specific CD8 T cells is induced following an RSV infection in human infants. In addition, RSV-specific memory CD8 T cells diminish to almost undetectable frequencies in the blood of the elderly. The lack of long-lasting immunity against RSV may be explained by an absence or low frequency of memory CD8 T cells within the lung following infection. However, I determined that the majority of effector CD8 T cells reside within the lung tissue following infection with either RSV or influenza A virus (IAV), both of which replicate primarily in the airways. In addition, approximately 70% of antigen-experienced memory CD8 T cells persist in the lung tissue at day 30 following RSV infection. In contrast, the majority of CD8 T cells remain in the pulmonary vasculature following intranasal infection with either of the systemically replicating viruses lymphocytic choriomeningitis virus or vaccinia virus. Therefore, the tissue tropism of a virus will determine if CD8 T cells preferentially accumulate in the lung tissue following infection of the respiratory tract. An experimental formalin-inactivated RSV (FI-RSV) vaccine caused enhanced respiratory disease in vaccinated children following a natural RSV infection. Incomplete knowledge of the underlying immunological mechanisms that were responsible for mediating the enhanced disease has greatly hampered vaccine development. Previous studies have indicated that eosinophils, non-neutralizing antibodies, and CD4 T cells may be required to elicit FI-RSV vaccine-enhanced disease. I determined that distinct CD4 T cell subsets mediate individual disease parameters. The Th2-biased immune response, but not eosinophils specifically, was responsible for induction of airway hyperresponsiveness and mucus hypersecretion. On the other hand, the Th1-associated pro-inflammatory cytokine TNF-α was required to mediate baseline pulmonary dysfunction and weight loss. Lastly, while depletion of CD4 T cells ameliorated all disease parameters evaluated, the antibody titers remained unaltered in depleted mice. Thus, antibodies induced by FI-RSV immunization were not required for vaccine-enhanced disease. My data demonstrate that discrete disease manifestations associated with FI-RSV immunization are orchestrated by distinct subsets of CD4 T cells. The CD8 T cell response is believed to contribute to both pathogen clearance and immunopathology following an acute RSV infection. However, it is unclear if robust memory CD8 T cell responses will protect against an RSV infection. I determined that induction of a high-magnitude, epitope-specific memory CD8 T cell pool mediated increased viral clearance following RSV challenge. However, mice with robust secondary CD8 T cell responses exhibit increased airway dysfunction, weight loss, and mortality as compared to mock-immunized mice undergoing an acute RSV infection. The enhanced disease severity was unique to the context of an RSV infection as similarly immunized mice were protected from chge with a lethal dose of a recombinant IAV engineered to express an RSV-derived epitope. In addition, the increased morbidity and mortality was associated with an elevated amount of both IFN-γ and TNF-α in the serum of immunized mice. Neutralization of either IFN-γ or TNF-α led to a significant reduction in disease severity and survival of all mice. These results demonstrate that robust memory CD8 T cell responses enhance viral clearance, but also lead to severe pulmonary immunopathology following RSV infection. Overall, I establish that the majority of effector CD8 T cells are localized within the lung tissue following a respiratory infection, and determine that either memory CD4 or CD8 T cell responses elicits severe immunopathology following a RSV infection.

Thesis (M. Med. Sc.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 42-47).

Respiratory syncytial virus (RSV) infection causes ~64 million cases of respiratory disease and 200,000 deaths annually worldwide, yet there is no broadly effective prophylactic or treatment regimen. RSV can produce acute respiratory illness in patients of all ages but strikes the age extremes, infants and the elderly, with highest frequency presumably due to innate immune deficiencies. A higher morbidity and mortality has been reported for the elderly above 65 years of age, which has been attributed to immune senescence. Efforts to generate an effective vaccine have thus far been unsuccessful. The innate immune system provides the first line of defense against viral pathogens with a repertoire of anatomical barriers, phagocytic immune cells, pattern recognition receptors (PRRs) and antiviral cytokines like interferons (IFNs). The precise mechanism of subversion of innate immunity in young and aged is poorly understood. A better understanding of innate immune pathways is expected to aid in the development of appropriate vaccines or prophylactics for these high-risk groups. Previously, the RSV nonstructural protein 1 (NS1) was shown to antagonize IFN responses by disrupting components of the innate immune system, although the mechanism is not well defined. We hypothesized that NS1 targets constituents of the PRR pathways to evade innate immunity and thus ensure viral survival. Using microscopy and co-immunoprecipitation assays, we found that NS1 localizes to the mitochondria and binds to the mitochondrially associated adaptor protein MAVS, thus preventing MAVS interaction with the RNA helicase, RIG-I. Expression of NS1 was also correlated with upstream IFN-response regulator, LGP2, and its expression was inducible in the absence of a viral infection. Tetracycline-inducible expression of recombinant NS1 in a cell model also promoted viral replication and emphasizes the key contribution of NS1 to RSV survival. Through this study, we demonstrated a mechanism for RSV NS1 in the disruption of early innate responses through mitochondrial localization and alteration of the RLH signaling. Whereas the above studies showed the importance RSV-induced innate immune pathways, whether the expression and signaling of innate immune pathways were adversely affected upon RSV infection in the high-risk groups remains unknown. Since elderly individuals are at an increased risk for severe bronchiolitis and RSV-induced pneumonia, often resulting in hospitalization and medical intervention and adaptive immune cell functionality and responsiveness reportedly decline with age, we hypothesized a similar age-related deterioration of the innate antiviral system. In this investigation, we used an aged mouse model to correlate age-associated changes in innate immune gene expression with RSV pathology. Of 84 antiviral genes examined, five genes including RIG-I, IFNAR1, TLR8, IL-1Β, and osteopontin (OPN) were associated with both age and infection. In response to RSV infection, aged mice had delayed induction of antiviral genes and diminished ability to secrete IL-6 in response to TLR7/8 agonist in primary alveolar macrophages. Lungs from aged, RSV-infected mice had increased cellular infiltration and prolonged infection as compared to young mice. In summary, age-related decline in expression and functionality of antiviral defenses were correlated with enhance RSV-induced lung disease in aged mice. In the absence of infection, aged mice chronically overproduced IL-1Β and OPN relative to young mice. Upon infection, aged mice had impaired ability to secrete higher levels of IL-1Β and mucus. In contrast, OPN secretion remained high and prolonged in aged mice throughout infection. The age-related decline in host antiviral gene induction and delayed cytokine production correlated with enhanced disease pathology. Using a transgenic strain of mice deficient in OPN (OPN-KO), we observed greater resistance to RSV and enhanced secretion of mucus, but unaltered cellular infiltration into the lungs. Therefore, OPN overproduction and defective mucus production likely contribute to pathology in aged mice. These findings demonstrate that RSV targets the innate virus recognition and antiviral cytokine activation pathways but also that the antiviral defense system is significantly affected by age. Consequently, efforts to generate vaccines or develop therapies that stimulate IFN induction may prove unsuccessful in the elderly given that RSV virulence factors and age weaken these responses. This study contributes to our understanding of how aging relates to the RSV subversion of the host antiviral response and should help with the development of better antiviral therapies suited to the growing elderly population.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

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