The spread of pandemic influenza A (2009 H1N1 influenza) virus resulted in a global influenza pandemic in 2009. During the early stages of the pandemic, population surveillance was crucial. However, officials around the world realized that many of our surveillance and reporting systems were not prepared to respond in a coordinated, integrated way, which made informed public health decision-making very difficult. More accurate estimates of the total number of hospitalized 2009 H1N1 influenza cases were required to calculate population-based 2009 H1N1 influenza-associated mortality, morbidity and hospitalization rates. For instance, how many people were hospitalized with 2009 H1N1 influenza in Massachusetts? Of these, how many were admitted to the ICU and how many died? Compared to seasonal influenza, were some race/ethnic and age groups affected more than others, and what types of characteristics led to more severe manifestations of 2009 H1N1 influenza among these groups in Massachusetts? To address the above questions, I proposed a retrospective cohort study using data from the Hospital Discharge Database (HDD), which contains data for all inpatients discharged from 76 acute care hospitals in Massachusetts, as well as Census information to provide a measure of socioeconomic status (SES). My specific aims are as follows: 1. Develop methods to identify influenza cases precisely and describe characteristics of those hospitalized with ILI in MA between April 26-Sept 30, 2009; 2. Conduct analyses to identify race/ethnicity-related trends in reference to 2009 H1N1 influenza-related hospitalizations; 3. Conduct analyses to identify age-related trends in reference to 2009 H1N1 influenza-related hospitalizations. First, I established influenza case selection criteria using hospital discharge data. I addressed limitations in the published methods on defining cases of influenza using administrative databases, and evaluated ICD-9 codes that correspond with common and relatively serious respiratory infections and influenza using a ‘maximum’ and ‘minimum’ approach. Results confirmed that 2009 H1N1 influenza affected a younger population, and disproportionately affected racial minorities in Massachusetts. There were also higher rates of ICU admission compared to seasonal influenza. I then presented epidemiological data indicating race/ethnic disparity among 2009 H1N1 influenza cases in Massachusetts. I found that Hispanics had significantly lower odds of 2009 H1N1 influenza-related ICU stay. SES gradients calculated using five-digit zip code information did not account for these differences. Within race/ethnic strata, Hispanics Finally, I presented epidemiological data indicating differences among 2009 H1N1 influenza cases by age group in Massachusetts. I calculated measures of Diagnostic Cost Group (DxCG) comorbidity for the study population to provide a comorbidity measure at baseline. Main results indicate that although comorbidity scores were similar between the 2009 H1N1 influenza and seasonal influenza groups, 2009 H1N1 influenza caused more severe disease in younger age groups. This is the first study to report population-based statewide outcomes in all acute care centers in MA. In this dissertation I address challenges surrounding influenza surveillance to create case selection criteria within an administrative database. Using my case selection criteria, I then provide data related to fatality and severity of 2009 H1N1 influenza in Massachusetts in reference to sociodemographic variables such as racial/ethnicity and age groups, and provide evidence for patient-level interventions to those hardest hit by influenza. These findings provide valuable information about using large administrative databases to describe pandemic influenza cases and guide resource allocation to reduce disparities in relation to pandemic influenza preparedness.

Influenza is a severe disease in humans and animals, causing upwards of 40,000 deaths every year in America alone. Influenza A virus (IAV) also causes periodic pandemics every 10 to 50 years, killing millions of people. Despite this, very few effective therapies are available. All strains of IAV are prone to developing resistance to antibodies due to the high mutation rate in the viral genome. Because of this mutation rate, a yearly vaccine must be generated before every flu season, and efficacy varies year to year. IAV has also mutated to escape several of the clinically-approved small molecule inhibitors. A therapeutic agent that targets a highly conserved region of the virus could bypass resistance and also be effective against multiple strains of IAV. IAV attachment is mediated by many individually weak hemagglutinin–sialic acid interactions that all together make a strong attachment to a host cell. Polymerized sialic acid analogs can recreate these interactions and block infection. However, they are not ideal therapeutics due to solubility issues and in vivo toxicity. We used liposomes as a novel means for delivery of the sialic acid-containing glycan, sialylneolacto-N-tetraose c (LSTc). LSTcbearing decoy liposomes form multivalent, polymer-like interactions with IAV. Decoy liposomes competitively bind IAV in hemagglutination inhibition assays and inhibit infection of target cells in a dose-dependent manner. LSTc decoy liposomes co-localize with IAV, while control liposomes do not. Inhibition is specific, as inhibition of Sendai virus and respiratory syncytial virus is not observed. In contrast, monovalent LSTc does not bind IAV or inhibit infectivity. LSTc decoy liposomes prevent the spread of IAV during multiple rounds of replication in vitro and extend survival of mice challenged with a lethal dose of virus. Considering the conservation of the hemagglutinin binding pocket and the ability of decoy liposomes to form high-avidity interactions with IAV hemagglutinin, our decoy liposomes have potential as a new therapeutic agent against emerging strains.

Orientador: Hélio José Montassier / Banca: Manoel Victor Franco Lemos / Banca: Ricardo Luiz Moro de Souza / Resumo: A nucleoproteína (NP) do Vírus da Influenza Aviária (VIA) é um importante alvo antigênico no imunodiagnóstico desta doença, devido à sua baixa variabilidade entre as diferentes estirpes do VIA, resultando em uma elevada reatividade cruzada, e por ser também uma proteína altamente imunogênica para hospedeiros vertebrados. Neste estudo, o gene codificador da NP do VIA foi parcialmente clonado e expresso em Escherichia coli como uma proteína recombinante fusionada ao polipeptídeo SUMO e uma etiqueta de poli-histidina para seu uso no desenvolvimento de um ensaio de ELISA indireto para a detecção de anticorpos específicos contra o VIA. A NP recombinante foi expressada na fração solúvel e foi mais facilmente purificada. Após análise em relação aos seus principais sítios de antigenicidade e caracterização por meio de Western blotting, a NP recombinante foi utilizada como uma preparação antigênica no ELISA indireto para detecção de anticorpos contra o VIA presentes em amostras de soro de galinha. A análise comparativa do teste desenvolvido no presente estudo com um ELISA comercial apresentou valores de 95%, 97% e 96,7% de sensibilidade, especificidade e acurácia, respectivamente e um índice κappa de 0,88. Os resultados permitem concluir que a NP recombinante do VIA desenvolvida neste estudo possui características favoráveis para ser aplicada como antígeno no ELISA indireto, constituindo-se em um método sensível e específico para o imunodiagnóstico da Influenza Aviária em galinhas / Abstract: The nucleoprotein (NP) of Avian Influenza Virus (AIV) is an important antigenic target for immunodiagnosis of this disease, due to its low variability among different AIV strains, resulting in high cross-reactivity, and the also highly immunogenic for vertebrate hosts. In this study, the gene enconding NP of AIV was cloned and expressed in Escherichia coli as a recombinant protein fused to SUMO polypeptide with a polyhistidine tag and used to develop an indirect ELISA for the detection of AIV-specific antibodies. The recombinant NP was expressed in the soluble fraction and easily purified. After Analysis of the main sites of antigenicity and characterization in Western-Blotting, the recombinant NP was optimized as an antigen preparation for indirect ELISA to detect anti-AIV antibodies in chicken serum samples. The comparative analysis of this ELISA with a commercial ELISA showed values of 95%, 97%, 96.7% of sensitivity, specificity and accuracy, respectively, and an agreement of k=0.88. In conclusion, the results indicated that the recombinant NP of AIV produced in this study is a good source of antigen for indirect ELISA and provides a sensitive and specific method for the immunodiagnosis of Avian Influenza in chickens / Mestre

Le virus Influenza pandémique de 2009 (p2009/H1N1) a été remarquable de par l’inflammation incontrôlée qu’il produisait et la production de cytokines dérégulée qui a été observée chez certains individus jeunes et en santé. Nous avons évalué l’impact de ce virus pandémique sur la réponse immunitaire en la comparant à celle obtenue avec un virus conventionnel (PR8/H1N1) pour tester l’hypothèse selon laquelle l’effet de l’inflammation exagérée sur la réponse cellulaire cause la morbidité et la mortalité observées chez les individus infectés avec le virus Influenza pandémique de 2009. Le virus p2009 a d’abord été adapté par 8 passages successifs dans la souris. La réponse antivirale, la libération de cytokines inflammatoires ainsi que les réponses immunitaires cellulaires innée, adaptative et mémoire ont été comparés entre ce virus adapté (MAp2009) et PR8. À charge virale initiale égale, il a été déterminé qu’avec MAp2009, la morbidité et la mortalité sont accrues et que certaines cytokines (IFN-β, TNF-α, IL-6) sont exprimées plus fortement à jour 2 post-infection (p.i.) Les titres viraux pulmonaires étaient aussi 100 fois plus élevés et persistaient 2 jours de plus. Une infiltration cellulaire précoce, dès le 2e jour p.i. et soutenue, jusqu’à 10 jours p.i. ainsi que des dommages pulmonaires exacerbés ont été observés avec ce même virus. Environ 20 fois plus de cellules NK ont été observées au site de l’infection chez les souris infectées avec le virus pandémique à jour 7 p.i., ces mêmes cellules qui sont connues pour contribuer grandement à l’immunopathologie dans les poumons. Trois fois moins de lymphocytes T CD8[indice supérieur +] activés et spécifiques au virus ont été retrouvés dans les poumons des souris infectées avec MAp2009 comparativement au virus conventionnel à jour 8 p.i. De plus, les cellules dendritiques inflammatoires (iDCs) étaient triplées dans les poumons lors de l’infection pandémique à jour 3 p.i. Aussi, il semblait y avoir un débalancement dans le recrutement des DCs tissulaires au ganglion du médiastin (MLN) ce qui peut contribuer à une activation moins efficace des lymphocytes T. Ainsi, la trop forte réponse innée et l’inflammation exagérée compromettent la survie en induisant de graves dommages pulmonaires. De plus, les DCs peuvent suractiver les lymphocytes T CD8[indice supérieur +] qui déjà, sont produits en moins grand nombre et deviennent anergiques avant d’avoir détruit les cellules infectées par le virus.

The segmented negative-sense vRNA genome of influenza A virus is replicated via a complementary RNA (cRNA) intermediate by the viral RNA-dependent RNA polymerase (RdRP). The replicative intermediate has been thought to exist as a complementary ribonucleoprotein (cRNP) complex. Development of an RNA-based affinity-purification strategy for the isolation of viral ribonucleoprotein complexes enabled the structural and functional characterisation of the previously uncharacterised cRNP complex. The cRNP exhibits a filamentous double-helical organisation with defined termini, containing the viral RdRP at one end and a loop structure at the other end. In vitro characterisation of cRNP activity led to a proposal of a model of vRNA synthesis that relies on a trans-activating RdRP. The viral RdRP is an important host range determinant indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Affinity-purification followed by mass spectrometry was performed to identify cellular proteins that interact with the viral RdRP. 171 cellular proteins were found to co-purify with the viral RdRP, the most abundant of which were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, mitochondrial and ribosomal proteins. Among the phosphatases, three subunits of the cellular serine/threonine-protein phosphatase 6 (PP6) were identified. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and siRNA-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. Taken together, these results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP.

Chaque année, la grippe provoque des centaines de milliers de décès dans le monde. Dans le cas d’infections sévères, il a été démontré que la génération excessive de molécules inflammatoires telles que les cytokines et les chimiokines, la sécrétion d’espèces réactives dérivées de l'oxygène ainsi que l’afflux massif de cellules immunitaires innées et adaptatives dans les voies respiratoires contribuent à la génération de dommages pulmonaires aigus et contribuent à l'immunopathologie reliée à l’infection. Tenant compte de ce fait, le défi actuel dans le traitement de la grippe est de contrôler la réponse inflammatoire tout en inhibant la réplication virale afin de permettre à l'organisme de se défendre contre les infections sévères à l'influenza. Des études récentes ont montré que l’activation du récepteur nucléaire PPARγ par ses ligands, tel que la 15d-PGJ[indice inférieur 2], diminuait l’inflammation pulmonaire et améliorait la survie des souris infectées avec des doses létales du virus influenza. Mis à part ses effets sur PPARγ, le ligand 15d-PGJ[indice inférieur 2] est aussi connu pour activer le facteur nucléaire antioxydant Nrf2. Il a été montré que Nrf2 réduit la réplication du virus influenza. Cependant, son mode d'action dans cette fonction nécessite une clarification. De manière intéressante, une étude a montré que Nrf2 réduit l’inflammation pulmonaire en régulant l’expression de PPARγ et ceci dans un modèle murin du syndrome de détresse respiratoire aigu. Les résultats de ces études précédentes mènent à l’hypothèse que les voies de PPARγ et Nrf2 interagissent fonctionnellement et qu'elles sont impliquées dans la réduction de l’inflammation induite lors d'infections sévères causées par l'influenza. L’objectif général de cette étude est donc de mieux comprendre les mécanismes protecteurs de PPARγ et Nrf2 dans la régulation de l’inflammation et la réplication virale suite à une infection par le virus influenza. Nos résultats ont démontré premièrement que le fait de cibler les deux voies moléculaires PPARγ et Nrf2, permet une inhibition significative de l’inflammation et de la morbidité liée à l’infection. Dans un deuxième temps, nos résultats dévoilent le mécanisme antiviral de Nrf2 et démontrent que l’activation de cette voie réduit la réplication du virus influenza d’une façon dépendante de l’expression de l’antiprotéase SLPI.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Pathology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

published_or_final_version / Community Medicine / Master / Master of Public Health