Cold-adapted (ca) live attenuated influenza vaccines (LAIVs) have been introduced as alternatives to existing inactivated influenza vaccines. The influenza A components of the FDA-approved ca LAIVs (Flumist®; Medimmune) have common internal genes derived from the donor strain A/Ann Arbor/6/60 ca and surface genes derived from current wild-type (wt) epidemic strains. The aim of this thesis was to investigate determinants of immunogenicity for reassortants of A/Ann Arbor/6/60 ca, using a range of immunological assays, including recently developed MHC tetramer techniques. From the study, the extent of viral replication in the respiratory tract of mice, the primary site of inoculation, was a key factor in determining ca vaccine immunogenicity. Replication was shown to be influenced by both viral surface Ags and the host MHC. The H3 ca reassortants CR6, CR18, CR29 and CR6-35* exhibited greater replication efficiency (as determined by their PFU:HAU ratios) than the H1 ca reassortants CR35 and CR6-35. The H3 ca reassortant CR6 caused a 3.79% loss in body weight but no losses were observed for the H1 ca reassortant CR35 and the ca H2N2 donor strain A/Ann Arbor/6/60 ca. Higher HI responses were detected after 3 weeks in groups infected with the H3 ca reassortant CR6 (GMT 80) than with the H1 reassortant CR35 (GMT 10) and the H2 ca donor strain A/Ann Arbor/6/60 ca (GMT 13). Recently developed techniques were used to evaluate specific T-cell response to ca LAIVs. Fluorescent-labelled tetramer is the key reagent for use in tetramer-based flow cytometry assays. The NP366-374 peptide of influenza A viruses comprises an immunodominant epitope that is highly conserved between subtypes. Tetramers developed for A/PR/8/34 (H1N1) were able to detect NP-specific cytotoxic T lymphocytes (CTLs) induced by A/Ann Arbor /6/60 ca (H2N2). An attempt to prepare the A/Ann Arbor/6/60 ca-specific-NP-tetramer is described. H-2Db monomers were successfully refolded with the peptide, but only 20% were able to form tetramers through biotin-streptavidin linkage, resulting in a poor capacity to stain. By contrast, an IFN-γ ICC assay developed in parallel demonstrated that peptide NP366-374 was able to restimulate A/Ann Arbor/6/60 NP ca-specific CTLs and secrete IFN-γ when tested in vitro. Specific-B and T cell responses induced in the lungs in response to infection by ca reassortants exhibited great variability that was determined by the growth characteristics of different viruses. Type I (CTL) responses were induced by low yielding ca reassortants, such as CR35 (H1N1). Viruses with enhanced growth characteristics, such as CR6 (H3N2), produced higher Type II (HA-specific Ab) responses. In addition, host factors, such as MHC type, were found to play an important role in responses to the same viruses. Susceptible mouse strains, such as C57BL/6, showed higher CTL but lower serum Ab responses than more resistant strains, such as BALB/c. Throughout this PhD project, a fine balance between the humoral and CMI, local and systemic immune responses induced by ca LAIVs was demonstrated. The need to assess local immune responses, in addition to serum antibody levels, for the evaluation of vaccine efficacy was an important conclusion of the thesis.

Made available in DSpace on 2016-03-10T13:20:27Z (GMT). No. of bitstreams: 2 paola_silva_ioc_dout_2015.pdf: 5954809 bytes, checksum: 02b143793e3dbd017a0d95c668d3108c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A primeira detecção do vírus Influenza A (H1N1)pdm09 no Brasil aconteceu em maio de 2009, e foi seguida de uma extensa disseminação por toda a população brasileira, com grande impacto em morbidade e mortalidade. Para entender a dinâmica molecular do Influenza A (H1N1)pdm09 no país, a presente tese reuniu sete trabalhos que abordaram a análise filogenética deste agente viral durante e após o período pandêmico (2009 a 2014) e buscou indentificar polimorfismos virais associados à virulência e à resistência ao antiviral Oseltamivir (OST). Para isso, as metodologias realizadas foram o sequenciamento dos genes de hemaglutinina (HA) e neuraminidase (NA) utilizando a metodologia de Sanger e a metodologia de pirosequenciamento para detectar polimorfismos de base única (SNPs). Nossos resultados revelaram a circulação de nove grupos filogenéticos ao longo dos cinco anos do estudo, indicando uma substituição temporal dos grupos e ocasionalmente uma estratificação geográfica. No entanto, nenhum dos grupos filogenéticos identificados foram associados com um pior prognóstico da infecção por influenza. Ao contrário do que foi observado em estudos anteriores, as mutações K-15E e Q310H no gene HA não se associaram ao aumento de virulência, mesmo na infecção de indivíduos imunocomprometidos. Por outro lado, polimorfismos no resíduo 222 da HA, que caracterizaram a presença de quasispecies virais, mostraram uma forte associação com a gravidade da infecção, especialmente em gestantes. Nesta tese, também realizamos a vigilância de marcadores de resistência no gene NA. Entre as amostras analisadas encontramos sete vírus com a mutação H275Y e dois com S247N, esses marcadores estão relacionados com a diminuição de sensibilidade ao antiviral OST Entre as amostras resistentes, a grande maioria foi detectada na região Sul do Brasil, em pacientes que não receberam OST. Isto sugere uma possível transmissão sustentada do vírus resistentes no país. Embora variantes H275Y resistentes apresentem baixa aptidão viral, a propagação deste vírus pode ocorrer com o ganho de mutações permissivas nos genes HA ou NA. No Brasil, o vírus que circulam desde 2011 apresentaram mutações V241I e N369K no gene NA, que foram, teoricamente, associadas a uma maior aptidão viral da variante resistente. Diante disso, cinco anos após a pandemia observamos que o vírus A (H1N1)pdm09 está estabelecido na população humana com várias substituições genéticas quando comparado com a sequencia da cepa vacinal A/California/07/2009. A maioria destas substituições parecem não ocasionar uma maior gravidade da infecção, e esta pode ser atribuída a outros fatores, tais como fatores genéticos do hospedeiro. Por fim, considerando o risco de surgimento e disseminação de vírus resistentes é importante reforçar o monitoramento viral e também realizar estudos para novas drogas antivirais / The Influenza A (H1N1)pdm09 virus was first detecte d in May 2009 in Brazil and later resulted in an extensive spread throughout the Brazilian pop ulation with a severe impact on morbidity and mortality. To understand the molecular dynamic of ( H1N1)pdm09 virus in Brazil this thesis grouped seven papers which approached the phylogenetic reco nstruction of the virus during and after the pandemic period (2009 to 2014) and the genomic iden tification of viral polymorphisms associated with virulence or antiviral resistance to Oseltamivir (O ST). For this, we performed genome sequencing, focusing especially on the hemagglutinin (HA) and n euraminidase (NA) genes using conventional Sanger sequencing and PyroMark 96ID to detect singl e nucleotide polymorphisms (SNPs). Our results showed that in Brazil nine (H1N1)pdm09 phylogenetic groups circulated along the five years of the study, indicating a temporal repl acement of groups and ocasionally a geographic stratification. However, no phylogenetic group seem ed to be associated with a worse clinical outcome. The increased virulence observed in previous studie s with a 2009 group bearing the genetic markers K-15E and Q310H was not confirmed in our analyses, even evaluating an immunocompromised population. On the other hand, polymorphysms at pos ition 222 of HA gene, which characterized the presence of viral quasispecies, showed an associati on with increased virulence in brazilian samples, especially in pregnant women. In this study we also performed surveillance of resistance markers at the NA gene. From the analysed samples we found sev en viruses with H275Y and two with S247N mutation, that diminish the sensibility to oseltami vir (OST). Among the resistant samples, the large majority was detected in the Southern region of Bra zil in patients that did not receive OST. This suggests a possible sustained transmission of resis tant virus in the country. Although resistant H275Y variants have low viral fitness, the spread of this virus can occur with the gain of permissive mutati ons in the HA or NA genes. In Brazil, viruses circulati ng since 2011 presented mutations V241I and N369K in the NA gene, which were theoretically associated with greater viral fitness. Five years after the pandemic we observed that A (H 1N1)pdm09 is established in the population with genetic substitutions in all gene s egments when compared to the vaccine strain A/California/07/2009. We demonstrated that the majo rity of those substitutions did not increase the severity of infection. The worst clinical outcomes may be attributed to other factors, such as genetic host factors. Finally, considering the risk of emer gence and spread of resistant viruses it is importa nt to strengthen viral surveillance and also carry out studies for new antiviral drugs

Influenza virus is a pleiomorphic human pathogen which causes self-limiting respiratory illness lasting one-two weeks in most individuals. However, in immunologically compromised individuals, influenza infection may lead to severe morbidity and fatality. Annual epidemics cause 250,000-500,000 deaths worldwide and remain a major public health threat. The virus has evolved mechanisms of antigenic ‘drift’ and ‘shift’ to evade the host immune response. Hence, current influenza vaccines need to be updated every few years. Moreover, the currently available inactivated/live attenuated vaccines entail virus culture in embryonated chicken eggs hindering rapid scale-up. The aforementioned limitations of the current vaccines has had debilitating effect when strain mismatch between vaccine formulation and influenza viruses circulating within the population has occurred in the past, despite intensive monitoring. Public health is further compromised when an unpredictable mixing event among influenza virus genomes leads to antigenic shift, facilitating a potential pandemic outbreak. These concerns have expedited efforts towards developing a ‘universal’ flu vaccine. Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. The precursor polypeptide, HA0, is assembled into a trimer along the secretory pathway and transported to the cell surface. Cleavage of HA0 generates the mature, disulfide linked HA1 and HA2 subunits. Mature HA has a globular head domain which mediates receptor binding and is primarily composed of the HA1 subunit while the stem domain predominantly comprises of the HA2 subunit. The HA stem is trapped in a metastable state and undergoes an extensive low-pH induced conformational rearrangement in the host-cell endosomes to adopt the virus-host membrane fusion competent state. The ‘antigenic sites’ on the immunodominant globular head of HA are subjected to heightened immune pressure resulting in escape variants, thereby limiting the breadth of head-directed neutralizing antibodies (nAbs). As opposed to the highly-variable head domain, the HA stem is conserved and targeted by several broadly neutralizing antibodies (bnAbs) with neutralizing activity against diverse influenza A virus subtypes. Although several bnAbs bind to the conserved HA stem, focusing the immune response to this conserved, subdominant stem domain in presence of the variable head domain of HA has been challenging. Alternatively, mimicking the epitope of these stem-directed bnAbs in the native, pre-fusion conformation in a ‘headless’ stem immunogen capable of eliciting a broadly protective immune response has been difficult because of the metastable conformation of HA. Addressing the aforementioned challenges, we describe the design and characterization of novel influenza immunogens by HA protein minimization. Chapter 1 gives an overview of the influenza virus life cycle, and outlines the structural organization and function of viral proteins. The conventional vaccines that are currently used and their limitations are described in this chapter. Recent improvements in influenza vaccine production focusing on recombinant HA as an alternate solution are discussed. Painstaking efforts of several groups in the recent past has led to the isolation of bnAbs that recognize novel ‘antigenic signatures’ within the globular head and the HA stem domains. Attempts to focus the immune response to these ‘cross-protective’ epitopes are described. The design and characterization of trimeric HA stem-fragment immunogens from influenza A Group-1 viruses which mimic the native, pre-fusion conformation of HA are described in Chapter 2. We engineered ‘headless’ HA stem immunogens based on influenza A/Puerto Rico/8/34 (H1N1) subtype. H1HA10-Foldon, a trimeric derivative of our parent construct (H1HA10), bound conformation sensitive stem-directed bnAbs such as CR6261, F10 and FI6v3 with high affinity (equilibrium dissociation constant [KD] of 10-50nM). The designed immunogens elicited broadly cross-reactive antiviral antibodies which neutralized highly drifted influenza virus strains belonging to both Group-1 (H1, H5 subtypes) and 2 (H3 subtype) in vitro. Significantly, stem immunogens designed from unmatched, highly drifted influenza strains conferred protection against a lethal (2LD90) heterologous A/Puerto Rico/8/34 virus challenge in mice. Our immunogens conferred robust subtype-specific and modest heterosubtypic protection in vivo. In contrast to previous HA stem domain immunogens, the designed immunogens described here were purified from the soluble fraction in E.coli. These HA stem-fragment immunogens do not aggregate even at high concentrations and are cysteine-free which eliminates the complications arising from incorrect disulfide-linked, misfolded conformations. The aforementioned properties of the HA stem-fragment immunogens make it amenable for scalability at short notice which is vital during pandemic outbreaks. The detailed mechanism(s) by which our ‘headless’ stem immunogens provide protection need further investigation. The long central α-helices (LAH) located in the HA stem assemble together into a parallel, trimeric coiled-coil. Immunization with the wt-LAH (76-130 of HA2) derived synthetic peptide designed from an H3 subtype (H3N2 A/Hong Kong/1/68) and conjugated to keyhole limpet hemocyanin (KLH) was shown previously to elicit antibodies reactive in ELISA with multiple hemagglutinin subtypes and to confer protection against challenge with H3N2, H1N1 and H5N1 virus strains. The LAH peptide sequence was chosen based on maximal binding to the monoclonal antibody (MAb), 12D1, which has broad neutralizing activity against influenza viruses of the H3 subtype. These results motivated us to rationally design stabilized derivatives of wt-LAH and test their protective capacity in a mouse challenge model of influenza. This work is described in Chapter 3. Additionally, to understand the contribution towards protection conferred by the two distinct surface exposed patches on LAH, we designed constructs spanning different stretches of LAH. The biophysical characterization of the LAH-derived constructs indicates that most of them were well-folded. All these constructs were moderately immunogenic in mice but at best, conferred limited protection from lethal viral challenge. In contrast to previously reported results, our data suggests that the LAH in the absence of other regions of HA may require not only strong, but also specific adjuvantation to induce a robust and functional immune response in vivo. Chapter 4 describes an immunogen design (H1pHA9) based on the globular head domain of pandemic H1N1 HA which can be produced using a prokaryotic expression system. The HA-fragment, H1pHA9, stably refolds to mimic the conformation sensitive neutralizing epitopes in the globular head domain of HA. We have also successfully engineered the HA head domain to delineate the epitope of antibodies neutralizing the pandemic H1N1 virus using a yeast cell-surface display platform. In this direction, we report the isolation of a novel, neutralizing murine MAb, MA2077, against the pandemic H1N1 virus. The epitope of this MAb has been mapped onto the ‘Sa’ antigenic site. The ability of the head domain fragment, H1pHA9, which binds MA2077 with high affinity to elicit such neutralizing antibodies in vivo needs to be further explored. Structural analysis has shown that elements of the HA stem diverge between the two phylogenetic groups. Therefore, to mitigate the threat of circulating influenza A viruses from these distinct structural classes (H1 and H3 belonging to Groups 1 and 2 respectively), in lieu of a ‘universal’ vaccine, a combination of immunogens derived from both the groups is a practical alternative. In Chapter 5 we describe the design of stem-fragment immunogens from an influenza A Group-2 virus strain. We report the characterization of engineered ‘headless’ HA stem immunogens based on influenza A/Hong Kong/1/68 (H3N2) subtype. The designed immunogens were expressed in E.coli and purified from the soluble fraction with abundant yields (~15mg/lt). The HA stem-fragment immunogens could be concentrated to high concentrations without aggregation. While, H3HA10-IZ and H3HA10-Foldon, the trimeric derivatives of our parent construct (H3HA10) which were folded, conferred modest protection against a lethal homologous virus challenge in mice, there is considerable scope to improve our immunogen design. Analyzing the results from our previous work (Chapter 2), we speculate that structural elements at the N-terminus of A-helix are critical for helix initiation. We therefore extended the design to include residues from the start of the A-helix. We designed the extended stem immunogens from both H3 and H7 subtypes. The proteins were purified from the soluble fraction of the E.coli cell culture lysate. Preliminary studies suggest that extension of the A-helix has aided proper folding. These proteins need to be further characterized and evaluated in an animal model.

Orientador: Maria Rita Donalísio Cordeiro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T16:23:15Z (GMT). No. of bitstreams: 1 Freitas_AndreRicardoRibasde_D.pdf: 4270845 bytes, checksum: 09078cb28a971b59a0103ccfbe6a3bee (MD5) Previous issue date: 2014 / Resumo: Introdução e objetivos: As infecções respiratórias estão entre as mais importantes causas de morbimortalidade no mundo. A sua alta incidência tem relevante impacto nos óbitos, como também na sobrecarga do sistema de saúde e absenteísmo no trabalho e escola Todas as faixas etárias são acometidas, porém, as mais afetadas são as crianças e os idosos. Também são particularmente susceptíveis os imunocomprometidos e os portadores de doenças crônicas em geral. Os vírus são os agentes responsáveis pela maior parte das infecções respiratórias, os principais vírus causadores de infecções respiratórias são o influenza A e B e o Vírus Sincicial Respiratório (VSR). Estes vírus têm comportamento biológicos distintos e o conhecimento de como estes vírus afetam a saúde da população é fundamental para embasar as ações de prevenção, profilaxia e tratamento de pacientes permitindo uma alocação adequada de recursos em quantidade e tempo adequados. No Brasil, no ano 2000, para monitorar a ocorrência destes vírus foi implantada a vigilância de síndromes gripais SIVEP-GRIPE, que através de 128 unidades sentinelas distribuídas em todas as regiões do país coletam semanalmente amostras de secreção de nasofaringe por semana de pacientes com síndromes gripais. Neste trabalho estudamos o impacto do influenza na mortalidade no estado de São Paulo, nas diferentes faixas etárias no período entre 2002 e 2011, incluindo o período da pandemia de 2009. Estudamos também a sazonalidade do VSR nas 5 diferentes regiões brasileiras e o impacto deste vírus nas internações por doenças respiratórias entre menores de 5 anos. Metodologia: Para o estudo da mortalidade associada ao influenza utilizamos o método de regressão de Serfling adaptado para dados semanais extraindo da série histórica os períodos de maior circulação viral a partir dos resultados do sistema de vigilância sentinela SIVEP-GRIPE. Comparar a mortalidade associada à pandemia de influenza de 2009, às epidemias prévias anuais de influenza nas diferentes faixas etárias e com diferentes subtipos de vírus influenza circulantes no estado de São Paulo. Para o estudo da sazonalidade do VSR utilizamos análise de Wavelets, análise de Fourier, análise simplificada de estações anuais comparando os resultados nas 5 regiões administrativas do Brasil. Para identificar possíveis correlações temporais entre a circulação dos vírus respiratórios utilizamos métodos de regressão de ranque de Spearman e de regressão parcial. Resultados e conclusões: A mortalidade por pneumonia e influenza associada à pandemia de 2009 no estado de São Paulo foi ligeiramente mais alta que nos outros anos de influenza sazonal, considerando a mortalidade geral, sem distinção de faixa etária. Houve diferenças no risco de morrer entre as faixas etárias. Entre os indivíduos de 5 a 19 anos, a mortalidade associada à pandemia de 2009 foi 2,6 maior (0,6 óbitos/100.000hab) que a de anos não pandêmicos. Na faixa etária de 20 a 59 anos, a mortalidade associada à pandemia de 2009 foi 5,1 maior (2,8 óbitos/100.000hab) que nos anos não pandêmicos. As taxas de mortalidade entre menores de 5 anos foi 0,9 óbitos/100.000hab e na população de mais 60 anos foi 13,1 óbitos/100.000hab, ou seja, foram menores que nos anos não pandêmicos. O método de análise utilizado permitiu a diferenciação entre a mortalidade associada a subtipos virais (A(H3N2), B ou sazonal A(H1N1) e A(H1N1) pdm 2009). Foi possível a comparação entre a mortalidade associada à pandemia de influenza de 2009 em São Paulo, às epidemias anuais de influenza nas diferentes faixas etárias e com diferentes subtipos de vírus influenza circulando. Isto é, o impacto da circulação do vírus pandêmico influenza A(H1N1) foi maior na mortalidade em adultos e jovens, enquanto em maiores de 65 anos foi discreto. Por outro lado, o excesso de mortalidade foi expressivo em maiores de 65 anos, nos anos de circulação do influenza A(H3N2). O modelo de Serfling adaptado a dados semanais com validação por meio de dados da vigilância sentinela de síndromes gripais (SIVEP-GRIPE) mostrou-se confiável para detectar picos de maior circulação viral do Influenza e supostos reflexos na mortalidade em diferentes faixas etárias em período pandêmico, epidêmico e de circulação sazonal do vírus Influenza. Sobre o VSR foi possível identificar padrões sazonais do VSR em todas as regiões administrativas do Brasil utilizando-se dados da vigilância de síndromes gripais (SIVEP-GRIPE). Houve diferenças entre os momentos de maior circulação do vírus em algumas das cinco regiões administrativas do Brasil. Os padrões sazonais de internação por doenças sabidamente relacionadas com o VSR [Pneumonia devida a vírus respiratório sincicial, Bronquite aguda devida a vírus sincicial respiratório, Bronquiolite aguda devida a vírus sincicial respiratório, Bronquiolite (aguda, não especificada),] foram semelhantes aos encontrados pela análise da circulação do VSR por meio de dados da vigilância de síndromes gripais (SIVEP-GRIPE). Houve correlação temporal entre a circulação do VSR e as taxas de internação por doenças do aparelho respiratório em geral (Capítulo-X da CID-10) entre menores de 5 anos, nas cinco regiões administrativas do Brasil. Houve correlação temporal entre as taxas de internação entre menores de 5 anos por doenças sabidamente relacionadas com o VSR [Pneumonia devida a vírus respiratório sincicial, Bronquite aguda devida a vírus sincicial respiratório, Bronquiolite aguda devida a vírus sincicial respiratório, Bronquiolite (aguda, não especificada),] e as taxas de internação por doenças respiratórias em geral nesta faixa etária nas cinco regiões administrativas do Brasil, indicando que este é o principal vírus associado às internações de crianças até 5 anos por doenças respiratórias. De acordo com as evidências encontradas neste estudo, os esquemas de profilaxia contra o VSR hoje utilizados precisam ser revistos e particularizados para cada região do país. Entre as ações a serem revistas estão a disponibilização do palivizumabe, bem como medidas de prevenção à circulação do VSR na comunidade / Abstract: Introduction and Objectives: Respiratory infections are amongst the most important causes of morbidity and mortality worldwide. Its high incidence has significant impact on deaths, but also burdens the health system and leads to absenteeism from work and school All age groups are affected, but the most affected are children and the elderly. Are also particularly susceptible immunocompromised and patients with chronic diseases in general. Viruses are the agents responsible for most respiratory infections, the main cause of respiratory virus infections are influenza A and B and Respiratory Syncytial Virus (RSV). These viruses have distinct biological behavior and knowledge of how these viruses affect people's health is fundamental to support the prevention, prophylaxis and treatment of patients allowing an adequate allocation of resources in quantity and adequate time. In Brazil, in 2000, to monitor the occurrence of these viruses was established surveillance of influenza-like syndromes SIVEP-FLU, which through 128 sentinel units distributed in all regions of the country collect weekly samples of nasopharyngeal secretions of patients per week with influenza-like illness. In this work we study the impact of influenza on mortality in the state of São Paulo , in different age groups between 2002 and 2011 , including the period of the 2009 pandemic. We also studied the seasonality of RSV in 5 different Brazilian regions and the impact of this virus in hospitalizations for respiratory diseases among children under 5 years. Methods: To study the mortality associated with influenza used the regression method of Serfling adapted for extracting weekly data of the time series periods of increased viral movement from the results of sentinel surveillance system SIVEP - FLU . Compare the mortality associated with pandemic 2009 influenza , annual epidemics of influenza prior at different ages and with different subtypes of influenza viruses circulating in the state of São Paulo . To study the seasonality of RSV , we use wavelet analysis , Fourier analysis , simplified analysis of annual seasons comparing the results in five administrative regions of Brazil . To identify possible temporal correlations between the circulation of respiratory viruses use regression methods of Spearman rank and partial regression. Results and Conclusions: The mortality from pneumonia and influenza associated with the 2009 pandemic in the state of São Paulo was slightly higher than in the other years of seasonal influenza, considering the overall mortality, irrespective of age. There were differences in the risk of dying between age groups. Among individuals 5-19 years, the mortality rate associated with the 2009 pandemic was 2.6 higher than that of non-pandemic years. (0.6 deaths /100,000 inhabitants) In the age group 20-59 years, the rate associated with the 2009 pandemic mortality was 5.1 higher than in non-pandemic years. (2.8 deaths /100,000 inhabitants). Mortality rates among children under five years was 0.9 deaths /100,000 inhabitants and in persons over 60 years was 13.1 deaths /100,000 inhabitants, ie were lower than in non- pandemic years . The method of analysis used allowed the differentiation between mortality associated with viral subtypes (A(H3N2), A(H1N1) and B or seasonal A(H1N1) pdm 2009) . It was possible to compare the mortality associated with the 2009 influenza pandemic in Sao Paulo, annual influenza epidemics in different ages and with different subtypes of influenza viruses circulating. That is, the impact of the circulation of influenza A(H1N1) pandemic virus was higher mortality in adults and children, while in adults over 65 years was low . On the other hand, the excess mortality was significant in adults over 65 years ago, in circulating influenza A H3N2. The Serfling model adapted to weekly data validation using data from sentinel surveillance of influenza-like illness (SIVEP - GRIPE) was reliable for detecting peaks of higher viral circulation of influenza and alleged impacts on mortality in different age groups in pandemic period , epidemic and seasonal circulation of influenza viruses . About RSV was possible to identify seasonal patterns of RSV in all administrative regions of Brazil using surveillance data of influenza syndromes (SIVEP -GRIPE). There were differences between the moments of greatest circulation of the virus in some of the five administrative regions of Brazil. Seasonal patterns of hospitalization for known diseases with RSV [ Pneumonia due to respiratory syncytial virus , acute bronchitis due to respiratory syncytial virus , acute bronchiolitis due to respiratory syncytial virus bronchiolitis ( acute , unspecified ) ] were similar to those found by analysis of the movement of data through RSV surveillance of influenza-like syndromes ( SIVEP - GRIPE) . There was a temporal correlation between the circulation of RSV and the rates of hospitalization for respiratory diseases in general (Chapter X of ICD- 10) among children under 5 diseases in the five administrative regions of Brazil . There was a temporal correlation between the rates of hospitalization among children under 5 years for known diseases with RSV [ Pneumonia due to respiratory syncytial virus , acute bronchitis due to respiratory syncytial virus , acute bronchiolitis due to respiratory syncytial virus bronchiolitis ( acute , unspecified ) ] and rates of hospitalization for respiratory diseases in general in this age group in the five administrative regions of Brazil , indicating that this is the main virus associated with hospitalizations of children under 5 years due to respiratory diseases . According to the evidence found in this study , the schemes of prophylaxis against RSV used today need to be reviewed and individualized for each region of the country . Among the actions to be reviewed are the availability of palivizumab , as well as measures to prevent the circulation of RSV in the community / Doutorado / Epidemiologia / Doutor em Saude Coletiva

Memoria para optar al Título Profesional de Médico Veterinario / El virus influenza A (FLUAV) ha sido ampliamente estudiado debido a su gran importancia en salud pública y animal. Una de las razones de esto es su gran variabilidad genética otorgada en gran medida por el proceso de reassorment o reordenamiento genético. Este estudio consistió en la determinación de la diversidad genética de los genes que componen el complejo polimerasa del virus, es decir, los genes que codifican para la polimerasa básica 2 (PB2), la polimerasa básica 1 (PB1) y la polimerasa ácida (PA) de virus aislados de planteles porcinos de producción intensiva en Chile y evidenciar procesos de reordenamientos genéticos independientes de dichos genes. Para esto, se analizaron 2824 muestras desde 33 planteles porcinos mediante diagnóstico viral por RT-PCR en tiempo real, aislamiento viral, amplificación de segmentos virales por RT-PCR multisegmento y secuenciación por Illumina. Se obtuvieron un total de 89 secuencias genómicas de PB2 y 92 tanto de PB1 como de PA. Luego se realizó un análisis filogenético con estas secuencias nucleotídicas, más secuencias de referencia mediante el método Maximum likelihood. Todas las secuencias para los tres genes estudiados pertenecen al linaje pandémico del 2009. Se constató además la presencia de reordenamientos de estos genes en aproximadamente un 35% de los casos. Estos resultados permitieron conocer sobre la diversidad genética de los genes que componen el complejo polimerasa de FLUAV en Chile y pesquisar fenómenos de reordenamientos de estos. Además, permitieron evidenciar posibles quiebres de bioseguridad en parte de las producciones porcinas intensivas en Chile, debido a las relaciones filogenéticas encontradas entre virus de diferentes planteles. Futuros estudios son necesarios para confirmar y entender estos hallazgos / The influenza A virus (FLUAV) has been extensively studied for the importance in public and animal health. The virus presented high genetic diversity, due to in part, by the reassortment events. The aim of this study it was to determinate the genetic diversity of the segments 1 (PB2), 2 (PB1) and 3 (PA) of FLUAV isolated from swine in intensive production farms in Chile and too evidence reassortment events in these genes. For this, 2824 samples were collected from 33 pig farms. Real time RT-PCR, viral isolation, and full genome sequencing were performed. A total of 89 genomic sequences of PB2 and 92 genomic sequences for PB1 and PA were obtained. Using phylogeny, we determinate that all segment belonged into the pandemic 09 cluster. The results suggest reassortment events of these genes in approximately 35% of the cases. This is the first study about the genetic diversity of the PB2, PB1 and PA, internal genes and to evidence reassortment events of them in swine population. In addition, the phylogeny suggests direct or indirect transmission between farms. Further studies are needed to better understand the viral dynamic of polymerase genes / FONDECYT 11170877 y ANILLO ACT 1408

No description available.

Problématique : Une évaluation économique, commandée par le ministère de la Santé et des Services sociaux du Québec, vise à évaluer le rendement de deux programmes d'immunisation, soient la vaccination primaire et la vaccination antigrippale. Un des volets de cette étude économique nécessitera l'utilisation d'un questionnaire auto-administré auprès de personnes vaccinées contre l'influenza plusieurs semaines après leur vaccination. Objectif : Vérifier la fiabilité et la validité d'un questionnaire auto-administré sur l'efficacité populationnelle et les coûts assumés lors de la vaccination par les adultes vaccinés contre l'influenza. Cet outil sera utilisé pour une évaluation économique selon une perspective sociétale du programme d'immunisation contre l'influenza chez les adultes. Méthode : Un test-retest a été réalisé en soumettant le questionnaire à un échantillon de convenance de 499 personnes âgées de [supérieur ou égal à]50 ans de la Montérégie. Un premier questionnaire a été administré dans les CLSC en novembre 2004 (test) durant le temps d'attente après l'injection du vaccin. Le deuxième questionnaire a été envoyé 10 semaines plus tard par la poste aux participants en janvier 2005 (retest). Les variables testées sont de nature qualitative (variables sociodémographiques, statut vaccinal, lieu de vaccination, moyen de transport, absence du travail) ou quantitative (date et durée de la vaccination, distance parcourue, revenu annuel, coût du transport, autres coûts). La fiabilité a été évaluée par la comparaison des réponses déclarées entre les deux questionnaires alors que la validité de certaines variables a été évaluée par la comparaison entre les réponses fournies par les participants et des sources de référence. Les principaux tests statistiques utilisés sont: l'accord observé (P[indice inférieur omicron]), le kappa de Cohen ([kappa]) et le coefficient de corrélation intra-classe (CCI). Résultats : Le taux de participation a été de 95%. Les variables sociodémographiques obtiennent un P[indice inférieur omicron] entre les deux questionnaires variant de 82% à 98% et un [kappa] de 0,556 à 0,936. Les autres variables qualitatives affichent un P[indice inférieur omicron] de 89% à 100% mais un [kappa] variant de -0,007 à 0,862. Quant aux variables quantitatives transformées en catégorie, P[indice inférieur omicron] varie de 45% à 100% et [kappa] de 0,031 à 1,000. La divergence entre les indices de concordance [Kappa] et P[indice inférieur omicron] est attribuable aux paradoxes de kappa. Les variables de temps, soient la date de vaccination et la durée de vaccination, présentent une faible fiabilité avec respectivement un P[indice inférieur omicron] de 57% et 49% et un CCI de 0,305 et 0,690. Un grand nombre de réponses manquantes (38%) est constaté pour la variable date de vaccination. La validité des variables sélectionnées (statut vaccinal, lieu et date de vaccination, taille de la ville et moyen de transport) est jugée relativement bonne avec un P[indice inférieur omicron] variant de 58% à presque 100%. Conclusion : À l'exception des variables de temps, le questionnaire est assez fiable. La validité des variables évaluées est bonne. Le taux de participation et l'effectif élevés assurent une puissance statistique adéquate pour les analyses. La participation volontaire des sujets peut être une source de biais de sélection. La fiabilité et la validité étant étroitement liées, elles sont défavorablement influencées par le biais de mémoire. Celui-ci peut être engendré par le délai de réponses entre les deux questionnaires. Malgré tout, ce questionnaire pourra être utilisé dans l'étude économique après quelques modifications.

INTRODUCTION: With the rise of non-traditional providers offering influenza vaccination, it is becoming more of an endeavor to obtain documentation on vaccination. Researchers are relying more on self-report of vaccination, but the accuracy of differing definitions of self-reported vaccination status in the context of inpatient vaccine effectiveness studies is not well understood. OBJECTIVES: The first objective of this study was the assess agreement among four different definitions of self-reported influenza vaccination compared against documented influenza vaccination in a group for whom documented vaccination is expected to be available. For the definition with best agreement, enrollee characteristics were examined to assess which were associated with agreement between definitions. The second objective was to assess the effect of ten different vaccination status definitions, including variations of self-report and documented vaccination, on vaccine effectiveness (VE). METHODS: We used data from the inpatient component of the US Flu VE Network study of VE in patients hospitalized with acute respiratory illness (ARI). Variations of self-reported vaccination status were defined and compared to documented vaccination status. Agreement was assessed using the kappa statistic of agreement and other measures of agreement. VE was measured using logistic regression controlling for certain patient demographics and illness characteristics. RESULTS: Self-report with date had the highest percent agreement and kappa statistic (79.5%; 57.7%, 95% CI: 50.1, 65.2). Those in the oldest age groups (18-49 and ≥65 years) and those influenza positive were more likely to have agreement between self-reported vaccination with date and documented vaccination status (p < 0.05 for both age group and case status). Estimated VE differed three-fold depending on definition of vaccination, with documented vaccination having the lowest VE (10%, 95% CI: -54, 48) and self-reported vaccination with date and location having the highest VE (37%, 95% CI: -10, 64). CONCLUSION: Defining vaccination status using self-reported vaccination with date provides the most accurate classification of vaccination status. Older adults and those with lab-confirmed influenza were more accurate in their self-report of influenza vaccination. Differing definitions have an impact on estimated VE, and understanding how VE is influenced by choice of vaccination status definition is important to examine and report in studies of influenza VE.

The influenza virus has a large impact on global health; however, it is difficult to formulate vaccines and influenza therapies that are effective against influenza. The influenza virus mutates rapidly, has the ability to emerge as novel strains with pandemic potential and can quickly become resistant to any given drug. Therefore, the generation of novel anti-influenza therapeutics that are effective against multiple strains would be highly beneficial. To date, the majority of anti-influenza research has focused on targeting specific components of the virus in order to interfere with its replication. However, it has been proposed that host proteins and signaling pathways may be essential components to viral replication and could also become novel anti-influenza drug targets. Therefore, this study utilized a large proteomic screen to identify host proteins that were up- and down-regulated in response to influenza infection. Collectively, these proteins clustered into five specific cell pathways and processes including interferon signaling, purine metabolism, cell death, ubiquitin-like signaling and mitochondrial oxidoreductases. Overall, this project identified potential novel anti-influenza targets in primary airway epithelial cells. / May 2015

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy