Thesis (M.S.)--Brigham Young University. Dept. of Microbiology and Molecular Biology, 2005. / Includes bibliographical references.

Dissertation (D.P.H.)--University of Michigan.

No description available.

INTRODUCCIÓ L’abril de 2009 s’identifica un nou virus de la grip, l’A (H1N1) pdm09, en humans. El juny del mateix any, l’Organització Mundial de la Salut declara l’estat de pandèmia a nivell mundial. La malaltia pel nou virus va afectar centenars de nens al nostre medi durant la temporada 2009-2010. OBJECTIUS - Descriure l'espectre de malaltia per grip A (H1N1) pdm09 moderat i greu (aquells casos que requeriren ingrés en un hospital pediàtric de tercer nivell) en la població pediàtrica en el nostre medi. - Descriure la malaltia per grip A (H1N1) pdm09 en pacients pediàtrics en tractament per leucèmia limfàtica aguda, tant els ingressats com els que es va optar per tractar i seguir ambulatòriament. - Descriure els valors de càrrega viral de grip A (H1N1) pdm09 al moment del diagnòstic en relació amb variables epidemiològiques i clíniques en els pacients ingressats amb clínica respiratòria. PACIENTS I MÈTODES - Es dissenyen tres estudis amb recollida prospectiva de dades epidemiològiques, clíniques, analítiques i microbiològiques de nens amb malaltia confirmada amb detecció del material genètic del virus de la grip A (H1N1) pdm09 en aspirat nasofaringi. Els tres estudis es realitzen en un hospital pediàtric de tercer nivell (Hospital Sant Joan de Déu, Universitat de Barcelona) i els resultats es presenten en la memòria d’aquesta tesi. La recollida de dades es porta a terme durant la temporada pandèmica 2009-2010. S'efectuen els procediments estadístics pertinents per al tractament de dades. RESULTATS - El perfil del nen ingressat amb malaltia per grip A (H1N1) pdm09 és el d'un nen prèviament sa preescolar o bé el d'un nen d'edat escolar amb malaltia de base. La dificultat respiratòria i la hipoxèmia són el motiu principal d'ingrés, encara que també s'observen manifestacions extrapulmonars (neurològiques i cardíaques principalment). Les malalties cròniques pulmonars i neurològiques són els grups més importants de pacients que tenen malaltia de base i que requereixen ingrés. D'entre ells, els pacients amb malalties neurològiques suposen el principal grup de malalties cròniques d'entre els que requereixen ingrés a la Unitat de Cures Intensives Pediàtriques (UCIP). En els pacients que requereixen ingrés en UCIP trobem un major temps d'evolució de la malaltia abans d'iniciar el tractament amb oseltamivir i aquest retard en l'inici del tractament antiviral es relaciona amb una major risc d'ingrés en UCIP en el model multivariant. - Els nens amb leucèmia limfàtica aguda en fases de tractament més intensiu presenten una malaltia per grip més greu (broncopneumònia). Els nens en tractament de manteniment no presenten cap complicació amb tractament amb oseltamivir. - Els valors de càrrega viral al diagnòstic es correlacionen negativament amb el temps de durada de la clínica en el moment de fer la recollida de la mostra. Tenir una càrrega viral alta havent passat 5 o més dies des de l'inici de la clínica es relaciona amb un major risc de malaltia greu per grip A (H1N1) pdm09 (necessitat de tractament amb ventilació mecànica invasiva o no invasiva). / “INFLUENZA A(H1N1)PDM09: MODERATE AND SEVERE DISEASE IN THE PEDIATRIC PATIENT. VIRAL LOAD AT DIAGNOSIS AS A BIOMARKER OF SEVERITY.” TEXT: INTRODUCTION A new influenza virus was identified in April 2009 in humans. The influenza A (H1N1) pdm09 disease affected hundreds of children in our country during the pandemic season (2009-2010). OBJECTIVES - To describe the moderate and severe influenza A (H1N1) pdm09 disease (cases requiring for admission in a tertiary pediatric hospital) in children of our setting. - To describe the influenza A (H1N1) pdm09 disease in pediatric patients with acute lymphatic leukemia. - To describe the influenza A (H1N1) pdm09 viral load values at diagnosis in hospitalized children with respiratory symptoms and their relations with epidemiological and clinical variables. PATIENTS AND METHODS - Three different studies were designed and their results are presented. The studies were performed in a tertiary pediatric hospital (Hospital Sant Joan de Déu, University of Barcelona). Data collection was carried out during the pandemic season (2009-2010) in children with confirmed infection with a real-time RT-PCR. RESULTS - A previously healthy infant or a school-aged patient with underlying disease was the profile of the hospitalized child with influenza A (H1N1) pdm09 infection. Respiratory distress and hypoxemia were the main reasons for admission, although extrapulmonary manifestations were also observed (mainly neurological and cardiac). Children with chronic pulmonary diseases or with neurological disorders were the most important groups of patients with an underlying disease of those who required hospitalization. Patients with neurological chronic diseases more often required admission to the Pediatric Intensive Care Unit (PICU). Delays in starting treatment with oseltamivir were associated with an increased risk of admission to PICU in a multivariate model. - Children with acute lymphatic leukemia in intensive treatment phases developed a more severe influenza disease. Children in maintenance treatment phase had not complications. All of them were treated with oseltamivir. - The values of viral load at diagnosis were correlated negatively with the duration of the symptoms at the moment of sampling. To have a high viral load after 5 or more days of the onset of clinical symptoms was associated with an increased risk of severe illness (requiring for mechanical ventilation) due to influenza A (H1N1) pdm09 infection.

Orientador: Maria Rita Donalisio Cordeiro / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-07T11:53:53Z (GMT). No. of bitstreams: 1 Francisco_PriscilaMariaStolsesBergamo_D.pdf: 1732128 bytes, checksum: e40308a72ae69b30999c757a5f111ac4 (MD5) Previous issue date: 2006 / Resumo: O aumento da proporção de idosos no país associado à maior longevidade modificou o perfil de saúde da população com considerável aumento da demanda por prevenção e assistência à saúde nas faixas etárias mais avançadas. Neste contexto, destaca-se a importância relativa das doenças respiratórias nos idosos, com impacto econômico e social. O objetivo deste estudo foi investigar os fatores associados à vacinação contra influenza e doença pulmonar em idosos, com a utilização de diferentes técnicas de análise em estudos transversais de delineamento complexo. Os dados foram obtidos do "Inquérito de saúde de base populacional em municípios do Estado de São Paulo" (ISA-SP), cuja coleta foi realizada entre 2001 e 2002, e se referem a 1.958 registros de indivíduos de 60 anos e mais, analisados por meio de regressão logística e regressão de Poisson. Os resultados desta tese são apresentados em capítulos que se referem a três artigos publicados em revistas científicas. No primeiro artigo, "Fatores associados à vacinação contra influenza em idosos", os achados apontaram maior adesão à vacinação contra influenza entre os indivíduos com idade igualou superior a 70 anos (OR = 1,47; IC 95%: 1,09 -:- 1,99) e entre os hipertensos (OR = 1,39; IC 95%: 1,03 - 1,87), enquanto os idosos com nove anos ou mais de estudo referiram menor adesão à vacinação (OR = 0,64; IC 95%: 0,41 - 0,98). No segundo trabalho, "Vacinação contra influenza em idosos por área de residência: prevalência e fatores associados", pôde-se verificar diferenças no perfil do idoso quanto à referência de vacinação segundo subgrupos específicos e locais de residência. Nos municípios mais populosos apenas a menor escolaridade esteve associada à vacinação referida (RP = 1,26; IC 95%: 1,02-1,54), já na área composta pelos municípios menos populosos, idade mais avançada (RP = 1,15; IC 95%: 1,02-1,31), hipertensão arterial (RP = 1,21; IC 95%: 1,02-1,45), diabetes (RP = 1,16; IC 95%: 1,01-1,33) e doença crônica de pulmão (RP = 1,30; IC 95%: 1,03-1,64) referidas, estiveram associadas. No estudo, "Fatores associados à doença pulmonar em idosos", os resultados indicaram associação independente entre doença pulmonar referida e tabagismo (RP = 2,03; IC 95%: 1,39 - 2,97), uso de medicamentos (RP = 2,05; IC 95%: 1,11 - 3,79), auto-avaliação do estado de saúde atual como ruim ou muito ruim (RP = 1,89; IC 95%: 1,20 - 2,96) e depressão, ansiedade ou problemas emocionais (RP = 1,86; IC 95%: 1,11 - 3,10). Com o envelhecimento e a crescente importância das doenças respiratórias entre os idosos, os achados desse estudo apontam para a necessidade de ações que priorizem a manutenção da saúde do idoso, por meio de programas de promoção e prevenção que considerem as especificidades desse segmento / Abstract: The increase in the proportion of elder1y persons in Brazil, associated to increased longevity, has modified the health profile of the population, with a considerable increase in the demand for prevention and healthcare among the older age groups. In this context, the relative importance of respiratory diseases among the elder1y, along with its social and economic impact, is worthy of note. The aim of the present study was to investigate the factors associated with influenza vaccination and pulmonary disease in the elder1y population, using different analytical techniques in cross-sectional studies with complex design.The data were obtained from the Population-based survey of municipalities of the State of Sao Paulo (ISA-SP), whose data collection took place between 2001 and 2002 and inc1uded 1.958 records of subjects aged 60 years or older, and were analyzed using logistic regression and Poisson regression.The results of the present thesis are presented as chapters that refer to artic1es published in scientific journals. In the first article, "Factors associated with influenza vaccination among elderly persons", our findings indicate greater adherence to influenza vaccination among subjects aged 70 years or older (OR = 1.47; 95%CI: 1.09 - 1.99) and among subjects with hypertension (OR = 1.39; 95%CI: 1.03 - 1.87), whereas elder1y subjects with nine or more years of schooling reported lesser adherence to vaccination (OR = 0.64; 95%CI: 0.41- 0.98). In the second paper, "Influenza vaccination among elderly persons according to place of residence: prevalence and associated factors", we observed differences in subject profile in tenns of self-reported vaccination according to specific subgroups and places of residence. In more populous municipalities, only lesser schooling (PR = 1.26; 95%CI: 1.02-1.54) was associated with vaccination. Among the less densely populated municipalities, older age (pR = 1.15; 95%CI: 1.02-1.31) and 'reported hypertension .(PR = 1.21; 95%CI: 1.02-1.45), diabetes (pR = 1.16; 95%CI: 1.01-1.33), and chronic pulmonary disease (PR = 1.30; 95%CI: 1.03-1.64) were associated with the outcome. In the paper entitled "Factors associated with pulmonary disease among the elderly," our results indicate independent associations between self-reported pulmonary disease and smoking (pR = 2.03; 95%CI: 1.39 - 2.97), medication use (pR = 2.05; 95%CI: 1.11 - 3.79), self-perception of current health status as poor or very poor (RP = .1.89; 95%CI: 1.20 2.96), and depression, anxiety, or emotional disorders (pR = 1.86; 95%CI: 1.11 - 3.10).With the aging of the population, and the increasing importance of respiratory diseases among the elderly, the findings of the present study indicate a need for measures that prioritize the maintenance of health among this population, through promotion and prevention programs that take into account the specificities of this segment / Doutorado / Epidemiologia / Mestre em Saude Coletiva

Influenzavirus (IV)-Infektionen der unteren Atemwege induzieren virale Pneumonien, die häufig in akutem Lungenversagen resultieren. Merkmale einer IV-induzierten Pneumonie sind Schädigungen des Alveolarepithels und eine Ansammlung von Ödemflüssigkeit im Alveolarraum, wodurch der Gasaustausch beeinträchtigt wird. In Abhängigkeit eines Natriumgradienten, aufgebaut durch die basolaterale Na,K-ATPase (NKA) und den apikalen epithelialen Natriumkanal (ENaC), wird unter normalen Bedingungen die Ödemflüssigkeit aus dem Alveolarraum entfernt. In Folge einer IV-Infektion werden verschiedene Membranionenkanäle dysreguliert und eine verringerte alveoläre Flüssigkeitsresorption (AFC) beobachtet. Eine IV-Infektion führt u.a. zu einer reduzierten NKA-Expression in nicht-infizierten Nachbarzellen, sowie zu einer Dislokation der NKA zur apikalen Zellmembran in infizierten Zellen. Co-Immunopräzipitationsstudien identifizierten das virale M2-Protein als NKA-Bindepartner. Mittels Mutationsanalyse konnten drei Aminosäuren im zytoplasmatischen Teil von M2 als kritisch für die NKA-Bindung identifiziert werden. Rekombinante IV mit gestörter NKA Bindung zeigten im Vergleich zu IV WT in polarisierten Calu 3 Zellen in vitro sowie in Mäusen in vivo eine verbesserte AFC. Eine mutationsbedingte Glykosylierung des M2-Proteins führte jedoch unerwartet zu einer verstärkten Immunantwort in vivo, die trotz verbesserter AFC zu einem schwereren Krankheitsverlauf führte. Grund dafür könnte eine Aktivierung der Unfolded Protein Response aufgrund der Glykosylierung sein. Die Erkenntnis, dass M2 ein wichtiger Modulator in der Regulation der alveolären Flüssigkeitshomöostase ist, könnte dennoch helfen, neue therapeutische Ansätze für IV-induzierte Pneumonien zu definieren. Darüber hinaus unterstreicht es die Relevanz einer in der vorliegenden Arbeit durchgeführten Surfactome-Analyse zur Identifizierung neuer potentieller Angriffspunkte an der Zelloberfläche IV-infizierter Zellen, die in der antiviralen Therapie von Bedeutung sein könnten. / Influenza Virus (IV) infections of the lower respiratory tract can induce viral pneumonia resulting in acute lung injury (ALI/ARDS) with fatal outcome. Characteristics of an IV-induced pneumonia are alveolar epithelial cell (AEC) damage and accumulation of protein-rich edema fluid in the alveolar compartment impairing gas exchange. Depending on a sodium gradient established by the basolateral Na,K-ATPase (NKA) and the apical epithelial sodium channel (ENaC) edema fluid is removed from the alveolar space under normal conditions. However, after IV-infection various ion channels are dysregulated and reduced alveolar fluid clearance (AFC) is observed. An IV-infection leads to a reduced NKA expression in the non-infected neighbouring cells and to a mistargeting of the NKA to the apical cell membrane in IV-infected cells. Co immunoprecipitation (co-IP) studies identified the viral M2 protein as NKA binding partner and mutational analysis presented three amino acids in the cytoplasmic tail of M2 directly abutting the transmembrane domain as critical for NKA binding. A recombinant IV mutant with disrupted NKA binding showed in comparison to IV WT an increased fluid transport in polarized Calu 3 cells in vitro as well as in mice in vivo. However, mutation-induced glycosylation of the M2 protein unexpectedly led to an enhanced immune response in vivo, resulting in a more severe disease course despite improved AFC. The reason for this could be an activation of the unfolded protein response by the glycosylation of M2. Nevertheless, the finding that M2 appears to be an important modulator in the regulation of alveolar fluid homeostasis might provide new potential approaches for therapeutics of an IV induced pneumonia. Moreover, it highlights the relevance of a surfactome analysis performed in the present work to identify novel potential targets on the cell surface of IV-infected cells which could play an important role in antiviral therapy.

Les virus influenza aviaires faiblement pathogènes (IAFP) ciblent principalement les épithéliums des voies respiratoires et intestinales chez les poulets (Gallus gallus) infectés. Cependant, les virus influenza aviaires hautement pathogènes (IAHP) mènent à une maladie systémique fatale avec une localisation particulière aux endothéliums. L’objectif de cette thèse a été d’explorer les relations entre la réplication des virus influenza aviaires (IA) et la réponse antivirale de l’hôte dans deux modèles cellulaires originaux obtenus chez le poulet : des cellules épithéliales pulmonaires (CLEC213) et des cellules endothéliales d’aortes (chAEC). Les résultats clés sont les suivants : (i) la réplication productive des virus IA dans les chAEC dépend du clivage de l’hémagglutinine et de l’échappement viral à la réponse immunitaire innée ; (ii) les CLEC213 sont très permissives aux virus IA et présentent une faible réponse antivirale médiée par la signalisation TLR3 et MDA5 ; (iii) les fonctions régulatrices de SOCS1 et SOCS3, sur le signal des interférons et des cytokines, sont conservées chez le poulet. Nous proposons que certains virus IA peuvent exploiter les fonctions pro-virales de SOCS1 et SOCS3 à leur avantage de manière spécifique au type cellulaire. / Low pathogenic avian influenza (LPAI) viruses essentially target the epithelia of the respiratory and intestinal tract in the infected chicken host (Gallus gallus). However, highly pathogenic avian influenza (HPAI) viruses induce a peracute fatal systemic disease and exhibit a striking endothelial cell tropism. The objective of the present thesis was to explore the interdependencies of AI virus replication and the antiviral host response in two novel avian cell culture models: chicken lung epithelial cells (CLEC213) and chicken aortic endothelial cells (chAEC). The salient findings from this study are that (i) productive AI virus replication in chAEC is dependent on hemagglutinin cleavability and appears to be related to innate immune escape; (ii) CLEC213 are highly permissive to AI virus infection, due to a cell type-specific diminished TLR3- and/or MDA5-mediated antiviral signaling response; (iii) the interferon and cytokine regulatory functions of SOCS1 and SOCS3 are conserved in the chicken. Based on our data, we propose a model that predicts that certain AI viruses may exploit the proviral functions of SOCS1 and SOCS3 in a cell type-specific manner.

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.