Os pacientes com doenças reumáticas crônicas exibem um risco aumentado de contrair infecções. Consequentemente, sua vacinação é indispensável. Há relatos da produção de anticorpos antifosfolípides e tromboses após infecções e vacinação nesta população, exceto em síndrome antifosfolípide (SAF) primária. O objetivo principal deste estudo foi avaliar a curto e longo prazos um painel de anticorpos antifosfolípides após a vacinação contra influenza A/H1N1 (sem adjuvante) em SAF primária e controles saudáveis. Quarenta e cinco pacientes com SAF primária e 33 controles saudáveis foram imunizados e prospectivamente avaliados antes da vacinação e 3 semanas e 6 meses após a vacinação. Os anticorpos antifosfolípides foram determinados por ensaio imunoenzimático (ELISA) e incluíram os anticorpos IgG e IgM a seguir: anticardiolipina (aCL), anti-beta2glicoproteína I (anti-beta2GPI), anti-anexina V, anti-fosfatidilserina e anti-protrombina. O anticorpo anti-Sm foi igualmente determinado por ELISA e o anti-DNA dupla hélice, por imunofluorescência indireta. Avaliamos clinicamente à ocorrência de tromboses arterial e venosa. A frequência pré-vacinação de pelo menos um anticorpo antifosfolípide foi significativamente maior nos pacientes com SAF primária comparados aos controles (58% vs 24%, p = 0,0052). A frequência global de anticorpos antifosfolípides pré-vacinação e 03 semanas e 06 meses após a vacinação permaneceu inalterada tanto em pacientes (p = 0,89) como em controles (p = 0,83). A frequência de cada anticorpo específico nos dois grupos permaneceu estável nas três avaliações (p > 0,05). A frequência de cada anticorpo mantevese invariável nos pacientes tratados com cloroquina (p > 0,05). Em 3 semanas, 2 pacientes com SAF primária deselvolveram um anticorpo antifosfolípide novo porém transitório (aCL IgG e IgM), enquanto que em 6 meses novos anticorpos foram observados em 6 pacientes e nenhum apresentou altos títulos. Anti-Sm e anti-DNA dupla hélice foram negativos e nenhuma nova trombose arterial ou venosa foi observada durante o estudo. Este foi o primeiro estudo a demonstrar que a vacina contra influenza pandêmica em pacientes com SAF primária não induz tromboses e uma produção significante de anticorpos antifosfolípides a curto e longo prazos. (ClinicalTrials.gov, #NCT01151644). / Chronic rheumatic disease patients exhibit an increased risk for infections. Therefore, vaccination is imperative. Antiphospholipid antibodies (aPL) and thrombosis triggering after infections and vaccination in this population were reported, except for primary antiphospholipd syndrome (PAPS). Study\'s main objective was short and long-term evaluation of a panel of antiphospholipid autoantibodies following pandemic influenza A/H1N1 non-adjuvant vaccine in primary antiphospholipid syndrome patients and healthy controls. Forty-five PAPS and 33 healthy controls were immunized with A/H1N1 pandemic influenza vaccine. They were prospectively assessed at pre-vaccination, 3 weeks and 6 months after vaccination. aPL autoantibodies were determined by an enzyme-linked immunosorbent assay (ELISA) and included IgG/IgM: anticardiolipin (aCL), anti-beta2GPI; anti-annexin V, anti-phosphatidyl serine and antiprothrombin antibodies. Anti-Sm was determined by ELISA and anti-dsDNA by indirect immunfluorescence. Arterial and venous thrombosis were also clinically assessed. Pre-vaccination frequency of at least one aPL antibody was significantly higher in PAPS patients versus controls (58% vs. 24%, p=0.0052). The overall frequencies of aPL antibody at pre-vaccination, 3 weeks and 6 months after immunization remained unchanged in patients (p=0.89) and controls (p=0.83). The frequency of each antibody specificity for patients and controls remained stable in the three evaluated period (p > 0.05). The frequency of each antibody kept invariable in PAPS patients under chloroquine treatment (p > 0.05). At 3 weeks, 2 PAPS patients developed a new but transient aPL antibody (aCL IgG and IgM), whereas at 6 months new aPL antibodies were observed in 6 PAPS patients and none had high titer. Anti-Sm and anti-dsDNA autoantibodies were uniformly negative and no new arterial or venous thrombosis were observed throughout the study. This was the first study to demonstrate that pandemic influenza vaccine in PAPS patients does not trigger short and long-term thrombosis or a significant production of aPL related antibodies. (ClinicalTrials.gov, #NCT01151644)

Les virus influenzae sont des pathogènes importants ayant un impact à la fois écologique et en santé publique humaine. En effet chez l’Homme, ces virus sont responsables de la grippe, qu’elle soit saisonnière, pandémique ou zoonotique. Face à ces infections très peu d’options thérapeutiques sont disponibles : seuls les inhibiteurs de neuraminidases (INA) sont recommandés par l’OMS mais des résistances ont été décrites. Les substitutions H274Y, E119V et R292K sont les substitutions de résistance retrouvées le plus fréquemment dans les neuraminidases des virus influenzae humains. Dans notre travail, l’étude des résistances aux INA a été organisée autour de deux axes, d’une part via le suivi clinique et biologique de la résistance chez les patients et d’autre part via l’étude de l’impact des substitutions de résistance dans des neuraminidases aviaires. La surveillance de ces résistances au sein du CNR des virus des infections respiratoires a permis la caractérisation du génome de virus influenza A(H1N1)pdm09 dans un contexte d’excrétion chronique en présence d’un traitement par INA chez un patient atteint de déficit immunitaire combiné sévère mais aussi le développement et l’évaluation de la PCR digitale pour le diagnostic de la substitution H274Y des virus A(H1N1). Au niveau mécanistique, notre travail a permis l’analyse des substitutions (H274Y, R292K, E119V±I222L) sur l’ensemble des sous-types de neuraminidases des virus influenzae A. La substitution H274Y se retrouve préférentiellement dans les N1 mais est responsable d’une diminution de la sensibilité à l’oseltamivir pour toutes les neuraminidases du groupe 1 (N1, N4, N5 et N8). La substitution E119V entraine une diminution de la sensibilité à l’oseltamivir variable en fonction des neuraminidases avec un impact plus important pour les N2, N7, N9 et N5. De plus l’association des substitutions E119V+I222L entraine un effet synergique sur le phénotype de résistance à l’oseltamivir. Enfin la substitution R292K entraine une diminution de la sensibilité à tous les INA dans toutes les neuraminidases du groupe 2 (N2, N3, N6, N7 et N9). La production d’une N9 recombinante portant la substitution R292K a montré un impact majeur de la substitution sur l’activité enzymatique. Un développement technologique toujours en cours au laboratoire va nous permettre d’aller plus loin dans l’analyse des mécanismes qui sous-tendent l’apparition de ces résistances / Influenza viruses are important human pathogens that are responsible for flu, whether seasonal, pandemic or zoonotic. Very few therapeutic options are available against these pathogens and neuraminidase inhibitors (NAI) are the only antiviral agents recommended by the WHO for treatment and prophylaxis of influenza virus infections. NAI resistance has already been described and the H274Y, E119V and R292K neuraminidase substitutions are the most frequently encountered substitutions responsible for oseltamivir resistance. During this work, we focused the NAI resistance study around two main objectives: first, we monitored clinical and biological resistance in treated patients and then we studied the impact of substitutions responsible for NAI resistance using avian neuraminidases. For NAI resistance monitoring, viral genomic diversity of a child's influenza A(H1N1)pdm09 was characterized in the context of a severe combined immunodeficiency and a chronic viral excretion despite antiviral treatment. For a better detection of H274Y substitution in A(H1N1) influenza viruses, a digital droplet PCR was developed and evaluated. At a more fundamental level, resistance substitutions (H274Y, R292K, E119V ± I222L) were analysed using all neuraminidase subtypes of influenza A viruses. To summarize, H274Y substitution is preferentially isolated in N1 but also decreases oseltamivir susceptibility in all group 1 neuraminidases (N1, N4, N5 and N8). The E119V substitution impact on oseltamivir susceptibility depends on the neuraminidase and decreases oseltamivir susceptibility especially within N2, N7, N9 and N5. Moreover, the E119V+I222L substitutions has a synergistic effect on oseltamivir resistance profile. The R292K substitution decreases all NAI susceptibility for all group 2 neuraminidases (N2, N3, N6, N7 and N9). The production of a recombinant N9 bearing the R292K substitution allows to highlight the substitution impact on the sialidasic activity. Development of new technological tools are still in progress to allow a more accurate analysis of the mechanisms that underlie the NAI resistance

Influenza A virus infections are a major cause of morbidity and mortality in the United States and throughout the world. The current vaccine elicits primarily a humoral response that is specific for the external glycoproteins hemagglutinin (HA) and neuraminidase (NA). However, these are the viral proteins that are most susceptible to antigenic shift and drift, and can evade the humoral response. Cytotoxic T lymphocytes (CTL) recognize and lyse virus-infected cells and are important in clearing influenza A virus infections. CTL can recognize epitopes on both the external glycoproteins and the more conserved internal viral proteins. This thesis investigates the hypothesis that there is a broad CTL memory response in humans, and, if boosted by vaccines, these CTL may help clear influenza A virus strains of different subtypes. The CTL repertoire specific for influenza A viruses reported in inbred mice is extremely limited and has focused on a few immunodominant epitopes. We perfonned preliminary bulk culture chromium release assays using human peripheral blood mononuclear cells (PBMC) stimulated with influenza virus strain A/PR/8/34 (H1N1) in vitro. CTL activity was observed against autologous B-lymphoblastoid cell lines (B-LCL) infected with vaccinia constructs that expressed several influenza A viral proteins, including nucleoprotein (NP), matrix (M1), nonstructural 1 (NS1) and polymerase (PB1). This was more diverse than the limited response reported in inbred mice. To further characterize the CTL repertoire in humans, PBMC from healthy adult donors were stimulated and CTL were cloned by limiting dilution. Isolated cell lines were further characterized by their CD4/CD8 surface expression, histocompatibility leukocyte antigen (HLA) restriction, cross-reactive or subtype-specific influenza A subtype recognition, and epitope recognition. CTL lines isolated from three donors recognized epitopes on many different influenza virus proteins. The ELISPOT assay was used to identify the number of IFN-γ- secreting cells and determine the precursor frequency of the CTL specific for the epitopes that were mapped. The precursor frequency of IFN-γ producing CTL ranged from 1 in 4,156 PBMC to 1 in 31,250 PBMC. The precursor frequency for one epitope was below the level of detection of this assay, but most of the memory CTL were readily detected. The cross-reactive or subtype-specific recognition of various human influenza A subtypes by these T cell lines was determined by chromium release assays. Most of the CTL lines recognized B-LCL infected with any of the three influenza A subtypes that have caused epidemics in the last century (H1N1, H2N2, and H3N2) and recognized epitopes on conserved internal influenza viral proteins. Most of the subtype-specific cell lines recognized the surface HA or NA glycoproteins, which are not well conserved between influenza subtypes. Although most of the T cell lines that were characterized were cross-reactive with influenza viruses of human origin, infection of humans with a divergent swine or avian derived strain could cause a global pandemic. To study the human CTL responses to non-human influenza viruses, B-LCL were infected with an Hsw1N1 influenza A virus of swine origin, and cell lines were tested for recognition of these targets in a chromium release assay. Most cell lines lysed the targets infected With the Hsw1N1 subtype to the same degree as targets infected with the human H1N1 strain. Two influenza viruses of duck origin were also tested and were recognized by many of the cell lines. The subtypes of these duck strains were Hav1N1 and H5N2. The isolates of influenza A virus from the Hong Kong outbreak of 1997 were also used to infect targets and analyze recognition by these CTL. We found that approximately 50% of the human T cell lines tested recognized both of the Hong Kong isolates, 25% recognized at least one isolate, and 25% recognized neither isolate to the same degree as the A/PR/8/34 (H1N1) virus. We analyzed the amino acid (aa) changes in the epitopes of the T cells lines from the 25% of cell lines that did not recognize either Hong Kong virus isolate. Non-conservative mutations were found in all of the epitopes that lost recognition by the human CTL lines. Bulk cultures of PBMC from three donors that were stimulated with A/PR/8/34 (H1N1) influenza A virus of human origin recognized all of the non-human virus strains tested. Thus, humans have memory CTL that recognize influenza viruses of avian and swine species. This may provide a second line of defense against influenza infection in case of exposure to a novel influenza A virus derived from these species. These results made it clear that humans have broad CTL memory to influenza A virus. In order to determine whether these T cells could be boosted in a vaccine, immune-stimulatory complexes (Iscom) incorporating inactivated influenza particles were tested in vitro. Iscoms containing inactivated influenza A vaccine (Flu-Iscom) were used to pulse autologous B-LCL overnight that were then used as targets in chromium release assays with human CTL lines as effectors. A CD8+ HA-specific CTL line lysed these targets, but not targets pulsed with Iscoms alone or with inactivated influenza A vaccine alone. An NS1-specific cell line recognized targets pulsed with NS1 protein and Iscoms, but not targets pulsed with Iscoms or NS1 protein alone. Therefore, CTL could recognize in vitrotarget cells that were exposed to the Iscom vaccines containing their specific epitope. Flu-Iscom and Iscom mixed with inactivated influenza virus particles (Flu-Iscomatrix) were then used as vaccines in a clinical trial to test CTL and neutralizing antibody induction against influenza. Fifty-five donors were bled pre-vaccination, and on days 14 and day 56 post-vaccination. Bulk culture chromium release assays were performed using targets infected with live vaccine strain viruses. There were significantly more increases in the influenza A specific CTL activity in the PBMC of donors that were vaccinated with the Flu-Iscom and Flu-Iscomatrix vaccines than in recipients of the standard vaccine. In order to determine whether these increases in cytotoxicity were due to an increase in the precursor frequency of influenza specific CTL, the PBMC were used in ELISPOT assays to assess the changes pre-and post-vaccination. When there was an increase in the level of cytotoxicity detected in bulk culture CTL, there was often also an increase in the precursor frequency of influenza-specific CTL. Peptide-specific increases in the number of CTL that recognize epitopes such as M1 aa 58-66 were detected in several donors confirming the increase in influenza-specific CTL post-vaccination. Another type of T cell that may be involved in defense against viruses is the γδ T cell. T cells expressing the γδ T cell receptor (TCR) have been found extensively in mucosal tissues in mice and humans. Influenza A viruses enter via the airway tract, infecting the epithelial cells at the mucosal surface. These epithelial cells have been shown in vitro to be targets for influenza-specific cytolytic recognition of αβ T cells. To analyze whether γδ T cells can respond to influenza A-infected APCs, PBMC were stimulated with influenza A virus. Intracellular IFN-γ staining was used to determine whether γ/δ T cells can secrete IFN-γ in response to the influenza A virus infection. We observed an increase in the percentage of γ/δ T cells secreting IFN-γ post-influenza A virus infection of PBMC compared to uninfected or allantoic fluid-stimulated cultures. These T cells also upregulated CD25 and CD69 in response to live influenza A virus. We focused on the responses in the CD8- population of γδ T cells, which are the majority of γδ T lymphocytes. Furthermore, the increases in IFN-γ production and activation marker expression were much more clear in the CD8- γδ+ T cells. The level of CD8- γδ T cell activation with inactivated influenza A virus was much less, and in some cases no higher than uninfected PBMC. The CD8+ αβ and γδ responses could be partially blocked by anti-class I antibodies, but the CD8- γδ responses could not. Vaccinia virus infection did not activate the CD8- γδ T cells to the same degree as influenza virus infection. γδ T cells are thought to have a regulatory role that includes the secretion of cytokines and epithelial growth factors to help restore tissue back to health. Humans have broad multi-specific T lymphocyte responses by αβ T cells to influenza A viruses and those responses are cross-reactive with human, avian, and swine virus strains. These CTL can be activated in vitro and boosted in number in vivo by Iscom incorporating vaccines. There is also a population of γδ+ T lymphocytes in humans that responds to influenza virus infection by producing cytokines and becoming activated. Increasing memory CTL as a second line of defense against influenza A viruses may be important in future vaccine development.

Estudos recentes têm apresentado resultados in vitro satisfatórios em pacientes com Imunodeficiência Comum Variável (ICV) que receberam vacinas contra tétano, influenza e meningococo. No entanto, existem poucos ensaios clínicos que avaliem a resposta clínica e laboratorial após a exposição a antígenos específicos. O presente estudo tem como objetivo avaliar a resposta clínica à imunização contra antígenos protéicos e polissacarídicos (influenza, H1N1 e pneumococo) em pacientes com diagnóstico de ICV seguidos no ambulatório de Imunodeficiências Primárias do Serviço de Imunologia Clínica e Alergia do HC-FMUSP. O diagnóstico dos pacientes foi estabelecido de acordo com os critérios da OMS / PAGID / ESID. Um grupo de 37 pacientes foi vacinado contra a influenza A (H2N3), gripe H1N1 e pneumococo e outro grupo com 16 pacientes, não foi vacinado. A avaliação clínica foi realizada através da aplicação de um score com avaliação dos seguintes parâmetros clínicos: pneumonia, sinusite, otite média, infecções de vias aéreas superiores (IVAS), amigdalites, diarréia, bronquiectasias, hospitalizações, uso de antibióticos, uso de antibióticos profiláticos, sepse e meningite. O score foi aplicado durante os 12 meses que precederam a vacinação e 12 meses posteriores à administração das vacinas. O mesmo score foi aplicado ao grupo controle, com os pacientes que não foram vacinados. A determinação da IgG contra os sorotipos do pneumococo foi feita por ELISA. A determinação da IgG específica H1N1 foi feita por hemaglutinação indireta, enquanto que a dosagem da IgG específica para influenza, por ELISA, utilizando o kit comercial RIDASCREEN ® Influenza. O grupo de pacientes vacinados incluiu 37 pacientes (51% mulheres), com idade entre 20 e 78 anos (mediana= 33 anos). Observou-se uma mediana de 7 anos de atraso no diagnóstico de ICV. A mediana de idade do grupo de pacientes (n=16, 37,5% mulheres) que não receberam a vacina foi de 41 anos e a mediana de atraso no diagnóstico foi de 8 anos. Observamos que as infecções de vias aéreas superiores (IVAS), sinusites e pneumonias foram as manifestações mais freqüentes no grupo controle. IVAS seguida por pneumonia e sinusite foram as manifestações infecciosas mais freqüentes em mulheres (80%, 78% e 55%, respectivamente). Entretanto, em homens observamos IVAS seguido por sinusite e pneumonia (78%, 65% e 35%, respectivamente). Observou-se redução significativa no score relativo ao número de infecções respiratórias superiores, sinusites e pneumonias um ano após a administração das vacinas (p <0,001). Os dados foram comparados com pacientes ICV não vacinados e neste grupo não houve diferença entre os scores dos dois períodos de 12 meses . Após a vacinação, observou-se uma tendência a aumento no título de anticorpos específicos para a H2N3, mas sem resultado significativo. Em relação aos resultados obtidos com as sorologias para o H1N1 e o pneumococo, não se observou resposta após a vacinação. Concluindo, houve redução do número de infecções, principalmente das IVAS, sinusites e pneumonias em pacientes com ICV após a vacinação contra a influenza, H1N1 e pneumococo. Embora não tenhamos encontrado correlação entre a redução do número de infecções e os títulos de anticorpos específicos para as vacinas testadas, a melhora clínica observada nos pacientes com ICV reforça o benefício da vacinação / Recent studies have shown satisfactory in vitro results in patients with CVID who received immunization against tetanus, influenza and meningococcus. However, there are only a few studies that evaluate the clinical and laboratory response after exposure to specific antigens in these patients. This study aims to evaluate the clinical response to immunization with protein and polysaccharide antigens (influenza, H1N1 and pneumococcus) in CVID patients followed at the Primary Immunodeficiency outpatient clinic of the Division of Clinical Immunology and Allergy, Hospital das Clínicas, FMUSP. CVID patients were diagnosed according the WHO/PAGID/ ESID criteria. Thirty-seven patients were immunized against influenza (H2N3), H1N1 and pneumococcal polysaccharide vaccine while another group with 16 CVID patients were not vaccinated. Clinical evaluation was performed through a score with assessment of the following parameters: pneumonia, sinusitis, otitis media, upper respiratory infections (URI), tonsillitis, diarrhea, bronchiectasis, hospitalizations, use of antibiotic therapy, and use of prophylactic antibiotics, sepsis and meningitis. The score was applied during the 12 months prior to immunization and one year after the administration of vaccines. The same score was applied to the group of CVID patients who weren´t immunized. Determination of IgG antibodies to pneumococcal serotypes was made by ELISA. H1N1-specific IgG was detected by indirect hemagglutination while the determination of influenzaspecific IgG was performed by ELISA, using the RIDASCREEN ® Influenza kit. The group of patients who were vaccinated included 37 patients (51% women), aged 20 to 78 years (mean 33 years). This group presented a median delay in the diagnosis of 7 years. The control group consisted of 16 patients (37.5% females) who were not immunized. Their median age was 41 years and the median delay in the diagnosis was 8 years. URI followed by pneumonia and sinusitis were the most frequent infections in women (80%, 78% and 55% respectively). However in men, URI followed by sinusitis and pneumonia were the most frequent (78%, 65% and 35% respectively). We observed a significant reduction in the score of URI, sinusitis and pneumonias in the year post administration of the vaccines (p <0.001). Conversely, there was no difference in the infections pre and post supposed vaccination scores in the group of CVID patients who were not immunized. There was no significant change in specific antibody titers to influenza and pneumococcus after vaccination. Regarding H1N1, there was no statistically significant production of antibodies to H1N1, although we observed a slight non-durable increase in antibody titers. In conclusion, there was a reduction in the number of infections, mainly sinusitis, URIs and pneumonias in patients with CVID vaccinated against influenza, H1N1 and pneumococcus. While we found no correlation between the reduction in the number of infections and specific antibody titers for the vaccines administered, the clinical improvement observed in CVID patients reinforces the benefit of vaccination

La grippe porcine est une infection enzootique touchant 50% du cheptel français. Elle passe parfois inaperçue, mais peut également induire une forte morbidité au sein des lots d’animaux touchés, entraînant une baisse des performances zootechniques et des pertes économiques importantes. La sévérité de l’infection à virus influenza A chez le porc peut dépendre de divers facteurs, comme les virus eux-mêmes, les pratiques d’élevage, le statut immunitaire des animaux, les infections concomitantes par d’autres pathogènes respiratoires, etc. De la même manière, diverses formes épidémiologiques de la grippe existent en élevage. Ainsi, des infections peuvent se répéter à un âge déterminé, sur toutes les bandes successives d’un élevage, notamment chez des jeunes présentant une immunité passive. Afin de mieux comprendre cette diversité clinique et épidémiologique de la grippe porcine, et aider à l’élaboration de stratégies d’intervention adéquates pour le contrôle de la maladie, nous avons cherché à apporter de nouvelles connaissances quant à certains facteurs pouvant favoriser l’exacerbation du syndrome grippal et/ou son caractère récurrent, et plus généralement aux mécanismes sous-jacents à la pathogenèse des virus influenza A chez le porc, en relation avec les réponses de l’hôte infecté. Nous avons d’abord comparé, suite à inoculations expérimentales de porcs exempts d’organismes pathogènes spécifiés, la pathogénicité des deux virus influenza porcins les plus fréquemment rencontrés chez le porc en France, l’un du lignage européen « avian-like swine H1N1 » (H1avN1), l’autre du lignage européen « human-like reassortant swine H1N2 » (H1huN2), seuls ou en association avec Mycoplasma hyopneumoniae (Mhp), autre pathogène respiratoire répandu en élevage. Nous avons montré que l’infection H1huN2 induit une pathologie plus marquée que l’infection H1avN1, et que la pré-infection des porcs par Mhp induit une exacerbation de l’infection H1avN1, mais pas H1huN2. Nous avons utilisé le modèle de co-infection Mhp/H1avN1 pour évaluer deux approches alternatives qui permettraient de diminuer l’impact des infections grippales et de leurs complications : l’apport de composés aux propriétés antioxydantes via l’alimentation ; et la restriction alimentaire de courte durée. Dans ces deux cas nous avons montré des effets bénéfiques sur les paramètres zootechniques pendant les jours suivant l’infection grippale. Ce travail a également apporté de nouvelles connaissances quant aux modifications des marqueurs plasmatiques de stress oxydant, ainsi que sur les modifications métaboliques faisant suite à la co-infection Mhp/H1avN1. La sévérité des manifestations cliniques de la grippe étant liée à la qualité de la réponse immunitaire mise en place chez l’hôte infecté, nous avons entrepris d‘étudier les réponses immunitaires du porc touché par la grippe et d’évaluer l’impact de facteurs tels que la présence de Mhp ou d’anticorps d’origine maternelle sur ces réponses. Nous avons ainsi montré que l’infection virale induit une inflammation et une réponse interféron. La pré-infection par Mhp exercerait un effet additif sur cette inflammation et pourrait favoriser la persistance du virus dans le poumon. Nous avons également montré que la présence d’immunité passive protège cliniquement le porcelet mais n’empêche pas l’excrétion du virus, retarde la réponse lymphocytaire T et inhibe la réponse humorale post-infectieuse. Malgré la réponse immunitaire humorale défaillante, les animaux étaient totalement protégés d’une seconde infection homologue lorsque les anticorps maternels avaient disparus. Ces travaux ont permis d’apporter de nouvelles connaissances sur les facteurs influençant l’infection grippale en élevage porcin ainsi que sur les mécanismes sous-jacents, ce qui est une prérequis pour l’amélioration des mesures de lutte et de maitrise de la maladie. Ils permettent, d’envisager d’améliorer la santé des animaux en agissant sur leur régime alimentaire. / Swine influenza is an enzootic infection affecting 50% of the French livestock. The infection can be unnoticed but can also induce high morbidity among batches of affected animals, resulting in lower production performance and significant economic losses. The severity of influenza A virus in pig is influenced by many factors such as the virus strain, husbandry practices, the immune status of animals, concomitant infections with other respiratory pathogens, etc. In the same way, various epidemiological forms of influenza exist in farms. Thus, infections can be repeated in all successive batches within a farm, especially among young animals with passive immunity. In order to better understand the clinical and epidemiological diversity of the swine flu, and help develop appropriate strategies to control the disease, we tried to bring new knowledge about factors that promote the exacerbation of the flu syndrome and/or its recurrence, and more generally to give new information about the mechanisms underlying the pathogenesis of influenza viruses in pigs, in relation to the response of the infected host. Firstly, we compared, through experimental infections of specific pathogen free pigs, the pathogenicity of the two swine influenza viruses mostly detected in pigs in France, i.e. one from the European “avian-like swine H1N1” lineage (H1avN1) and the other one from the European “human-like reassortant swine H1N2” lineage (H1huN2), each one alone or in co-infection with Mycoplasma hyopneumoniae (Mhp), another respiratory pathogen widespread in French farms. We showed that the H1huN2 infection induced a more marked pathology than the H1avN1 infection, and that Mhp pre-infection induced the exacerbation of the H1avN1, but not the H1huN2, infection. Then, we used the Mhp/H1avN1 co-infection model to evaluate alternative approaches that could reduce the impact of influenza infections and their complications: firstly, a supply of compounds with antioxidant properties in food; and secondly, a feed restriction of short duration. In both cases, we showed beneficial effects on zootechnical parameters the days following influenza infection. This work has also brought new knowledge on modulation of oxidative stress markers in plasma, as well as metabolic changes following the co-infection with Mhp and H1avN1 in pigs. The severity of flu clinical manifestations being related, among other, to the quality of the immune responses developed by the infected host, we studied these responses in pigs experimentally infected by H1avN1 and assessed the impact of factors such as the presence of Mhp or maternal derived antibodies on these responses. We showed that the viral infection induced inflammation and interferon response. The Mhp pre-infection exerted an additive effect on inflammation of lung tissue and may promote the virus persistence in the lung. Finally, we have shown that the presence of maternally-derived immunity protected the piglets clinically but did not prevent viral shedding, delayed the T cell response and strongly inhibited the post-infectious humoral response. However, despite the failed humoral immune response, animals were completely protected from a second infection occurring when maternal antibodies had disappeared. Therefore, this work have brought new knowledge on factors influencing influenza infection in pig as well as the underlying mechanisms, which is a prerequisite for improving disease control. They allow, between-other, to consider improving the health and welfare of animals by acting on their diet.

La grippe, infection respiratoire virale fréquente, est due aux virus Influenza. Leur génome est constitué par huit molécules d’ARN de polarité négative retrouvés sous la forme de complexe ribonucléique (RNPv). Au cours du cycle viral, il a été démontré que les régions terminales des segments de gène étaient cruciales pour l’incorporation sélective des huit RNPv à l’intérieur des particules virales. Par des techniques d’interaction in vitro et de tomographie électronique, nous avons montré que les segments de gène du virus H3N2 interagissaient entre eux par des interactions ARN/ARN impliquant leurs régions de packaging. Nos résultats suggèrent que la mise en place de ce réseau permettrait la formation d’un complexe supra macromoléculaire multi-segmenté permettant l’incorporation d’un jeu complet des huit RNPv dans les particules virales néosynthetisées. En raison de la nature segmentée du génome viral, des phénomènes de réassortiment génétique peuvent avoir lieu lors d’une co-infection. Afin de définir les mécanismes responsables de la restriction observée lors de ce phénomène, nous avons évalué le taux de réassortiment génétique in vitro entre le virus humain H3N2 et le virus aviaire H5N2. Nos résultats suggèrent que le mécanisme gouvernant l’incorporation sélective des segments de gènes, régulerait le réassortiment génétique. Nous avons montré que la modulation de l’interaction ARN/ARN entre les segments de gènes HA et M permet d’augmenter le taux d’incorporation du segment de gène HA H5 dans le fond génétique du virus humain, prérequis pour l’émergence de virus pandémique / The Flu is a frequent viral infectious disease caused by the Influenza viruses. Their genomes are composed by eight negative single-stranded RNA organised as vRNPs. During the viral cycle, the terminal non-coding and coding regions of viral genome have been shown to be crucial for the selective incorporation of a complete set of the eight vRNPs into influenza viral particles. Band shift assay and electron tomography allowed us to show that all gene segments interact together by RNA/RNA interactions involving their packaging region. Our results suggest that the eight genomic vRNAs are selected and packaged as an organized supramolecular complex held together between identified packaging regions into neosynthesized virions. Due to genome segmented nature, genetic reassortment can occur during co-infection. In order to identify molecular mechanisms responsible for the observed restriction during the genetic reassortment, we have developed a new competitive reverse genetic strategy allowing us to evaluate the genetic reassortment between H3N2 and H5N2 viruses. Our results suggest that mechanism controlling the packaging should regulate genetic reassortment. We have shown that the modulation of RNA/RNA interaction between HA and M gene segment have allowed us to increase HA H5 gene segment incorporation rate into a viral human genetic background, prerequisite for pandemic virus emergency

Les huit ARN viraux (ARNv) génomiques des influenzavirus de type A sont sélectivement empaquetés dans les virions, vraisemblablement sous la forme d’un complexe supramoléculaire maintenu par des appariements ARN/ARN entre signaux d’empaquetage. Afin d’améliorer la compréhension des règles qui gouvernent ce mécanisme, nous avons déterminé le réseau d’interactions formé entre les ARNv de la souche modèle de laboratoire A/Puerto Rico/8/34 (H1N1). Nous avons ensuite défini, au nucléotide près et par deux approches in vitro, les séquences des ARNv impliquées dans la formation de certaines interactions. Le rôle fonctionnel de deux d’entre elles a été testé en contexte infectieux. Nous avons également poursuivi l’étude d’une interaction identifiée au laboratoire entre deux ARNv de la souche A/Moscou/10/99 (H3N2) circulante. Enfin, nous avons collaboré avec le Dr L. Brown (Australie) sur l’étude du rôle d’une interaction entre deux ARNv de la souche A/Udorn/307/72 (H3N2). / The genome of influenza A viruses comprises eight viral RNAs (vRNAs) likely to be selectively packaged into progeny virions as organized supramolecular complexes where vRNAs are held together by base pairing within the packaging signals. To better understand the rules governing this mechanism, we investigated the vRNA/vRNA interaction network of the A/Puerto Rico/8/34 (H1N1) strain. We then identified, at the nucleotide level, using two in vitro approaches, the vRNA sequences involved in several of the interactions. Two of them were functionally tested in an infectious context. We also studied an interaction previously identified in the laboratory between two vRNAs belonging to the circulating A/Moscow/10/99 (H3N2) strain. Finally, we collaborated with Dr L. Brown (Australia) in order to assess the role of an interaction between two vRNAs of the A/Udorn/307/72 (H3N2) strain.

Orientadores: Maria Laura Costa do Nascimento, José Guilherme Cecatti / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T01:47:22Z (GMT). No. of bitstreams: 1 Pfitscher_LuciaChaves_M.pdf: 6366056 bytes, checksum: 9a70e9992cbec443ea7d924bdc77f7bb (MD5) Previous issue date: 2015 / Resumo: Introdução: A infecção representa importante causa de morbidade e mortalidade materna, sendo uma preocupação crescente no mundo todo. As doenças respiratórias, especialmente as virais, têm se destacado justamente pelo potencial de epidemia com que ameaçam a saúde da população mundial e pela vulnerabilidade identificada durante a gestação. Objetivo: Avaliar o impacto da morbidade materna grave (MMG) atribuível à infecção (sepse, meningite e doença respiratória) e os fatores associados ao pior resultado materno (near miss e óbito), entre mulheres da Rede Brasileira de Vigilância da Morbidade Materna Grave. Métodos: análise secundária de um estudo transversal, multicêntrico, que incluiu 27 centros de referência obstétrica das cinco regiões do Brasil no período de 2009 e 2010. A vigilância prospectiva dos casos de infecção grave foi realizada utilizando os critérios da OMS de condições potencialmente ameaçadoras da vida (CPAV) e near miss materno (NMM). Os principais focos de infecção foram identificados e comparados a outras causas de MMG. Mulheres com complicação devido à doença respiratória também foram avaliadas em dois grupos: com e sem suspeita de A(H1N1)pdm09 e também comparadas a outras causas de MMG. Casos com suspeita de A(H1N1)pdm09 foram revisados e separados em três grupos: não-testados, confirmados e não confirmados para A(H1N1)pdm09 e os seus resultados foram comparados. Complicações devidas à infecção e a doenças respiratórias foram comparadas com complicações devidas a outras causas de MMG. Os fatores associados com desfecho materno grave (DMG) foram avaliados para os casos de infecção e doença respiratória. Resultados: Dentre os 9555 casos de MMG, apenas 502 (5,3%) apresentaram infecção grave, entretanto foram responsáveis por cerca de um quarto dos casos de NMM e quase metade dos casos de morte materna (MM). Os indicadores de saúde avaliados demonstram maior gravidade dos casos complicados por infecção, com índice de mortalidade (IM) superior a 26% em comparação com 11% para as demais causas de MMG. Para doença respiratória, 206 mulheres apresentaram suspeita de A(H1N1)pdm09, cerca de 60% foram testados para a doença e 49 mulheres apresentaram resultado positivo. A gravidade dos desfechos maternos foi pior entre os casos de A(H1N1)pdm09 positivo, com uma taxa de NMM:MM abaixo de 1 (0,9:1), em comparação a 12:1 para outras causas de MMG. O IM para doença respiratória foi superior a 50% (7,4% outras causas de MMG). Demoras no atendimento foram associadas com pior prognóstico materno e estiveram presentes em mais de 50% entre os casos de infecção, aumentando em duas vezes o risco de DMG para doença respiratória. Resultados perinatais foram piores dentre os casos de doença respiratória, com aumento da prematuridade, morte fetal, baixo peso ao nascer e Apgar <7. HIV/AIDS, histerectomia, hospitalização prolongada, admissão em UTI e demoras no atendimento foram alguns fatores independentes associados DMG. Conclusão: complicações por infecção e em especial por influenza A(H1N1)pdm09 geram grande impacto sobre morbidade e mortalidade materna no Brasil e compreender os fatores associados à maior gravidade pode gerar medidas capazes de colaborar para a melhoria do cuidado obstétrico. Investir em intervenções específicas para gravidez, visando diagnóstico precoce e tratamento oportuno são essenciais para melhorar a saúde materna e reduzir o número de mortes maternas evitáveis no país / Abstract: Background: Infection represents the major cause of maternal morbidity and mortality, and a growing concern worldwide. Respiratory diseases, especially viral, have stood out because of their epidemic potential and the identified vulnerability towards infection during pregnancy. Objective: To assess the impact of severe maternal morbidity (SMM) due to infection (sepsis, meningitis and respiratory disease) and the factors associated with worse maternal outcome (near miss and death) among women of the Brazilian Network for the Surveillance of Severe Maternal Morbidity. Methods: secondary analysis of a cross-sectional, multicenter study that included 27 obstetric referral centers in five regions of Brazil between 2009 and 2010. Prospective surveillance of severe infection was performed using WHO criteria of potentially life threatening conditions (PLTC) and maternal near miss (MNM). The main sources of infection were identified and compared to other causes of SMM. Women with complications due to respiratory disease were also assessed in two groups: with and without suspected A(H1N1)pdm09 and also compared to other causes of SMM. Cases of suspected A(H1N1)pdm09 were reviewed and divided into three groups: non-tested, confirmed and unconfirmed for A(H1N1)pdm09 and their results were compared. Complications due to infection and respiratory disease were compared with complications due to other causes of SMM. Factors associated with SMO were assessed for cases of infection and respiratory disease. Results: Among the 9555 cases of SMM, only 502 (5.3%) had severe infection, however they were responsible for about a quarter of cases of MNM and almost half of the cases of maternal mortality (MM). The assessed health indicators demonstrate greater severity of cases complicated by infection, with a mortality index (MI) above 26% compared to 11% for other causes of SMM. For respiratory disease, 206 women had suspected A(H1N1)pdm09, about 60% were tested for the disease and 49 women were positive. The severity of the maternal outcomes was worse between the cases of A(H1N1)pdm09 positive, with a rate of MNM: MM below 1 (0.9: 1), compared to 12: 1 for other SMM causes. The MI among respiratory disease was superior to 50% (7.4% other causes SMM). Delays in care were associated with worse maternal prognosis and were present in over 50% of cases of infection. Perinatal results were worse in cases of respiratory disease, with increased prematurity, stillbirth, low birth weight and Apgar <7. HIV/AIDS, hysterectomy, prolonged hospitalization, ICU admission and delays in care were independent factors associated with severe maternal outcome. Conclusion: infections and especially those caused by A(H1N1)pdm09 presented great impact on maternal morbidity and mortality in Brazil and the identification of factors associated with the increased severity can contribute to the improvement of obstetric care. There is need for specific interventions during pregnancy, seeking early diagnosis and timely treatment of infections, which are essential for improving maternal health and to reducing the number of preventable maternal deaths in the country / Mestrado / Saúde Materna e Perinatal / Mestra em Ciências da Saúde

Ein vielversprechender Ansatz zur Verhinderung von Infektionen mit Influenzavirus ist die kompetitive Inhibition der Virusanhaftung an die Wirtszellen durch Behinderung der Bindung des viralen Hemagglutinin (HA) an sialylierte Glykanrezeptoren. Allerdings erschwert die hohe Variabilität des HA die Entwicklung von universellen Sialinsäure (SA)-basierten Virostatika. In dieser Arbeit wurde der antivirale Effekt von mit SA funktionalisierten Polyglycerolen (PGs) auf Influenza A Viren (IAV) evaluiert. SA-basierte PGs waren nur bei der Inhibition einer geringen Anzahl an IAV Stämmen effektiv. Um die molekulare Basis für diese Beschränkung zu ergründen, wurden mittels Serienpassagen IAV Mutanten selektiert, die gegen sialyliertes PG resistent waren. Es entwickelten sich drei unabhängige resistente Virusvarianten, die einen einfachen bzw. doppelten Aminosäuren-Austausch in der HA RBS aufwiesen. Durch Hemagglutinations-Elution, Einzel-Virus Kraft-Untersuchungen und Glykanarray Analysen konnte eine verringerte Rezeptorbindungsstabilität sowie ein verändertes Rezeptorbindeprofil für diese Virusvarianten gezeigt werden. Interessanterweise wurden drei unterschiedliche Fälle von Virusbindung und Inhibition beobachtet: 1) Virales HA wurde vom PG gebunden und die Virusreplikation inhibiert, 2) virales HA wurde vom PG gebunden ohne Inhibition der Virusreplikation und 3) Virales HA wurde nicht vom PG gebunden und es gab keine Inhibition. Diese Ergebnisse suggerieren, dass es eine Mindestanforderung an die Affinität oder Avidität für eine effektive kompetitive Inhibition von HA gibt. Durch modifizierte PGs, die Sialyllaktose statt SA und einen Amidlinker enthielten, konnte das Potential von PGs als breite IAV Inhibitoren demonstriert werden. Zusammenfassend bieten die Ergebnisse dieser Arbeit wertvolle Einblicke in die Entwicklung von Resistenzen in IAV gegen Inhibitoren des HA-Attachment und in das strategische Design von sialylierten mutlivalenten Inhibitoren gegen IAV. / A promising approach to block influenza virus infections is competitive inhibition of virus attachment to host cells by interfering with binding of the viral surface protein hemagglutinin (HA) to sialylated glycan receptors. However, the high structural and genetic variability of the viral HA has hampered the development of universal sialic acid (SA)-based antivirals. Here, the antiviral effect of biocompatible Polyglycerols (PGs) functionalized with SA on influenza A virus (IAV) was evaluated. PG compounds were only effective at inhibiting a narrow spectrum of IAV strains. To elucidate the molecular basis for this restriction, PG-resistant IAV mutants were selected using serial passaging. Three independent resistant variants developed with single or double amino acid changes mapping to the HA RBS. By employing hemagglutination elution, single-virus force measurements and glycan array analyses, a reduced receptor binding stability as well as an altered receptor binding profile of mutant viruses was shown. Intriguingly, three different cases of virus binding and inhibition were observed using Cy3-labeled compound: 1) viral HA was bound by the compound and resulted in inhibition of replication, 2) viral HA was bound by the compound but replication was not inhibited and 3) viral HA was not bound by the compound and no inhibition occurred. These results suggest that there is an affinity or avidity requirement for effective competitive inhibition of HA attachment. The suitability of PGs as IAV inhibitors with potential for broad activity was demonstrated by a modified PG incorporating sialyllactose instead of SA and an amide linkage covering an extended spectrum of inhibited IAV strains. Taken together, results described in this thesis provide valuable insights into the development of resistance against inhibitors of HA attachment in IAV and into the strategic design of sialylated, multivalent inhibitors aiming at broad activity against influenza viruses.

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.