Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 6 uppsatser.

Made available in DSpace on 2015-07-22T20:49:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Introdução: Isolados clínicos do Citomegalovirus (CMV) são facilmente propagados in vitro resultando em comprometimento da monocamada celular onde o vírus foi inoculado, evidenciando assim a presença ou ausência de infecção. A cultura celular é um método clássico para detecção do CMV e foi bastante utilizada no passado. O ensaio de antigenemia, que detecta o antígeno viral pp65 do CMV, é o método mais utilizado atualmente na prática clínica, por ser mais rápido e específico para detecção da infecção ativa. Recentemente a técnica de PCR em tempo real tem sido empregada no monitoramento da infecção por meio da quantificação da carga viral por ser um método de alta sensibilidade e especificidade ao DNA viral. Sendo assim, o objetivo do estudo foi empregar testes usados no diagnóstico e monitoramento da infecção clínica à cepa padrão do CMV como protocolo para implantação em experimentos in vitro. Métodos: Monocamada de células fibroblásticas humanas confluentes e em quiescência foram inoculadas com amostras de células infectadas pela cepa adaptada em laboratório do CMV AD169. O efeito do vírus sobre a cultura foi monitorado 1 hora, 24 horas, 48 horas e 72 horas após a infecção (h.p.i) através da observação do efeito citopático. As mesmas amostras foram analisadas por antigenemia estimando-se a média de células positivas em 2x105 células e por PCR em tempo real estimando-se a média de cópias de DNA viral/Log10 presente nas amostras. Resultados: Efeito citopático foi observado pela primeira vez 24 h.p.i, evidenciando que o início das mudanças morfológicas ocorreu precocemente. Esse efeito tornou-se mais intenso após 72h. O ensaio de antigenemia evidenciou presença de infecção ativa pelo padrão de marcação do antígeno viral pp65 encontrado no núcleo das células infectadas, enquanto que a PCR em tempo real evidenciou o número de cópias de DNA viral nos diferentes tempos de infecção. Antigenemia apresentou uma média 57 ±56 células positivas 1h.p.i. O pico da infecção foi alcançado 24h.p.i com um aumento significativo da média para 2.381 ±168 (P<0.05 versus 1h.p.i), mantendo-se elevado 48h.p.i, mostrando uma média de 2.012 ±352. Entretanto, os níveis de antigenemia diminuem significativamente 72h.p.i para 262 ±5 (P<0.05 versus 48h.p.i). Assim como na antigenemia, observou-se aumento significativo da carga viral de 1 h.p.i para 24 h.p.i, sendo uma média de DNA viral detectado 11.30 ±0.30 e 11.96 ±0.09, respectivamente (P<0.05 versus 1h.p.i). Os níveis de DNA viral se mantêm elevados 48h.p.i, sendo detectada uma média de 12.33 ±0,26. Após esse período, carga viral cai significativamente para 11.57 ±0.06 (P<0.05 versus 48h.p.i). Não foi encontrada correlação entre os métodos quantitativos de antigenemia e PCR em tempo real. Conclusão: Os três métodos utilizados, isolamento viral, antigenemia e PCR em tempo real evidenciaram o sucesso da infecção “in vitro” pelo CMV por meio de mudanças cito-morfológicas, detecção de antígeno viral específico e carga viral por detecção do DNA viral, respectivamente. A técnica de PCR se mostrou a mais sensível na detecção viral em relação às demais técnicas. Embora sejam métodos sensíveis e específicos, consideramos a necessidade da titulação viral em quaisquer ensaios experimentais in vitro. / Introduction: Clinical isolates of Cytomegalovirus (CMV) are easily spread in vitro resulting in impairment of the monolayer cell where the virus was inoculated, thus evidencing the presence or absence of infection. The cell culture is a classic method for detection of CMV and it was widely used in the past. Antigenemia assay, which detects CMV pp65 antigen, is the method most used currently in clinical practice, because it is faster and specific for detection of the active infection. Recently, the real-time PCR has been used in monitoring of the infection through the quantification of viral load for being a high sensitivity and specificity method to viral DNA. Therefore, the aim of the study was employing tests used in diagnosis and monitoring of infection to the standard CMV strain as a protocol for implantation in experiments in vitro. Methods: Quiescent human fibroblasts in confluent monolayer were inoculated with samples of infected cells by the adapted CMV AD169 strain. The effect of the virus on culture was monitored at 1 hour, 24 hours, 48 hours and 72 hours post infection (h.p.i) by observation of cytopathic effect. The same samples were analyzed by antigenemia being estimate the mean of positive cells in 2x105 cells and by real-time PCR being estimate the mean of copies of viral DNA/Log10 present in samples. Results: Cytopathic effect was first noticed 24 h.p.i, showing that the initiation of morphological changes occurred early. This effect became more intense after 72 h.p.i. Antigenemia assay showed the presence of active infection through pattern of labeling of the pp65 viral antigen found on nucleus of infected cells, while the real-time PCR showed the number of copies of viral DNA in different times of infection. Antigenemia showed an mean of 57 ±56 positive cells 1h.p.i. The peak of the infection was reached 24h.p.i with a significant increase in the mean 2.381 ±168 (P<0.05 versus 1h.p.i) and remained high 48h.p.i, showing an mean of 2.012 ±352 positive cells. However, the mean of antigenemia decrease 72h.p.i to 262 ±5 (P<0.05 versus 48h.p.i). As well as in antigenemia, a significant increase of th viral load was observed of 1h.p.i to 24h.p.i, being the mean of viral DNA detected 11.30 ±0.30 and 11.96 ±0.09, respectively (P<0.05). The levels of viral DNA stayed high 48h.p.i, being detected a mean of 12.33 ±0.26. After this period, viral load decreased significantly to 11.57 ±0.06 (P<0.05 versus 48h.p.i). No correlation was found between the quantitative methods of antigenemia and real-time PCR. Conclusion: The three methods, virus isolation, antigenemia and real-time PCR, showed the success of the CMV infection “in vitro” by cyto-morphological changes, detection of viral antigen specific and viral load by virus DNA detection, respectively. PCR method was more sensitive in detecting virus in relation the other methods. Although sensitive and specific, we consider the need for viral titration in any experimental studies in vitro. / TEDE / BV UNIFESP: Teses e dissertações

No presente trabalho, desenvolvemos uma estratégia vacinal baseada em vacinas de DNA que codificam proteínas do HPV-16, o tipo viral de maior relevância epidemiológica, fusionadas à glicoproteína D (gD) do vírus herpes simplex tipo 1 (HSV-1). A vacina que contêm o gene da E7 de HPV-16 fusionada à gD de HSV-1 (pgDE7), administrada em regime vacinal de quatro doses, foi capaz de gerar significativa ativação de células T CD8+ E7-específicas e apresentar 40% de efeito protetor anti-tumoral terapêutico em camundongos desafiados com células transformadas que expressam as proteínas E6 e E7 do HPV-16 (células TC-1). A partir das evidências geradas, desenvolvemos um novo vetor vacinal que codifica as proteínas E7, E6 e E5 do HPV-16 (pgD-E7E6E5). Em ensaios em modelo murino, apenas uma dose da vacina foi capaz de gerar ativação de células T CD8+ específicas e 70% dos camundongos previamente desafiados com células TC-1 e inoculados com 3 doses da vacina mantiveram-se livres de tumores. Como tentativa de potencializar o efeito protetor terapêutico encontrado, adotamos duas medidas: a co-administração de plasmídeos que codificam citocinas e a otimização de códons da sequência gênica que codifica a proteína quimérica. A combinação das vacinas pgDE7 ou pgD-E7E6E5 com plasmídeos que carregam os genes das citocinas IL-2, IL-12 ou GM-CSF foi capaz de aumentar a proteção terapêutica para 100% em regime vacinal de dose única. A adequação da sequência antigênica ao sistema de expressão humano, aumentou em cerca de 5 vezes o potencial terapêutico do vetor vacinal pgDE7. Em conjunto, os dados apresentados nesta tese demonstram a evolução do desenvolvimento de uma estratégia vacinal contra tumores induzidos por HPV-16 e encorajam seu potencial para uso em futuros ensaios clínicos. / The development of immunotherapeutic strategies against human papillomavirus (HPV) is a priority for the control of HPV-induced lesions and cervical cancer. In this study, we developed DNA vaccines encoding HPV-16 proteins fused to glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) as an approach to control HPV-16 induced tumors. The vaccine encoding HPV-16 E7 fused to HSV-1 gD (pgDE7), when administered in a four doses vaccine regimen, was able to generate significant activation of E7-specific CD8+ T cells and showed 40% of therapeutic anti-tumor effect in mice previously challenged with tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells). Following these evidences, we developed a new vaccine vector encoding HPV-16 E7, E6, E5 proteins fused to HSV-1 gD (pgD-E7E6E5). Only one vaccine dose generated antigen-specific CD8+ T cell responses and three doses conferred 70% protection to mice previously challenged with TC-1 cells. As an attempt to enhance the observed therapeutic anti-tumor effects, we tested two approaches: co-administration of cytokine-expressing plasmids and codon optimization of the gene encoding the chimeric protein. The combination of the vaccines pgDE7 or pgD-E7E6E5 with plasmids encoding the cytokines IL-2, IL-12 or GM-CSF increased the therapeutic protection to 100% of the vaccinated animals following a single dose. The gene sequence adaptation increased by a factor of 5 the therapeutic potential of the pgDE7 vaccine. In summary, the data presented in this thesis demonstrated the development of a vaccine strategy against HPV-16 induced tumors and reinforces its potential use in future clinical trials.

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. Despite decades of research, it is still unclear how these viruses establish a lifelong, asymptomatic infection in their rodent hosts while infection of humans often results in severe disease. Unable to enter a state of bona fide latency, the transcription and replication of the viral genomic RNA is likely highly regulated in time and subcellular space. Moreover, we hypothesize that the viral nucleoprotein (NP), responsible for the encapsidation of the viral RNA and the most highly expressed viral gene product, plays a key role in the regulation of the viral gene expression program. Further, exploring host-virus interactions may elucidate the basic aspects of arenavirus biology and how they cause such severe disease in humans. To explore these questions in greater detail, this dissertation has pursued three main avenues. First, to better understand lymphocytic choriomeningitis mammarenavirus (LCMV) genome replication and transcription at the single-cell level, we established a high-throughput, single-molecule (sm)FISH image acquisition and analysis pipeline and followed viral RNA species from viral entry through the late stages of persistent infection in vitro. This work provided support for a cyclical model of persistence where individual cells are initially transiently infected, clear active infection, and become re-infected from neighboring reservoir cells within the population. Second, we used FISH to visualize viral genomic RNA to describe the subcellular sites where LCMV RNAs localize during infection. We observed that, viral RNA concentrates in large subcellular structures located near the cellular microtubule organizing center and colocalizes with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane bound organelle as a site for the pre-assembly of viral components including genomic RNA and viral glycoprotein prior to their transport to the plasma membrane where new particles will bud. Last, we used mass spectrometry to identify human proteins that interact with the NPs of LCMV and Junín mammareanavirus (JUNV) strain Candid #1. We provided a detailed map of the host machinery engaged by arenavirus NPs, and in particular, showed that NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. We demonstrated that JUNV antagonizes the antiviral activity of PKR completely, effectively abrogating the antiviral activity of this surveillance pathway. In sum, the work composing this dissertation has given us fresh insight into how arenaviruses establish and maintain persistence; the nature of the subcellular site where viral genomic RNA is transcribed, replicated, and assembled with other viral components; and a global view of the cellular machinery hijacked by the viral nucleoprotein. This work improves our basic understanding of the arenavirus life cycle and may suggest novel antiviral therapeutic targets that could be exploited in the future.

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.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 98-104). Also available on the Internet.

No presente trabalho, desenvolvemos uma estratégia vacinal baseada em vacinas de DNA que codificam proteínas do HPV-16, o tipo viral de maior relevância epidemiológica, fusionadas à glicoproteína D (gD) do vírus herpes simplex tipo 1 (HSV-1). A vacina que contêm o gene da E7 de HPV-16 fusionada à gD de HSV-1 (pgDE7), administrada em regime vacinal de quatro doses, foi capaz de gerar significativa ativação de células T CD8+ E7-específicas e apresentar 40% de efeito protetor anti-tumoral terapêutico em camundongos desafiados com células transformadas que expressam as proteínas E6 e E7 do HPV-16 (células TC-1). A partir das evidências geradas, desenvolvemos um novo vetor vacinal que codifica as proteínas E7, E6 e E5 do HPV-16 (pgD-E7E6E5). Em ensaios em modelo murino, apenas uma dose da vacina foi capaz de gerar ativação de células T CD8+ específicas e 70% dos camundongos previamente desafiados com células TC-1 e inoculados com 3 doses da vacina mantiveram-se livres de tumores. Como tentativa de potencializar o efeito protetor terapêutico encontrado, adotamos duas medidas: a co-administração de plasmídeos que codificam citocinas e a otimização de códons da sequência gênica que codifica a proteína quimérica. A combinação das vacinas pgDE7 ou pgD-E7E6E5 com plasmídeos que carregam os genes das citocinas IL-2, IL-12 ou GM-CSF foi capaz de aumentar a proteção terapêutica para 100% em regime vacinal de dose única. A adequação da sequência antigênica ao sistema de expressão humano, aumentou em cerca de 5 vezes o potencial terapêutico do vetor vacinal pgDE7. Em conjunto, os dados apresentados nesta tese demonstram a evolução do desenvolvimento de uma estratégia vacinal contra tumores induzidos por HPV-16 e encorajam seu potencial para uso em futuros ensaios clínicos. / The development of immunotherapeutic strategies against human papillomavirus (HPV) is a priority for the control of HPV-induced lesions and cervical cancer. In this study, we developed DNA vaccines encoding HPV-16 proteins fused to glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) as an approach to control HPV-16 induced tumors. The vaccine encoding HPV-16 E7 fused to HSV-1 gD (pgDE7), when administered in a four doses vaccine regimen, was able to generate significant activation of E7-specific CD8+ T cells and showed 40% of therapeutic anti-tumor effect in mice previously challenged with tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells). Following these evidences, we developed a new vaccine vector encoding HPV-16 E7, E6, E5 proteins fused to HSV-1 gD (pgD-E7E6E5). Only one vaccine dose generated antigen-specific CD8+ T cell responses and three doses conferred 70% protection to mice previously challenged with TC-1 cells. As an attempt to enhance the observed therapeutic anti-tumor effects, we tested two approaches: co-administration of cytokine-expressing plasmids and codon optimization of the gene encoding the chimeric protein. The combination of the vaccines pgDE7 or pgD-E7E6E5 with plasmids encoding the cytokines IL-2, IL-12 or GM-CSF increased the therapeutic protection to 100% of the vaccinated animals following a single dose. The gene sequence adaptation increased by a factor of 5 the therapeutic potential of the pgDE7 vaccine. In summary, the data presented in this thesis demonstrated the development of a vaccine strategy against HPV-16 induced tumors and reinforces its potential use in future clinical trials.

Le virus T lymphotrope humain de type 1 (HTLV-1) est l'agent étiologique de la leucémie à cellules T de l'adulte (ATL) et de la paraparésie spastique tropicale/myélopathie associée à HTLV-1 (HAM/TSP). Chez les patients chroniquement infectés, le provirus d'HTLV-1 est majoritairement retrouvé dans les lymphocytes T CD4+. Ex vivo, on peut aussi retrouver le provirus dans les lymphocytes T CD8+, les lymphocytes B, les monocytes, les cellules dendritiques (DCs) myéloïdes, les DCs plasmacytoϊdes (pDCs) et les macrophages. In vitro, HTLV-1 est capable d'infecter productivement les cellules lymphoïdes et les cellules dendritiques dérivées de monocytes humains (MDDCs). De par leur fonction et leur distribution dans l'organisme, les DCs pourraient être les premières cellules à interagir avec HTLV-1 au cours de la primo-infection. Elles seraient ensuite capables de transmettre HTLV-1 aux lymphocytes T CD4+. Cette hypothèse est soutenue par les travaux de notre équipe qui ont montré que les MDDCs sont plus susceptibles que des lymphocytes T autologues à l'infection par HTLV-1. Ainsi, les DCs constitueraient des relais importants pour l'établissement de l'infection chronique. Dans ce contexte, nous nous sommes demandés si toutes les populations de DCs étaient également susceptibles à l'infection par HTLV-1 et si elles transmettaient similairement HTLV-1 aux lymphocytes T. Pour cela, nous avons différencié trois sous types de MDDCs après l'exposition de monocytes humains à divers cocktails de cytokines : - les IL4-DCs (pour interleukine 4 - DCs) miment les DCs immatures myéloϊdes du sang, - les TGF-β DCs (pour tumor-growth factor β - DCs) miment les DCs mucosales à phénotype tolérogène, - les IFN-α DCs (pour interféron α DC) miment les DCs activées et inflammatoires recrutées au niveau des sites d'inflammation. Nous avons aussi traité au lipopolysaccharide (LPS) des IL-4 DCs afin de générer des DCs qui sur-expriment les marqueurs de maturation CD80 et CD86. Nos résultats montrent que les IFN-α DC et les IL-4 DCs traités au LPS ne supportent pas une infection productive au contraire des TGF-β DCs et des IL4-DCs qui sont productivement infectés par HTLV-1. La restriction virale des IFN-α DC et les IL-4 DCs traitées au LPS n'est pas due à leur production d'IFN. Nous avons montré que la susceptibilité des IL4-DCs à l'infection productive par HTLV-1 est liée à leur phénotype immature. De plus, nos résultats montrent qu'HTLV-1 est internalisé par macropinocytose dans les IL-4 DCs alors qu'il est internalisé par endocytose médiée par la clathrine dans les IFN-α DCs. Enfin, nous avons pu restaurer partiellement la susceptibilité à l'infection productive des IL-4 DCs traités au LPS et celle des IFN-α DCs et nous avons pu restreindre celle des IL-4 DCs immatures en modulant le pH de leurs endosomes. Ces résultats suggèrent que le virus utilise le trafic vésiculaire pour infecter les DCs et que le pH des vésicules conditionne, au moins partiellement, le devenir de l'infection productive. De plus, parmi les IL-4 DCs, les IL-4 DCs traités au LPS et les IFN α DCs, seules les IL-4 DCs qui sont productivement infectées peuvent transmettre HTLV-1 aux lymphocytes T. En conclusion, nos résultats suggèrent que c'est le sous type de DC que rencontre HTLV- 1 lors de la primo-infection ainsi que le trafic viral d'HTLV-1 dans la DC qui conditionnent ou pas l'établissement de l'infection productive de la DC ainsi que la transmission aux lymphocytes T / HTLV-1 (Human T cell leukemia/lymphoma virus type 1) is the etiological agent of Adult T cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). In chronically infected patients, the provirus is mainly detected in the CD4 T-cell population and, to a lesser extent in myeloid dendritic cells (DCs), plasmacytoid DCs (pDCs), macrophages and monocytes. Among the different DCs subsets found in vivo, myeloid DCs from the blood, tolerogenic or inflammatory DCs from mucosa may first encounter HTLV-1 during blood transmission, breast-feeding or sexual transmission, respectively. They would then be able to transmit HTLV-1 to CD4 + T cells. This hypothesis is supported by the recent work of our team that showed that monocyte derived dendritic cells (MDDCs) are more susceptible to HTLV-1 infection in comparison to autologous T cells. We therefore asked whether all these DCs subsets were equally susceptible to HTLV-1 and whether the nature of the DC subset would impact HTLV-1 spread to T-cells. Human monocytes obtained from healthy blood donors were differentiated into IL-4 DCs, TGF-ß DCs or IFN-a DCs. In vitro-derived immature IL-4 DCs, TGF-ß DCs and IFN-a DCs mimic myeloid, tolerogenic and inflammatory DCs, respectively. We also generated LPS-matured IL-4 DCs that exhibited a strong maturation profile with over-expression of maturation markers. We observed HTLV-1 protein expression and provirus accumulation in IL-4 DCs and TGF-ß DCs but not in IFN-a DCs and LPS-matured IL-4 DCs. Despite their increased ability to capture HTLV-1 virion compared to IL-4 DCs and TGF-ß DCs, IFN-a DCs and LPS-matured IL-4 DCs restricted HTLV-1 productive infection. This was not due to the antiviral activity of type–I interferon produced by IFN-a DC or LPS-matured IL-4 DCs. In contrast, we showed that these differences in susceptibility to HTLV-1 infection might be linked to the maturation phenotype of the DCs subsets and to a different trafficking of HTLV-1 in IL-4 DC vs. IFN-a DC. Finally, using IL-4DCs, LPS-matured IL-4 DCs and IFN-a DCs, we demonstrate that productive infection rather than trans-infection is required for HTLV-1 transmission from DCs to CD4 T-cells. Thus, our results demonstrate that the nature of the DCs encountered by HTLV-1 during primo-infection and the trafficking route of the virus through the vesicular pathway of these cells determine the efficiency of viral transmission to T-cells

No description available.

Call number: LD2668 .T4 1963 G77 / Master of Science