L’infection à VIH-1 est associée à une forte déplétion des lymphocytes T CD4+ à polarisation Th17 au niveau des tissus lymphoïdes associés aux muqueuses intestinales (GALT, gut-associated lymphoid tissues). Ceci conduit à la translocation microbienne, qui est une cause d’activation immunitaire chronique et de progression de la maladie. Les cellules épithéliales (CE) jouent un rôle critique dans le maintien de l’intégrité et de l’homéostasie au niveau des muqueuses intestinales via le recrutement des cellules de l’immunité innée (e.g., neutrophiles) et adaptative (e.g., cellules Th17). Les neutrophiles produisent des molécules antivirales (e.g., défensines-) et ont la capacité de limiter la réplication virale au niveau des muqueuses. Les cellules Th17 jouent un double rôle lors de l’infection à VIH. Elles contribuent d’une part à la défense contre différents pathogènes opportunistes en augmentant, via la production d’IL-17, la capacité des CE à attirer les cellules Th17 et les neutrophiles. D’autre part, les cellules Th17 jouent un rôle délétère en tant que cibles de réplication virale et sources de cytokines pro-inflammatoires. La fréquence des cellules Th17 est diminuée dans les GALT mais pas dans les poumons des patients infectés par le VIH, suggérant qu’il existe des mécanismes différents par lesquels les cellules Th17 sont recrutées vers ces sites anatomiques. Nous avons testé l’hypothèse selon laquelle le VIH interfère avec la capacité des CE intestinales et non pas pulmonaires à produire des chimiokines (CK) responsables de l’attraction des cellules Th17 et des neutrophiles. Nous avons démontré que les CE intestinales et pulmonaires produisent des CK spécifiques pour les cellules Th17 (CCL20) et les neutrophiles (CXCL8) en réponse à des stimuli pro-inflammatoires tels que l’IL-1 et le TNF-. Le TNF- agit en synergie avec l’IL-17, un « signal de danger » récemment identifié, et augmente la capacité des CE intestinales mais pas pulmonaires à produire la chimiokine CCL20. Cette synergie s’explique par l’augmentation préférentielle de l’expression du récepteur à l’IL-17 à la surface des CE intestinales suite à la stimulation par le TNF-. L’exposition au VIH n’affecte pas la production de CCL20 et de CXCL8 par les CE intestinales, mais altère la capacité des CE alvéolaires à produire ces chimiokines en accord avec la permissivité sélective de ces dernières à l’infection par le VIH. En conclusion, nos résultats démontrent que (i) le VIH n’interfère pas directement avec la capacité des CE intestinales à recruter des cellules Th17 et des neutrophils et que (ii) la production de CCL20 par ces cellules est dépendantes de la synergie entre le TNF- et l’IL-17. Ainsi, la déplétion des cellules Th17 et la pénurie en IL-17 dans les GALT des sujets infectés pourrait causer de façon préférentielle des altérations fonctionnelles au niveau des CE intestinales, se traduisant par l’altération du recrutement des cellules Th17 en réponse au CCL20. / The HIV-1 infection is associated with a severe loss of CD4+ T-cells with Th17 polarization from the gut-associated lymphoid tissues (GALT). These alterations lead to microbial translocation, which is a cause of chronic immune activation and disease progression in HIV-infected subjects. Epithelial cells (EC) play a critical role in maintaining mucosal integrity and homeostasis in the GALT by mechanisms including recruitment of innate (e.g., neutrophils) and adaptive immunity cells (e.g., Th17 cells). Neutrophils produce antiviral molecules (e.g., -defensins) that may limit HIV replication at mucosal sites. Th17 cells play a dual role in HIV pathogenesis. Th17 cells contribute to the defence against different opportunistic pathogens by increasing the ability of epithelial cells to attract neutrophils in an IL-17-dependent manner. On the other hand, Th17 cells play a deleterious role in HIV pathogenesis as they are sites of productive viral replication and a source of pro-inflammatory cytokines. The frequency of Th17 cells is decreased in the GALT but not in the lungs of HIV-infected individuals, suggesting distinct mechanisms of Th17 recruitment in these anatomic sites in the context of HIV pathogenesis. In this manuscript we tested the hypothesis that HIV differentially interfere with the ability of intestinal but not pulmonary EC to produce chemokines that attract Th17 cells and neutrophils. We demonstrated that both intestinal and pulmonary EC produce chemokines that specifically attract Th17 cells (CCL20) and neutrophils (CXCL8) upon stimulation with the pro-inflammatory cytokines IL-1 and TNF- . TNF-α acted in synergy with IL-17, a recently identified « danger signal », and increases the capacity of intestinal but not pulmonary EC to produce CCL20. This synergistic effect can be explained by the preferential upregulation of IL-17 receptor expression on intestinal EC upon TNF- stimulation. The exposure of intestinal EC to HIV did not affect their ability to produce CCL20 and CXCL8; however, exposure to HIV altered the production of these chemokines by alveolar EC, consistent with their selective permissiveness to infection. In conclusion, our results demonstrate that (i) HIV does not interfere directly with the ability of intestinal EC to attract Th17 cells and neutrophils and that (ii) the ability of intestinal EC to recruit the Th17 cells via CCL20 production is selectively dependent on the synergy between TNF- and IL-17. Thus, the depletion of Th17 cells and the shortage in IL-17 in the GALT of HIV-infected subjects may preferentially lead to functional alterations of the intestinal barrier resulting by the alteration of Th17 recruitment in response to CCL20.

L’histoire naturelle et la progression des infections au VPH (virus du papillome humain) sont bien décrites. Cependant, la dynamique de transmission reste faiblement documentée. Une meilleure compréhension de la dynamique de transmission ainsi que de ses facteurs de risque permettrait d’optimiser les stratégies de prévention afin de réduire la prévalence de ces infections dans la population par la vaccination et les méthodes contraceptives. Notre étude vise à déterminer si la charge virale des infections au VPH influence leur transmission entre les partenaires sexuels. Pour ce faire, l’association entre la charge virale au niveau des organes génitaux et la concordance spécifique de type des infections prévalentes au VPH a été évaluée pour 250 couples hétérosexuels récemment formés. Les charges virales de VPH16 (r = 0.30), de VPH18 (r = 0.50) et de VPH51 (r = 0.19) étaient significativement corrélées (p < 0.05) entre les deux partenaires sexuels, contrairement à celles de VPH31 (r = 0.08) et de VPH42 (r = -0.1). Lorsqu’ajusté en fonction de l’âge des participants, une charge virale élevée augmentait significativement le taux de détection du même type chez le partenaire pour les types 16, 31 et 51. Ainsi, dans les couples hétérosexuels récemment formés, des charges virales élevées sont associées à une détection accrue du même type chez le partenaire sexuel. / The natural history and progression of genital HPV infection are well understood. However, less is known about transmission dynamics of HPV between sexual partners. A better knowledge of risk factors and dynamics of HPV transmission is needed to optimize prevention strategies through vaccination and contraceptive measures. Our study aims to determine if the viral load of HPV infection affects transmission between sexual partners. The association between human papillomavirus (HPV) loads in genital swabs and type-specific concordance of prevalent HPV infection was assessed in 250 heterosexual recently-formed couples to further characterize HPV transmission. Viral loads of HPV16 (r=0.30), HPV18 (r=0.50) and HPV51 (r=0.19) were significantly correlated (p<0.05) between partners in opposite to HPV31 (r=0.08) and HPV42 (r=-0.10). A higher HPV load increased significantly the rate of detection of HPV16, 31 and 51 in sexual partners (age-adjusted odds ratios from 1.64 to 7.71). In recently-formed heterosexual couples, higher HPV16, 31 or 51 load was associated with increased detection of the same HPV type in sexual partners.

Les cellules épithéliales pulmonaires constituent la première ligne de défense face aux virus respiratoires via la sécrétion de mucus, de peptides, de cytokines et chimiokines qui déterminent l'élimination ou la progression de l’infection. Les principales cytokines antivirales produites par les cellules épithéliales alvéolaires (AEC) sont les interférons (IFN) type I (α/β) et III (λ). La liaison d’IFNβ à son récepteur induit une voie antivirale bien caractérisée qui aboutit à l’activation du complexe ISGF3 (STAT1, STAT2 et IRF9) qui permet la transcription de multiples gènes codant pour des protéines à activité antivirale et immunorégulatrice. Il a récemment été démontré que la costimulation des cellules épithéliales pulmonaires par l’IFNβ et le Tumor Necrosis Factor α (TNFα), également produit lors d’une infection, synergisent pour induire un état antiviral tardif distinct. D’autre part, il a été montré que la synergie entre le TNFα et l'IFNβ induit une voie de signalisation impliquant STAT2 et IRF9, mais indépendante de STAT1 permettant l’expression du gène DUOX2. Notre but est de déterminer l’importance de cette nouvelle voie de signalisation induite par la costimulation du TNFα+IFNβ, impliquant STAT2 et IRF9 indépendamment de STAT1 dans la régulation d’un programme transcriptionnel antiviral et immunorégulateur tardif. Notre premier objectif est de déterminer si des gènes antiviraux et immunorégulateurs qui sont induits par la costimulation par TNFα+IFNβ sont dépendants de la voie STAT2/IRF9, indépendamment de STAT1. En utilisant la technique de qRT-PCR, nous avons identifié 3 gènes immunorégulateurs, CXCL10, IDO et APOBEC3G, induits de manière synergique en réponse à TNFα+IFNβ dans les cellules A549, un modèle de cellules épithéliales pulmonaires. Afin de confirmer que ces gènes sont induits indépendamment de STAT1, nous avons validé leur expression dans la lignée cellulaire U3A déficiente en STAT1. Par l'utilisation d'ARN interférants (ARNi) dirigés contre STAT2 et IRF9, nous avons confirmé que l’induction de ces gènes est dépendante de STAT2 et IRF9. Finalement, l’analyse de l’activité du promoteur de CXCL10 en réponse à TNFα+IFNβ par des essais rapporteurs luciférases a permis de montrer que la régulation se fait au niveau transcriptionnel. Notre deuxième objectif, est de déterminer si STAT6 pourrait remplacer STAT1 dans la voie de signalisation induite par TNFα+IFNβ. En effet, STAT6 est un inducteur connu de l’expression de DUOX2 en réponse à IL4+IL13. Contrairement à notre hypothèse, l’inhibition de STAT6 par ARNi augmente l’expression de DUOX2 en réponse à TNFα+IFNβ suggérant que STAT6 est un régulateur négatif. Nos résultats ont permis de comprendre de manière plus détaillée les mécanismes mis en place dans le développement d’une réponse antivirale. D’autre part, l’étude de l’effet de l’IFNβ et du TNFα est également pertinente pour les maladies chroniques inflammatoires et autoimmunes. De plus, nos résultats illustrent un nouveau paradigme concernant les mécanismes de signalisation cellulaire impliqués dans la synergie entre deux cytokines qui pourrait être applicable à des combinaisons de cytokines autres que TNFα+IFNβ. / Lung epithelial cells are the first line of defense against respiratory viruses via mucus secretion, peptides, cytokines and chemokines that determine the progression of the infection. The main antiviral cytokines produced by alveolar epithelial cells (AEC) are the interferons (IFN) type I (α / β) and III (λ). IFNβ binding to its receptor induces an antiviral pathway that is well characterized and leads to activation of the ISGF3 complex (STAT1, STAT2 and IRF9) which allows the transcription of multiple genes encoding proteins with antiviral and immunoregulatory activity. It has recently been shown that the costimulation of lung epithelial cells by IFNβ and Tumor Necrosis Factor α (TNFα), also produced during infection, induces a separate and late antiviral state, through synergy. On the other hand, it has been shown that the synergy between IFNβ+TNFα induces a signaling pathway involving STAT2 and IRF9 independently of STAT1 permitting the expression of the DUOX2 gene. Our goal is to determine the importance of this new signaling pathway induced by costimulation of TNFα+IFNβ involving STAT2 and IRF9 regardless of STAT1 in regulating the antiviral immunoregulatory and late transcriptional program. Our first objective is to determine whether antiviral and immunomodulatory genes that are induced by costimulation TNFα+IFNβ are dependent on the STAT2/IRF9 way, indenpant of STAT1. Using the technique of qRT-PCR, we identified 3 immunoregulatory genes, CXCL10, IDO and APOBEC3G, synergistically induced in response to TNFα+IFNβ in A549 cells, a model of pulmonary epithelial cells. To confirm that these genes are induced independantly of STAT1, we validated their expression in the STAT1 deficient cell line, U3A. By the use of interfering RNA (siRNA) directed against STAT2 and IRF9, we confirmed that the induction of these genes is dependent STAT2 and IRF9. Finally, the analysis of the activity of CXCL10 promoter in response to TNFα+IFNβ by luciferase reporter assays has shown that the regulation is at the transcriptional level. Our second objective is to determine whether STAT6 could replace the STAT1 in the signaling pathway induced by TNFα+IFNβ. Indeed, STAT6 is a known inducer of the expression of DUOX2 in response to IL4+IL13. Contrarily to our hypothesis, inhibition of STAT6 by RNAi increases the expression of DUOX2 in response to TNFα+IFNβ suggesting that STAT6 is a negative regulator. Our results allow the understanding of the mechanisms in the development of an antiviral response in more detail. On the other hand, the study of the effect of IFNβ and TNFα is also relevant for chronic inflammatory and autoimmune diseases. In addition, our results illustrate a new paradigm for cell signaling mechanisms involved in the synergy between two cytokines that may be applicable to combinations of cytokines other than TNFα+IFNβ.

No description available.

Influenza A viruses (IAV) are prominent human pathogens and understanding the molecular biology of their replication is of utmost importance for the development novel anti-viral treatments and surveillance practices. IAV genome replication proceeds in the nucleus of infecting cells; where the virus takes advantage of host cell transcription machinery, particularly mRNA processing enzymes. The role of nuclear localization in the viral replication cycle is not clear and the viral and host cell determinants that are required for efficient nuclear import and export are an active area of study in our laboratory and others. Specifically, we are studying the role of IAV Nuclear Export Protein (NEP) in nuclear export in virus replication, biogenesis, and pathogenesis. A previously identified host nuclear export protein, CRM-1, has been identified as a critical mediator of the nuclear egress of newly replicated viral ribonucleoproteins (vRNPs). To define the virus-host interactions required for pathogenesis in humans and ferrets, the preferred animal model for IAV infection, we have cloned and expressed human and ferret CRM-1 and fused them to GFP and RFP to allow in vitro analysis of NEP/CRM-1 interactions in the context of IAV replication and biogenesis. We have developed a mammalian two-hybrid system for co-transfection of human or ferret CRM-1 and NEP from a panel of human and highly pathogenic avian influenza virus strains. This mammalian two-hybrid system has allowed for the identification of critical amino acid residues required for efficient nuclear export and virus biogenesis in human or ferret cells with a focus on the role of these later steps in replication in the dissemination of virus in the infected host or in the transmission to other susceptible hosts. In addition, using microRNA (miRNA) targeting sequences of CRM-1 and/or NEP inhibiting their expression, we examined the interaction of these host and viral proteins in cells and the role of this interaction in replication efficiency. Our results are consistent with a critical role for these interactions in virus biogenesis and pathogenesis for influenza virus infection in both humans and ferrets providing novel anti-viral therapeutic targets for antiviral development / acase@tulane.edu

acase@tulane.edu

<p>Orthopoxviruses encode many genes that are not essential for viral replication, which often account for differences in pathogenesis among otherwise closely related orthopoxviruses. Although dendritic cells (DCs) are essential to the generation of an effective anti-viral immune response, the effects of different orthopoxviruses on DC function is poorly understood. The objective of these studies was to determine the effect of different orthopoxviruses on DCs. Cowpox virus (CPXV) is ideally suited to this purpose because it encodes the largest and most representative set of accessory genes among orthopoxviruses, it is endemic in mouse populations, and can infect humans. </p><p>We hypothesized that CPXV would have novel mechanisms of evading the immune response that other orthopoxviruses lack, which may exert maximal effect in the context of antigen presenting cells such as DCs, allowing for discovery of novel viral strategies of immune evasion. To test this, CPXV was used to infected mouse bone marrow-derived DCs (BMDCs), and the effect of the virus on DC survival, expression of T-cell costimulatory molecules and cytokine production was determined. The effects of vaccinia virus strain Western Reserve (VV), the prototype of the species, and modified vaccinia virus strain Ankara (MVA), a promising vaccine vector, on mouse BMDCs were also determined. Confirming the hypothesis that CPXV would have different effects on mouse BMDCs from other orthopoxviruses, BMDCs infected with CPXV survived longer in culture than those infected with MVA or VV. In addition, CPXV specifically downregulated MHC I, MHC II, CD40, and CD86, and induced production of significant levels of IL-6 and IL-10.</p><p>Because IL-10 has many suppressive effects on the immune system, inducing IL-10 may provide a selective advantage to CPXV in vivo. To examine the role of IL-10 in a CPXV infection, wild type and IL-10 deficient mice were infected intranasally with CPXV. The effect of CPXV infection on disease morbidity, viral loads, inflammation and the protective immune response was determined. As expected, IL-10 was important in controlling inflammation during CPXV infection, but there was no effect on viral replication or clearance. Surprisingly, IL-10 was important in generation of a protective memory response to CPXV, which may reflect a novel role for IL-10 in the immune response.</p> / Dissertation

<p>Herein we detail the progress made at understanding the overall process of CD8<super>+</super> T-lymphocyte noncytolytic antiviral response (CNAR). This response is comprised of 3 key components, the virus, the effector cell and the target cell, each of which contribute to noncytolytic suppression. During the course of CNAR, the effector cells express antiviral factors that induce intracellular events in the target cell resulting in host-pathogen interactions that inhibit HIV-1 gene expression. The goal of this work was to clarify each step of the process of noncytolytic suppression. </p><p>The effector cell was examined to understand the regulation of antiviral factors and to construct a profile of the factors expressed during CNAR. CD8<super>+</super> T-lymphocytes from HIV-1 infected individuals express unidentified factors that suppress viral replication by inhibiting HIV-1 gene expression. Understanding the regulation of these antiviral CD8<super>+</super> T cell-derived factors can provide important insights into how to elicit these factors with therapeutic regimens. For a small subset of human genes, histone deacetylases (HDACs) are epigenetic regulators that condense chromatin to repress transcription. We examined the role of epigenetics in modulating the HIV-1 suppressive factors expressed by primary CD8<super>+</super> T cells from subjects naturally controlling virus replication. HIV-1 suppression by CD8<super>+</super> T-lymphocytes from virus controllers was reversed up to 40% by the addition of an HDAC inhibitor. Therefore, histone deacetylation within CD8<super>+</super> T-lymphocytes was necessary for potent suppression of HIV-1 infection.</p><p>Blocking HDACs impairs the ability of CD8<super>+</super> T-lymphocytes to repress HIV-1 transcription, demonstrating the expression of the suppressive factors is regulated by epigenetics. We used this tool to identify the potential antiviral factors that result in decreased noncytolytic suppression. Through real-time PCR analysis of 164 genes we identified 4 genes in primary CD8<super>+</super> T-lymphocytes from a virus controller, and 12 genes in a CD8<super>+</super> T-cell line that were greatly downregulated in response to a HDAC inhibitor. Additionally, we analyzed the chemokine and cytokine profile of these two cell types to characterize what molecules these cells secrete during CNAR. MIP-1 Beta, MIP-1 Alpha, IP-10, and MIG correlated most strongly with the magnitude of CNAR (<italic>p</italic> < 0.0001). </p><p>The response of the target cell to the antiviral factors was analyzed to better understand how CD8<super>+</super> T cell antiviral factors exert suppressive activity on the HIV-1 genome in an infected cell. Noncytolytic suppression was not dependent on epigenetic changes within the target cells, as HDAC1 within the target cell was dispensable, and histone acetylation at the HIV-1 LTR remained unchanged in the presence of CD8+ T-lymphocytes. </p><p>The genetic elements within HIV-1 and the viral protein Tat were investigated to provide insight into resistance to CNAR. Two virus isolates from the same individual with contrasting sensitivities to CNAR were investigated to identify the genetic elements that confer these phenotypes. Sequence analysis of the two isolates identified mutations in the exon splicing silencers (ESS) 2 and 3 in these viruses. ESS2 and 3 are thought to control splicing of HIV-1 Tat, however levels of spliced Tat RNA levels did not differ between the two isolates. The introduction of the ESS2 mutation into a heterologous HIV-1 isolate moderately boosted resistance to CNAR, suggesting a function for the mutation apart from spliced Tat RNA levels. </p><p>In total, a comprehensive analysis of each component of CNAR is discussed here to enhance the overall understanding of the mechanisms of CNAR.</p> / Dissertation

Thesis (Ph.D.)--Brown University, 2008. / Vita. Advisor: Walter J. Atwood. Includes bibliographical references (leaves 172-175).

Réovirus, connu sous le nom REOLYSIN®, est présentement à l'étude à titre d'agent oncolytique. Or, la spécificité du virus pour les cellules cancéreuses pourrait être optimisée par une modification au niveau de la protéine d'attachement σ1. La présente étude vise à démontrer qu'une telle amélioration est possible par l'utilisation de la méthode nouvellement décrite de génétique inverse. Par cette technique, il est possible d'ajouter un polypeptide d'une longueur de quarante acides aminés à l'extrémité C-terminale de σ1. Il est aussi possible d'engendrer des virus mutés en leur site d'activité mucinolytique. Les virus nouvellement créés démontrent une efficacité de réplication diminuée, mais demeurent infectieux. Contrairement aux méthodes traditionnellement utilisées avec réovirus, la méthode de génétique inverse permet de conserver les mutations engendrées, par substitution ou addition, au cours des cycles de réplication. Une telle étude démontre qu'il serait possible de modifier le tropisme de réovirus. / Reovirus, also known under the tradename REOLYSIN®, is currently under clinical trial as an oncolytic agent. The specificity of the virus for transformed (cancerous) cells could be improved by modifications of the σ1 attachment protein. This study presents two ways to achieve this improvement by using a newly developed method of reverse genetics for double-stranded RNA viruses. As a proof of concept, we engineered the addition of an exogenous polypeptide of forty amino-acids to the C-terminal end of σ1. Subsequently, we created viruses with compromised mucinolytic activities by introducing amino-acids substitutions in the appropriate genetic sites. A decreased replicative efficacy was observed in all experimentally generated viruses, despite maintaining their infectious capacity. Reverse genetics allows the generation of viruses that retain their mutations throughout successive replicative cycles, which was impossible with traditionnal techniques. This project demonstrates the feasibility of tropism modification by using the reverse genetics method.