http://hdl.handle.net/2144/2448 2013-05-22T21:12:38Z 2013-05-22T21:12:38Z Pfefferle, Susanne Krähling, Verena Ditt, Vanessa Grywna, Klaus Mühlberger, Elke Drosten, Christian http://hdl.handle.net/2144/3388 2012-01-13T07:01:00Z 2009-08-24T00:00:00Z

Reverse Genetic Characterization of the Natural Genomic Deletion in SARS-Coronavirus Strain Frankfurt-1 Open Reading Frame 7B Reveals an Attenuating Function of the 7B Protein in-Vitro and in-Vivo Pfefferle, Susanne; Krähling, Verena; Ditt, Vanessa; Grywna, Klaus; Mühlberger, Elke; Drosten, Christian During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells). In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.

2009-08-24T00:00:00Z Dan, Jennifer M. Wang, Jennifer P. Lee, Chrono K. Levitz, Stuart M. http://hdl.handle.net/2144/3385 2012-01-13T07:00:59Z 2008-04-30T00:00:00Z

Cooperative Stimulation of Dendritic Cells by Cryptococcus neoformans Mannoproteins and CpG Oligodeoxynucleotides Dan, Jennifer M.; Wang, Jennifer P.; Lee, Chrono K.; Levitz, Stuart M. While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4+ T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.

2008-04-30T00:00:00Z Hanley, Timothy M. Blay Puryear, Wendy Gummuluru, Suryaram Viglianti, Gregory A. http://hdl.handle.net/2144/3386 2012-01-13T07:00:59Z 2010-07-01T00:00:00Z

PPARγ and LXR Signaling Inhibit Dendritic Cell-Mediated HIV-1 Capture and trans-Infection Hanley, Timothy M.; Blay Puryear, Wendy; Gummuluru, Suryaram; Viglianti, Gregory A. Dendritic cells (DCs) contribute to human immunodeficiency virus type 1 (HIV-1) transmission and dissemination by capturing and transporting infectious virus from the mucosa to draining lymph nodes, and transferring these virus particles to CD4+ T cells with high efficiency. Toll-like receptor (TLR)-induced maturation of DCs enhances their ability to mediate trans-infection of T cells and their ability to migrate from the site of infection. Because TLR-induced maturation can be inhibited by nuclear receptor (NR) signaling, we hypothesized that ligand-activated NRs could repress DC-mediated HIV-1 transmission and dissemination. Here, we show that ligands for peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor (LXR) prevented proinflammatory cytokine production by DCs and inhibited DC migration in response to the chemokine CCL21 by preventing the TLR-induced upregulation of CCR7. Importantly, PPARγ and LXR signaling inhibited both immature and mature DC-mediated trans-infection by preventing the capture of HIV-1 by DCs independent of the viral envelope glycoprotein. PPARγ and LXR signaling induced cholesterol efflux from DCs and led to a decrease in DC-associated cholesterol, which has previously been shown to be required for DC capture of HIV-1. Finally, both cholesterol repletion and the targeted knockdown of the cholesterol transport protein ATP-binding cassette A1 (ABCA1) restored the ability of NR ligand treated cells to capture HIV-1 and transfer it to T cells. Our results suggest that PPARγ and LXR signaling up-regulate ABCA1-mediated cholesterol efflux from DCs and that this accounts for the decreased ability of DCs to capture HIV-1. The ability of NR ligands to repress DC mediated trans-infection, inflammation, and DC migration underscores their potential therapeutic value in inhibiting HIV-1 mucosal transmission. Author SummaryHeterosexual transmission is the primary mode of HIV transmission worldwide. In the absence of an effective vaccine, there is an increasing demand for the development of effective microbicides that block HIV sexual transmission. Dendritic cells (DCs) play a critical role in HIV transmission by efficiently binding virus particles, migrating to lymph nodes, and transmitting them to CD4+ T cells, a process called trans-infection. In addition, DCs secrete proinflammatory cytokines that create a favorable environment for virus replication. DC maturation by pathogen-encoded TLR ligands or proinflammatory cytokines dramatically increases their capacity to capture HIV, migrate to lymphoid tissue, and trans-infect T cells. Here, we report that signaling through the nuclear receptors PPARγ and LXR prevents DC maturation and proinflammatory cytokine production, as well as migration. In addition, PPARγ and LXR signaling prevents efficient DC capture and transfer of infectious HIV by increasing ABCA1-mediated cholesterol efflux. Our studies suggest that PPARγ and LXR may be targets for drugs that can inhibit specific aspects of HIV mucosal transmission, namely inflammation, migration, and virus capture and transfer. These findings provide a rationale for considering PPARγ and LXR agonists as potential combination therapies with conventional anti-viral microbicides that target other aspects of mucosal HIV transmission.

2010-07-01T00:00:00Z Malherbe, DC Doria-Rose, N Misher, L Beckett, T Blay-Puryear, W Barnett, S Srivastava, I Richardson, B Stamatatos, L Haigwood, NL http://hdl.handle.net/2144/3387 2012-01-13T07:01:00Z 2009-10-22T00:00:00Z

P05-10. Sequential Immunization with a Subtype B HIV-1 Envelope Quasispecies Elicits Broader Neutralization than Vaccination with a Single Envelope Clone Malherbe, DC; Doria-Rose, N; Misher, L; Beckett, T; Blay-Puryear, W; Barnett, S; Srivastava, I; Richardson, B; Stamatatos, L; Haigwood, NL

2009-10-22T00:00:00Z