ABSTRACT: The innate immune response to 1st generation adenoviral vectors was ascertained through high level infection of primary cells from a inbred mouse strain, replete with an intact and functioning TLR system. Keywords: Immune Response
Project description:The innate immune response to 1st generation adenoviral vectors was ascertained through high level infection of primary cells from a inbred mouse strain, replete with an intact and functioning TLR system. Experiment Overall Design: Quiescent murine C57/Bl6 fibroblasts were Ad (E1-E3-LacZ at MOI=300) or Mock infected. Sixteen hrs post-infection, RNA was harvested, transcribe to cDNA and bitotin-labeled, and hybridized to Mu11k A and B arrays.
Project description:While recent studies of immunity have uncovered many aspects of the innate immune system, there has been precious little investigation of the cellular response to viruses in vivo. To this end, we exploited high titer adenoviral (Ad) vectors to investigate the cellular response to non-enveloped viral infection in vivo. Our results indicate a potent cellular transcriptome response early after infection, with global assessments revealing significant dysregulation in ~15% of measured transcripts derived from Ad infected tissue. Transcriptome analysis revealed a complex interplay between the innate and adaptive responses, with suppression of metabolism and mitochondrial genes akin to those observed when mice are challenged with LPS. But despite this common response profile, there were many unique aspects of the Ad dependent trancriptome response, including upregulation of several RNA regulatory mechanisms and apoptosis-related pathways, accompanied by suppression of lysosome, endocytic, Wnt, and Calcium signaling pathways. Investigations into the TLR pathway using MyD88KO mice revealed that Ad induction of the TLR, MAPK, and cytokine receptor genes are significantly dependent on MyD88, as well as five other functional gene modules (mitochondrial, RNA regulation, cell cycle and growth, extracellular, and immune response genes). Absence of MyD88 also resulted in a greatly diminished Th1 response and acute phase response at later time points, confirming the important role MyD88 plays as an anti-adenoviral immunity amplifier and regulator in vivo. However, continued activation of immune response genes in Ad infected MyD88KO mice indicates that MyD88 is not the only component of the Ad ‘sensing mechanism’ in vivo. Keywords: Infectious response, pathogen response
Project description:Understanding host responses to viral gene therapy vectors is necessary for the development of safe and efficacious in vivo gene transfer agents. We describe the use of high-density spotted complementary DNA microarrays in monitoring the in vivo host transcriptional responses in mouse liver upon administration of either a "first-generation"adenoviral (Ad) vector, a helper-dependent "gutless" adenoviral (HD) vector, or an adeno-associated viral (AAV) vector containing human factor IX (hFIX) expression cassettes. Since HD and AAV do not contain any viral genes, they allow us to assess the host response to the viral capsid and packaged nonviral DNA in whole animals. Comparison of the host response to Ad and HD helps assess the importance of leaky adenoviral gene expression. While all three vectors induced characteristic temporally sequenced programs of gene expression, the gene expression programs induced by the Ad and HD adenovirus vectors were remarkably similar, including the induction of a prominent type I interferon (IFN)-dependent cluster within 6 hours of administration. In contrast, the AAV-based vector caused far fewer alterations of host-gene expression. Our results indicate that recognition of the Ad capsid or double-stranded DNA (of nonviral origin) in the vector elicits a robust type I IFN response that is, however, not elicited by AAV-derived vector transduction.
Project description:Understanding host responses to viral gene therapy vectors is necessary for the development of safe and efficacious in vivo gene transfer agents. We describe the use of high-density spotted complementary DNA microarrays in monitoring the in vivo host transcriptional responses in mouse liver upon administration of either a "first-generation"adenoviral (Ad) vector, a helper-dependent "gutless" adenoviral (HD) vector, or an adeno-associated viral (AAV) vector containing human factor IX (hFIX) expression cassettes. Since HD and AAV do not contain any viral genes, they allow us to assess the host response to the viral capsid and packaged nonviral DNA in whole animals. Comparison of the host response to Ad and HD helps assess the importance of leaky adenoviral gene expression. While all three vectors induced characteristic temporally sequenced programs of gene expression, the gene expression programs induced by the Ad and HD adenovirus vectors were remarkably similar, including the induction of a prominent type I interferon (IFN)-dependent cluster within 6 hours of administration. In contrast, the AAV-based vector caused far fewer alterations of host-gene expression. Our results indicate that recognition of the Ad capsid or double-stranded DNA (of nonviral origin) in the vector elicits a robust type I IFN response that is, however, not elicited by AAV-derived vector transduction. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.
Project description:Non-coding small RNAs are involved in viral life cycles. Adenovirus-encoding small RNAs, virus-associated RNAs (VA RNAs), are transcribed throughout the replication process, and the transcript levels depend on the copy numbers of the viral genome. Although the VA RNA transcription starts immediate early phase, little is known about the function in the early phase. Here we applied replication-deficient adenovirus vectors (AdVs) and novel VA-RNA deleted AdVs to analyze expression change of cellular gene mediated by VA RNAs. Human lung cell, A549, were infected by VA(+) or VA(-) adenoviral vectors. After the designed preiod of infection, cells were harvested and RNA was extacted, followed by DNA microarray analysis.
Project description:Viral vectors are attractive vaccine platforms that elicit robust innate and adaptive immune responses; however, viral vector vaccine candidates vary greatly in their ability to induce protective immunity. Ad5 vectors elicit robust CD8+ T cell responses but typically characterized by an exhausted phenotype. The mechanisms by which Ad5 vectors induce dysfunctional CD8+ T cells have not been fully elucidated. Here we demonstrate that Ad5 vectors, but not Ad26 vectors, elicit exhausted antigen-specific IL-10+PD1+ CD4+ T cells with a dysfunctional transcriptional profile, and these cells effectively suppress CD8+ T cells responses in vivo. Induction of inhibitory CD4+ T cells by Ad5 vectors was associated with increased IL-27 expression, and IL-27 blockade improved CD4+ T cell polyfunctionality. Together our data highlight a novel role for IL-27 in regulating responses to viral vector vaccines.
Project description:Mice were injected intravenously with 1st generation or 2nd generation Ad vectors (each bearing a LacZ transgene) or mock infected (controls) to completely transduce the liver. At different time points post-injection (6 hours, 3 days, and 3 weeks (21 days)), the liver was harvested and the overall transcriptome response to viral infection was assayed to measure the cellular immune response. Keywords: Immune Response
Project description:Viral vectors are attractive vaccine platforms that elicit robust innate and adaptive immune responses; however, viral vector vaccine candidates vary greatly in their ability to induce protective immunity. Ad5 vectors elicit robust CD8+ T cell responses but typically characterized by an exhausted phenotype. The mechanisms by which Ad5 vectors induce dysfunctional CD8+ T cells have not been fully elucidated. Here we demonstrate that Ad5 vectors, but not Ad26 vectors, elicit exhausted antigen-specific IL-10+PD1+ CD4+ T cells with a dysfunctional transcriptional profile, and these cells effectively suppress CD8+ T cells responses in vivo. Induction of inhibitory CD4+ T cells by Ad5 vectors was associated with increased IL-27 expression, and IL-27 blockade improved CD4+ T cell polyfunctionality. Together our data highlight a novel role for IL-27 in regulating responses to viral vector vaccines. Splenic CD45.2+ OT-II TCR-Tg CD4 T cells from CD45.1+ B6 mice immunized with Ad5-OVA or Ad26-OVA were purified by FACS on day 10 post-immunization
Project description:human bronchial smooth muscle cells were either infected with empty adenoviral type 5 vector at 40 MOI for 4 days, or Mock infected. Each sample set was then stimulated with a mixture of 10ng/mL IL-1beta, TNF-alfa, and gamma-IFN for 20 hours prior to RNA harvest. RNA samples were split, and the series consists of 3 slides, 2 of which are dye flipped. Keywords: parallel sample
Project description:In order to identify targets for HDAC4, NRVM were infected with adenoviral vectors encoding beta-Galactosidase or Flag- HDAC4, and incubated in serum free or 10% fetal calf serum containing growth medium for 48 hrs. NRVM were infected with adenoviral vectors encoding beta-Galactosidase (control) or Flag- HDAC4 (experiment), and incubated in serum free or 10% fetal calf serum containing growth medium for 48 hrs. 2 biological samples of each condition were analyzed.