CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE214751 | GEO | 2023/02/01
REPOSITORIES: GEO
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Project description:Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.
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2019-05-09 | GSE127203 | GEO
Project description:CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX
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Project description:Many viruses potently inhibit host protein synthesis while employing unconventional strategies to sustain their own translation, but how and why certain cellular mRNAs continue to be translated remains unclear. Here, we show that during shutoff by Vaccinia Virus (VacV) several host mRNAs increase in polysome occupancy but few, topped by JUN, result in increased protein abundance. Translation of viral mRNAs depended on the small ribosomal protein, Receptor for Activated C Kinase 1 (RACK1) and the eukaryotic Initiation Factor, eIF3. Cryo-electron microscopy (cryo-EM) further showed that eIF3 bound to virus-modified RACK1-containing 40S subunits that exhibit altered head rotation during infection. By contrast, RACK1 and eIF3 were dispensable for JUN translation. Moreover, structurally distinct 5’ untranslated regions (5’UTRs) in viral versus JUN mRNAs conferred differential eIF3 dependencies. Altogether, we reveal how distinct modes of non-canonical initiation support the production of both host and viral proteins that facilitate poxvirus replication despite infection-induced shutoff.
2024-10-30 | GSE278320 | GEO
Project description:Viral infection both activates stress signaling pathways and redistributes ribosomes away from host mRNAs to translate viral mRNAs. The intricacies of this ribosome shuffle from host to viral mRNAs are poorly understood Here, we uncover a role for the ribosome associated quality control (RQC) factor, ZNF598, during vaccinia virus mRNA translation. ZNF598 acts on collided ribosomes to ubiquitylate 40S subunit proteins uS10 and eS10 initiating RQC-dependent nascent chain degradation and ribosome recycling We show that vaccinia infection in human cells enhances uS10 ubiquitylation indicating an increased burden on RQC pathways during viral propagation. Consistent with an increased RQC demand, we demonstrate that vaccinia virus replication is impaired in cells which either lack ZNF598 or express a ubiquitylation deficient version of uS10 Using SILAC-based proteomics and concurrent RNAseq analysis, we determine that translation and not transcription of vaccinia virus mRNAs is compromised in cells with deficient RQC activity. Additionally, vaccinia virus infection reduces cellular RQC activity, suggesting that co-option of ZNF598 by vaccinia virus plays a critical role in translational reprogramming that is needed for optimal viral propagation.
2021-04-14 | GSE165732 | GEO
Project description:Background Vaccinia virus (VACV) infection induces prominent changes in host cell metabolism. Little is known about the global metabolic reprogramming that takes place in the whole tissue during viral infection. Here, we performed an unbiased longitudinal metabolomics study in VACV-infected mice to investigate metabolic changes in the tissue during infection. We assessed metabolites in homogenized skin over time in the presence or absence of antigen-specific T cells using untargeted mass spectrometry. VACV infection induced several significant metabolic changes, including in the levels of nucleic acid metabolites (reflecting the impact of viral replication on the skin metabolome). Furthermore, monocyte- and antiviral T cell-produced metabolites, including itaconic acid, glutamine, and glutathione, were significantly increased following infection, highlighting the immune response’s contribution to the global skin metabolome. Additional RNA-Seq of infected skin tissue recapitulated transcriptional changes identified via metabolomics. Overall, our study reveals the metabolic balance of viral replication and the antiviral immune response in the skin and identifies metabolic pathways that could contribute to cutaneous poxvirus control in vivo.
2023-11-09 | GSE236603 | GEO
Project description:Human melanoma tumor cells (HS294T) and monocytes (THP-1) were infected with a double deleted (-VGF, -TK) oncolytic vaccinia virus expressing human DAI (DNA-dependent activator of interferon-regulatory factors). Total RNA was collected and gene expresson profiles were determined with Agilent microarray. An oncolytic vaccinia virus that does not express DAI was used to control the effect of DAI and uninfected cells (PBS treated) were used to control the effect of virus infection. In oncolytic virotherapy the ability of the virus to activate the immune system against tumors is nowadays generally understood to be a key mechanism in full eradication of cancer and for long-term anti-tumor effects. We armed an oncolytic vaccinia virus with DAI to increase the immunogenicity and the vaccine potency of the virus. The aim of this study was to study if the expression of DAI by a replicating vaccinia virus would alter the gene expression profile of infected cells and to study what are the differentially expressed genes. Three-condition experiment: vvdd-tdTomato-hDAI vs. vvdd-tdTomato vs. PBS treated cells. 2 cell lines: HS294T tumor cells and THP-1 monocytes. 3 biological replicates of virus infected cells per cell line and 2 uninfected replicates per cell line. HS294T and THP-1 cells were treated with vvdd-tdTomato-hDAI or vvdd-tdTomato control virus, or with PBS only to have an uninfected control. 16 hours after infection total RNA was extracted and whole genome gene pfofiles were analyzed and differentially expressed genes determined.
2016-03-01 | E-GEOD-76208 | biostudies-arrayexpress
Project description:Human melanoma tumor cells (HS294T) and monocytes (THP-1) were infected with a double deleted (-VGF, -TK) oncolytic vaccinia virus expressing human DAI (DNA-dependent activator of interferon-regulatory factors). Total RNA was collected and gene expresson profiles were determined with Agilent microarray. An oncolytic vaccinia virus that does not express DAI was used to control the effect of DAI and uninfected cells (PBS treated) were used to control the effect of virus infection. In oncolytic virotherapy the ability of the virus to activate the immune system against tumors is nowadays generally understood to be a key mechanism in full eradication of cancer and for long-term anti-tumor effects. We armed an oncolytic vaccinia virus with DAI to increase the immunogenicity and the vaccine potency of the virus. The aim of this study was to study if the expression of DAI by a replicating vaccinia virus would alter the gene expression profile of infected cells and to study what are the differentially expressed genes.
2016-03-01 | GSE76208 | GEO
Project description:Successful vaccination against smallpox with conventional vaccinia virus is usually determined by the development of a vesicular skin lesion at the site of vaccinia inoculation, called a ‘take’. Although previous vaccination is known to be associated with attenuation of the take, the immunology that underlies a no-take in vaccinia-naïve individuals is not well understood. We hypothesized that antibody profiling of individuals before and after receiving vaccinia would reveal differences between takes and no-takes that may help better understand the phenomenon. Using vaccinia proteome microarrays and recombinant protein ELISAs we first examined the antibody response in vaccinia-naïve individuals that failed to take after receiving different doses of the replication competent smallpox vaccines, DryVax® and APSV. Most that received diluted vaccine failed to respond, whereas 4 other no-takes receiving diluted vaccine, and 4 receiving undiluted vaccine, mounted an antibody response. Interestingly, their antibody profiles were not significantly different from controls that did show a take. However, we did find elevated antibody titers in no-takes prior to receiving DryVax® that was significantly different from takes. Although the sample size studied was small, we conclude the failure to take in responders correlates with pre-existing immunity of unknown etiology that may attenuate the skin reaction in a way similar to previous smallpox vaccination. Antibody profiling was peformed on sera from vaccinees receiving smallpox vaccines, either Wyeth DryVax or Aventis Pasteur Vaccine ('WetVax') in NIH-sponsored clinical trials. These samples comprise 23 vaccinia naïve no-takes, and 50 vaccinia naïve takes and 25 previoulsy vaccinated takes as controls. Samples from d0 (day of vaccination) and at the peak of the antibody repsonse (d28) were taken from each donor and probed in triplicate against the proteome arrays.
2012-01-09 | E-GEOD-34931 | biostudies-arrayexpress
Project description:Successful vaccination against smallpox with conventional vaccinia virus is usually determined by the development of a vesicular skin lesion at the site of vaccinia inoculation, called a ‘take’. Although previous vaccination is known to be associated with attenuation of the take, the immunology that underlies a no-take in vaccinia-naïve individuals is not well understood. We hypothesized that antibody profiling of individuals before and after receiving vaccinia would reveal differences between takes and no-takes that may help better understand the phenomenon. Using vaccinia proteome microarrays and recombinant protein ELISAs we first examined the antibody response in vaccinia-naïve individuals that failed to take after receiving different doses of the replication competent smallpox vaccines, DryVax® and APSV. Most that received diluted vaccine failed to respond, whereas 4 other no-takes receiving diluted vaccine, and 4 receiving undiluted vaccine, mounted an antibody response. Interestingly, their antibody profiles were not significantly different from controls that did show a take. However, we did find elevated antibody titers in no-takes prior to receiving DryVax® that was significantly different from takes. Although the sample size studied was small, we conclude the failure to take in responders correlates with pre-existing immunity of unknown etiology that may attenuate the skin reaction in a way similar to previous smallpox vaccination.
2012-01-09 | GSE34931 | GEO