Interferon-beta Gene Research Articles

Enteric coronavirus nsp2 is a virulence determinant that recruits NBR1 for autophagic targeting of TBK1 to diminish the innate immune response

ABSTRACT Non-structural protein 2 (nsp2) exists in all coronaviruses (CoVs), while its primary function in viral pathogenicity, is largely unclear. One such enteric CoV, porcine epidemic diarrhea virus (PEDV), causes high mortality in neonatal piglets worldwide. To determine the biological role of nsp2, we generated a PEDV mutant containing a complete nsp2 deletion (rPEDV-Δnsp2) from a highly pathogenic strain by reverse genetics, showing that nsp2 was dispensable for PEDV infection, while its deficiency reduced viral replication in vitro. Intriguingly, rPEDV-Δnsp2 was entirely avirulent in vivo, with significantly increased productions of IFNB (interferon beta) and IFN-stimulated genes (ISGs) in various intestinal tissues of challenged newborn piglets. Notably, nsp2 targets and degrades TBK1 (TANK binding kinase 1), the critical kinase in the innate immune response. Mechanistically, nsp2 induced the macroautophagy/autophagy process and recruited a selective autophagic receptor, NBR1 (NBR1 autophagy cargo receptor). NBR1 subsequently facilitated the K48-linked ubiquitination of TBK1 and delivered it for autophagosome-mediated degradation. Accordingly, the replication of rPEDV-Δnsp2 CoV was restrained by reduced autophagy and excess productions of type I IFNs and ISGs. Our data collectively define enteric CoV nsp2 as a novel virulence determinant, propose a crucial role of nsp2 in diminishing innate antiviral immunity by targeting TBK1 for NBR1-mediated selective autophagy, and pave the way to develop a new type of nsp2-based attenuated PEDV vaccine. The study also provides new insights into the prevention and treatment of other pathogenic CoVs. Abbreviations: 3-MA: 3-methyladenine; Baf A1: bafilomycin A1; CoV: coronavirus; CQ: chloroquine; dpi: days post-inoculation; DMVs: double-membrane vesicles; GABARAP: GABA type A receptor-associated protein; GFP: green fluorescent protein; GIGYF2: GRB10 interacting GYF protein 2; hpi: hours post-infection; IFA: immunofluorescence assay; IFIH1: interferon induced with helicase C domain 1; IFIT2: interferon induced protein with tetratricopeptide repeats 2; IFITM1: interferon induced transmembrane protein 1; IFNB: interferon beta; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; NBR1: NBR1 autophagy cargo receptor; nsp2: non-structural protein 2; OAS1: 2’-5’-oligoadenylate synthetase 1; PEDV: porcine epidemic diarrhea virus; PRRs: pattern recognition receptors; RIGI: RNA sensor RIG-I; RT-qPCR: reverse transcription quantitative polymerase chain reaction; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious doses; VSV: vesicular stomatitis virus.

Abstract LB116: Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors

Abstract In solid cancers, an immunosuppressive tumor microenvironment (TME) restrains immunotherapy. Induction of immunogenic cell death in tumor cells and simultaneously programming the TME from immunosuppressive to inflammatory employing dsRNA agonists for endosomal Toll-like receptor 3 (TLR3) represents an appealing in situ immunomodulation strategy. However, uptake of dsRNA by tumor cells is limited and systemic administration is linked to adverse side effects. Here we describe an exceptional therapeutically effect of systemically delivered TLR3 agonist RIBOXXOL using a recently developed antibody-guided nanoparticle-system designated “Rapid Inducer of Cellular Inflammation and Apoptosis” (RICIA) for targeting epidermal growth factor variant III (EGFRvIII) on tumor cells. We suggest that systemically applied monotherapy with RICIA might be effective in experimental solid tumors. This includes, (i) preferential enrichment of the immune-stimulatory RIBOXXOL and (ii) subsequent induction of a type-I interferon response in tumor cells expressing TLR3, and (iii) synergistic inflammation of the TME through TLR3 agonist thereby expediting anti-tumor immune responses. The effects of RICIA-treatment were comprehensively assessed in vitro and in murine syngeneic epidermal growth factor receptor variant III (EGFRvIII)-positive SMAvIII gliomas. Our in vitro studies confirm uptake of RICIA by EGFRvIII-mediated endocytosis in human and murine glioma cells and furthermore demonstrate TLR3-triggered type-I interferon release and mRNA signatures indicative of an innate immune response against virus. We show in the SMAvIII tumor model that monotherapy with picomolar amounts of RICIA induces regression of subcutaneous (s.c.) tumors leading to increase in survival time and results in complete tumor clearance in a significant fraction of mice accompanied by immune memory when compared to RICIAs equipped with an unspecific control antibody. Mechanistically, RICIA treatment reshapes the immunosuppressive tumor environment by mimicking viral infection leading to increased expression of the interferon beta (IFN-β) gene (Ifnb1), by subsequent induction of an inflammatory state characterized by RNA-expression signatures indicating induction of apoptosis, interleukin 1beta (IL-1β) signaling, and programming immunosuppressive M2 TAMs to activated pro-inflammatory M1 TAMs. In addition, a single stereotactic injection of RICIA into established intracranial glioma significantly improves mean survival time of treated mice and is able to induce complete tumor clearance thus showing therapeutic potency of RICIA in a relevant pre-clinical setting for glioma. Thus, we show that targeted activation of TLR3 represents a promising therapeutic strategy for immune-modulation of tumors with the prospect to elicit long-lasting anti-tumor immune responses. Citation Format: Achim Temme, Alexander Hagstotz, Isabell Schau, Michael Seifert, Duran Sürün, Stefan Zimmermann, Frank Buchholz, Luise Rupp, Rebekka Wehner, Marc Schmitz, Gabriele Schackert, Ilker Y. Eyüpoglu. Antibody-TLR3 agonist-conjugates induce innate immune responses and immune memory eradicating experimental EGFRvIII-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB116.

Abstract 2809: Bifidobacterium species BB_003 identified through metatranscriptomic analysis with experimentally validated antitumor actions

Abstract Introduction & Objectives: A host's microbiome influences both gene expression and phenotype. Whole transcriptome sequencing (WTS) provides information about the microbiome in addition to gene expression. This research probes the correlation between microbial abundance, host gene transcription, and therapeutic efficacy, pinpointing a beneficial microbial strain and its mechanism of action. Materials & Methods: Bladder tissue biopsies from NMIBC patients underwent WTS and subsequent bioinformatic analysis. After in silico evaluation, in vitro assays including WST, quantitative PCR, and immune cell profiling were performed on mouse splenocytes. These assays were followed by in vivo assessment in both subcutaneous and orthotopic murine tumor models. Results: Among 59 biopsies, non-recurrence correlated with heightened presence of Lactococcus species LC_001 and Bifidobacterium species BB_003. Notably, BB_003 was associated with an extended recurrence-free survival (RFS). Gene set enrichment analysis (GSEA) implicated BB_003 in mediating a pro-inflammatory cascade, accentuating macrophages and interferons. In vitro WST assays showed the antitumor potential of BB_003_β, characterized by upregulated pro-inflammatory gene transcription. This strain increased effector CD8 T cells and M2 macrophages. In vivo, direct live BB_003_β introduction into subcutaneous tumors curtailed tumor proliferation. An increase in effector CD8 T cell populations within tumors and elevated cDC1 and M1 macrophage counts in tumor-draining lymph nodes were observed. Intravesical administration of live bacteria markedly decreased bladder weight, amplifying interferon alpha and beta gene expressions compared to controls. Conclusions: According to this study, BB_003_β inhibits tumor growth and is involved in immune response. Our study confirmed the antitumor properties of BB_003_β and suggested that cytokine and immune cell stimulation play a key role in its mechanism of action. Augmented insights warrant further exploration, particularly in synergy with intravesical BCG treatment. Citation Format: Xuan-Mei Piao, Kyungchan Min, Young Joon Byun, Chuang-Ming Zheng, Seon-Kyu Kim, Seong-Hwan Park, Sungmin Moon, Kyeong Kim, Ho Won Kang, Won Tae Kim, Seok Joong Yun, Hansoo Park. Bifidobacterium species BB_003 identified through metatranscriptomic analysis with experimentally validated antitumor actions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2809.

Radiovirotherapy at twenty

Radiovirotherapy at twenty

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Establishment of Stable Chinese Hamster Ovary Cells Expressing Codon-Optimized Human Interferon-β Gene

Background: Codon optimization is an efficient approach to achieve a higher level of heterologous gene expression and generate productive recombinant mammalian cell lines. In our previous work, based on the codon usage preference of Chinese hamster ovary (CHO) cells, a codon-optimized human interferon-beta (opt-hIFN-β) gene was redesigned and transiently expressed in a suspension-adapted CHO (CHO-s) cell line. Our results indicated a 2.8-fold increase in the expression level of the codon-optimized gene compared to the unmodified sequence. Objectives: In the current work, based on our previous results, a stable CHO-K1 cell line expressing the opt-hIFN-β gene was engineered, in which the opt-hIFN-β gene expression was confirmed by dot and western blotting analyses. Methods: The designed opt-hIFN-β sequence was digested and cloned into a pcDNA3.0 shuttle vector downstream to the cytomegalovirus (CMV) promoter. The verified recombinant plasmid was then linearized and transfected into a CHO-K1 cell line to integrate the opt-hIFN-β gene into the CHO-K1 genome. The transfected cells were then grown under the selective pressure of 450 µg/mL of G418 to develop a stable CHO-K1 cell line expressing the opt-hIFN-β gene. The enzyme-linked immunosorbent assay (ELISA) and dot and western blotting analyses were carried out to verify hIFN-β protein expression. Results: ELISA and dot and western blotting analyses confirmed the expression of hIFN-β in the stably-transfected CHO-K1 cells. Conclusions: Stable expression of the opt-hIFN-β gene in the CHO-K1 cell line was verified by ELISA and dot and western blotting analyses. This study was a pioneering work for further production of recombinant hIFN-β in the bioreactor.

Anti-dengue activity of synthetic peptides increases antiviral interferon-beta genes in LLC-MK2 cells

Dengue virus (DV) causes dengue diseases in human via the Aedes mosquitoes. Fatal cases of dengue hemorrhagic fevers show a complication of acute kidney injury. Previous reports revealed that synthetic DV2(413-440), DV2(413-447), and DV2(419-447) peptides inhibit the dengue envelope proteins in Aedes albopictus C6/36 cells, but nothing is known about the effect of these peptides on mammalian cells in relation to adaptive and innate immunities. In this study, the anti-dengue activity of these synthetic peptides in Rhesus monkey kidney LLC-MK2 cells was investigated at the cellular and molecular levels. Moreover, the effects of these synthetic peptides on apoptotic caspase-10, pro-inflammatory interleukin-1beta, tumor necrosis factor-alpha, and antiviral interferon-beta genes in LLC-MK2 cells were also sought. The results revealed the maximum non-toxic doses of DV2 (413-440), DV2 (413-447), and DV2 (419-447) in LLC-MK2 cells, with values of 77.88± 0.52, 47.01±1.32, and 63.82±1.97 µM, respectively. At 25 µM concentration, synthetic DV2 (413-447) and DV2 (419-447) peptides showed 100% plaque inhibition in simultaneous treatment. By contrast, these peptides showed ≤58% plaque inhibition in pre- and post-treatment at 7 days post-incubation. These synthetic peptides also inhibited Dengue 2 virus, apoptotic caspase-10, pro-inflammatory tumor necrosis factor-alpha, and interleukin-1beta genes. On the other hand, these peptides upregulated the antiviral interferon-beta gene in innate immunity. This study is the first report to reveal the anti-dengue activity of synthetic DV2 (413-440), DV2 (413-447), and DV2 (419-447) peptides in adaptive and innate immunity.

Exploration of p53 plus interferon-beta gene transfer for the sensitization of human colorectal cancer cell lines to cell death

ABSTRACT While treatments for colorectal cancer continue to improve, some 50% of patients succumb within 5 years, pointing to the need for additional therapeutic options. We have developed a modified non-replicating adenoviral vector for gene transfer, called AdRGD-PG, which offers improved levels of transduction and transgene expression. Here, we employ the p53-responsive PG promoter to drive expression of p53 or human interferon-β (hIFNβ) in human colorectal cancer cell lines HCT116wt (wtp53), HCT116−/- (p53 deficient) and HT29 (mutant p53). The HCT116 cell lines were both easily killed with p53 gene transfer, while combined p53 and hIFNβ cooperated for the induction of HT29 cell death and emission of immunogenic cell death (ICD) markers. Elevated annexinV staining and caspase 3/7 activity point to cell death by a mechanism consistent with apoptosis. P53 gene transfer alone or in combination with hIFNβ sensitized all cell lines to chemotherapy, permitting the application of low drug doses while still achieving significant loss of viability. While endogenous p53 status was not sufficient to predict response to treatment, combined p53 and hIFNβ provided an additive effect in HT29 cells. We propose that this approach may prove effective for the treatment of colorectal cancer, permitting the use of limited drug doses.

Response of human melanoma cell lines to interferon-beta gene transfer mediated by a modified adenoviral vector

Since melanomas often retain wild type p53, we developed an adenoviral vector, AdRGD-PG, which provides robust transduction and transgene expression in response to p53. Previously, this vector was used for interferon-β gene transfer in mouse models of melanoma, resulting in control of tumor progression, but limited cell killing. Here, the AdRGD-PG-hIFNβ vector encoding the human interferon-β cDNA (hIFNβ) was used to transduce human melanoma cell lines SK-MEL-05 and SK-MEL-147 (both wild type p53). In vitro, cell death was induced in more than 80% of the cells and correlated with elevated annexinV staining and caspase 3/7 activity. Treatment with hIFNβ promoted cell killing in neighboring, non-transduced cells, thus revealing a bystander effect. In situ gene therapy resulted in complete inhibition of tumor progression for SK-MEL-147 when using nude mice with no evidence of hepatotoxicity. However, the response in Nod-Scid mice was less robust. For SK-MEL-05, tumor inhibition was similar in nude and Nod-Scid mice and was less efficient than seen for SK-MEL-147, indicating both cell type and host specific responses. The AdRGD-PG-hIFNβ vector provides extensive killing of human melanoma cells in vitro and a potent anti-tumor effect in vivo. This study provides a critical advance in the development of our melanoma gene therapy approach.

BVDV-1 induces interferon-beta gene expression through a pathway involving IRF1, IRF7, and NF-κB activation

The bovine viral diarrhea virus (BVDV-1) is a pathogen with the capacity to modulate the interferon type I system. To further investigate the effects of BVDV-1 on the production of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the IFNbeta expression profiles were analyzed.The results showed that cpBVDV-1 was able to induce the production of IFNbeta in a way similar to polyinosinic-polycytidylic acid, but with less intensity. Interestingly, all cpBVDV-1 activities were blocked by pharmacological inhibitors of the IRF-1, IRF-7, and NF-κB signaling pathway, and the level of IFNbeta decreased at the level of transcript and protein. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that cpBVDV-1 regulates IFNbeta expression in bovines through the activation of several key transcription factors. Collectively, the results suggest that during cpBVDV-1 infection, cross talk is evident between various signaling pathways involved in transcriptional activation of IFNbeta in cattle.

Abstract 5548: Indicators of immune activation upon p14ARF and interferon-beta gene transfer in a human melanoma cell line

Abstract Immunoevasion adopted by tumor cells is an important cancer hallmark and understanding of its mechanisms has generated promising therapeutic approaches. In this context, triggering immunogenic cell death (ICD) is a promising idea since it alarms the immune system by attracting a myriad of cell populations that promote innate and adaptive immune responses. Since melanomas preserve wild-type p53 in over 80% of cases, our group developed a gene therapy strategy that utilizes an adenoviral vector with a p53-responsive promoter (PG). Here we present a critical advance for our approach, examining the immune activating potential of combined p14ARF and IFN-beta gene transfer in human melanoma cells. Our results indicate that IFN-beta alone or in combination with p14ARF was able to induce massive cell death accompanied by caspase 3/7 activation in the human melanoma cell line SKMEL147. In our animal model of in situ gene therapy, s.c. SKMEL147 tumors were established in Nod-scid mice before intratumoral virus injection, revealing tumor inhibition and improved survival in response to IFN-beta alone or in combination with p14ARF. However, this model cannot reveal immune activation and so far the benefit of p14ARF has not been revealed. Strikingly, three markers of ICD (exposition of calrecticulin, secretion of ATP and IFN-beta) were induced only when cells were treated with combined p14ARF and IFN-beta gene transfer. We then used PBMCs derived from normal donors as a source of DCs and lymphocytes. Coculture of DCs with previously-transduced SKMEL147 exhibit a typical phenotype of activation. Finally, coculture of these exposed DCs with lymphocytes results in high-level secretion of proinflammatory cytokines, followed by the increase of IL-10 in the samples treated with combination of p14ARF and IFN-beta. Collectively, our results indicate that the advantage of p14ARF and INF-beta delivered by our adenoviral system is the induction of cell death in human melanoma cells by an immune-activating mechanism. Future studies will include examination of this phenomenon in tumors from melanoma patients and using their own PBMCS. In parallel, the use of PDX model for in situ treatment will also be explored. Citation Format: Otto Luiz Cerqueira, Maria Alejandra Salomon, Elaine Cristina Cardoso, Tharcisio Citrangulo Tortelli, Bryan Eric Strauss. Indicators of immune activation upon p14ARF and interferon-beta gene transfer in a human melanoma cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5548.

Development of the Bispecific Antibody in Fab-scFv Format Based on an Antibody to Human Interferon Beta-1 and Antibody to HER2 Receptor

The development of new therapies for malignant tumors is an urgent task. Currently, the humanized antibody trastuzumab is considered the “gold standard” in the complex treatment of breast tumors with overexpression of HER2, human epidermal growth factor receptor 2. However, in some cases, resistance to the specified preparation is observed. The search for new therapies for HER2-associated tumors seems to be an important area of research. A number of clinical studies are currently underway on the use of human interferon-beta (IFN-beta) in oncology. Most of these studies use viral vectors carrying the interferon-beta gene to reduce the systemic effect of this cytokine. The immunocytokine complex of the bispecific antibody and IFN-beta we developed can also avoid the systemic action of IFN-beta. Part of the development of such a complex is the creation of bispecific antibodies of various formats. Based on the neutralizing B16 antibody to IFN-beta and the trastuzumab (Tz) antibody specific for the HER2 receptor, we obtained various variants of bispecific antibodies in Fab-scFv format. It was shown that the proteins obtained bind and neutralize IFN-beta, and they also bind the HER2 receptor in tumor cell lysates and as a recombinant extracellular domain. Such molecules in the immunocytokine complex can be used as delivery vehicles of IFN-beta to HER2-positive tumor cells.

Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system.

Background and purpose:Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN- β) gene based on the codon preference of the CHO cell in a way to increase the GC content in the third position of each codon.Experimental approach:The nucleotide sequence of the codon-optimized rhIFN-β was synthesized in parallel with the wild-type and expressed transiently in CHO-s cells using Epstein-Bar virus (EBV)-based expression system. The protein expression of the rhIFN-β by codon-optimized and wild-type genes were quantified using ELISA test.Findings / Results:The results indicated a 2.8-fold increase in the expression level of the biologically active form of the rhIFN-β by codon-optimized sequence.Conclusion and implications:These results shed light on the capability of codon optimization to create a stable CHO cell for scaling up the production of recombinant therapeutics such as rhIFN-β.

Peste des Petits Ruminants Virus Nucleocapsid Protein Inhibits Beta Interferon Production by Interacting with IRF3 To Block Its Activation.

Peste des petits ruminants virus (PPRV) is the etiological agent of peste des petits ruminants, causing acute immunosuppression in its natural hosts. However, the molecular mechanisms by which PPRV antagonizes the host immune responses have not been fully characterized. In particular, how PPRV suppresses the activation of the host RIG-I-like receptor (RLR) pathway has yet to be clarified. In this study, we demonstrated that PPRV infection significantly suppresses RLR pathway activation and type I interferon (IFN) production and identified PPRV N protein as an extremely important antagonistic viral factor that suppresses beta interferon (IFN-β) and IFN-stimulated gene (ISG) expression. A detailed analysis showed that PPRV N protein inhibited type I IFN production by targeting interferon regulatory factor 3 (IRF3), a key molecule in the RLR pathway required for type I IFN induction. PPRV N protein interacted with IRF3 (but not with other components of the RLR pathway, including MDA5, RIG-I, VISA, TBK1, and MITA) and abrogated the phosphorylation of IRF3. As expected, PPRV N protein also considerably impaired the nuclear translocation of IRF3. The TBK1-IRF3 interaction was involved significantly in IRF3 phosphorylation, and we showed that PPRV N protein inhibits the association between TBK1 and IRF3, which in turn inhibits IRF3 phosphorylation. The amino acid region 106 to 210 of PPRV N protein was determined to be essential for suppressing the nuclear translocation of IRF3 and IFN-β production, and the 140 to 400 region of IRF3 was identified as the crucial region for the N-IRF3 interaction. Together, our findings demonstrate a new mechanism evolved by PPRV to inhibit type I IFN production and provide structural insights into the immunosuppression caused by PPRV.IMPORTANCE Peste des petits ruminants is a highly contagious animal disease affecting small ruminants, which threatens both small livestock and endangered susceptible wildlife populations in many countries. The causative agent, peste des petits ruminants virus (PPRV), often causes acute immunosuppression in its natural hosts during infection. Here, for the first time, we demonstrate that N protein, the most abundant protein of PPRV, plays an extremely important role in suppression of interferon regulatory factor 3 (IRF3) function and type I interferon (IFN) production by interfering with the formation of the TBK1-IRF3 complex. This study explored a novel antagonistic mechanism of PPRV.

Olea europaea leaves extract downregulate Newcastle disease virus gene expression in cancer cells

Many researches have been made to find antiviral agents from natural sources. Leaf of Olea europaea (OLE) is one of themost important medicinal plants, as it is a well-known source of different phytochemicals. This study was carried out toinvestigate the effect of using Olea europaea leaves extract on the level of viral gene expression (M and F) in Hela cell line byusing the qRT-PCR. Also, we examined the adding of interferon-beta ( IFN-b ) as an antivirus factor and analyzed the expressionof viral and IFN-b genes to identify viral targets for OLE extract. First, we propagated the virus in embryonated chickeneggs and showed to kill embryos in different times intervals (less than 72 hours) with marked sever hemorrhage in infectedembryos, and agglutination activity reflects titer (1024) and 1×10 6.4 (TCID50). Hela cells were cultured and different concentrationof OLE extract was added; cell viability was measured by MTT assay. Gene expression showed that the expression ofthe viral gene was downregulated in the present of OLE extract. Pretreatment with IFN-b downregulated viral gene expressionhigher than the extract, also evaluated the IFN-b concentration by ELISA assay. OLE can be considered as antivirus agent andused this property to help in control Newcastle disease.

Abstract 75: A phase I trial of mesenchymal stem cells transfected with a plasmid secreting interferon beta in advanced ovarian cancer

Abstract Background: Standard treatment for ovarian cancer with a platinum and taxane is effective initially but associated with significant relapse. Our group was first to demonstrate genetically modified MSC preferentially engraft and secrete gene products such as interferon beta (IFNB) at tumor sites, resulting in complete responses in animal models (Studeny M et al. Cancer Res 2002;62:3603; JNCI 2004;96:1593). Methods: We conducted a single-center phase 1 trial to evaluate the safety, feasibility tumor response of mesenchymal stem cells transfected with plasmid secreting IFNB administered via intraperitoneal infusion (IPI) for women with advanced refractory epithelial ovarian cancer. We electroporated MSC derived from male donors with a plasmid vector containing the IFNB gene (MSC-IFNB). MSC-IFNB were then administered on an outpatient basis via IPI. Pretreatment biopsy, peritoneal fluid sample, blood draw, and a CT were performed at enrollment. Results: Three patients were enrolled at dose level 1 (105 MSC/kg) and received 4 weekly IPI of MSC-IFNB between 6-10/2016. After the 4th dose we performed a restaging CT and a biopsy, which was examined by FISH for the presence of male Y-chromosome positive MSCs, for production of IFNB assessed by multispectral multiplexed IHC, presence of injected MSCs, changes in immune cell phenotypes, and tumor cell death. We demonstrated detectable production of IFNB in the serum of patient 1, and at week 4 detection of IFNB in the peritoneal fluid, suggesting injected MSC-IFNB produce IFNB locally. We performed multispectral analysis to identify and quantitate the constituents of the ovarian tumor microenvironment in pre- and post-treatment biopsy specimens. We focused on ovarian tumor cells (CK3/5+,AE1+), as well as stromal cells (αSMA+), and IFNB. We observed in all 3 patients colocalization of IFNB (red) with αSMA-(green,) suggesting MSC homing and production of IFNB at tumor stroma interface. To confirm the presence of injected MSC (derived from male donors), we then analyzed the pre- and post-treatment tumor biopsies for presence of the “XY” chromosome by FISH. We identified “XY” chromosomes MSC only in the post-tumor biopsy, suggesting incorporation of injected MSC at the tumor site. There were no infusion- or MSC-related adverse events and 1 patient had stable disease on CT scan at 4 months after treatment. Conclusion: We demonstrate, for the first time, proof of principle that MSC-IFNB home to and are incorporated into tumors and locally produce beta-interferon after IP injection in patients with ovarian cancer. We anticipate increased responses at higher MSC dose levels and envision MSC-IFNB may be used in combination with immunotherapies and chemotherapy, as we have shown in animal models that MSC-IFNB modulate the immune phenoptype of the tumor, recruit FOXp3 immune cells, and facilitate long-term control of tumor growth and metastases. Citation Format: Michael Andreeff, Frank C. Marini, Shannon N. Westin, Robert L. Coleman, Peter F. Thall, Vivian Aljahdami, Muzaffar H. Qazilbash, Katy Rezvani, Melissa Timmons, Lauren Heese, Rui-Yu Wang, Richard E. Champlin, Elizabeth J. Shpall, Amanda Olson. A phase I trial of mesenchymal stem cells transfected with a plasmid secreting interferon beta in advanced ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 75.

Abstract CT072: First in human (FIH) dose escalation studies of intravenous administration of VSV-IFNβ-NIS (Voyager-V1™) in Stage IV or recurrent endometrial cancer

Abstract Introduction. VSV-IFNβ-NIS is an oncolytic vesicular stomatitis virus (VSV; Rhabdovirus family) with rapid replication kinetics and potent antitumor activity. VSV-IFNβ-NIS encodes the human interferon beta (IFNβ) gene as a STING agonist and the human sodium iodide symporter (NIS) as a reporter gene for tracking the pharmacokinetics (PK) of virus replication in infected tumors. VSV replicates selectively in cancer cells and has promising preclinical antitumor activity across a broad spectrum of cancer types. We report here the safety and correlative data from an FIH trial of intravenous (IV) administration of VSV-IFNβ-NIS in patients with stage IV or recurrent endometrial cancer (EC). Methods. There are two ongoing IV FIH trials using VSV-IFN-NIS, in patients with EC (NCT03120624) and one in patients with hematological malignancies (NCT03017820). In EC, it is a classical 3+3 phase I trial, starting at 5x109 TCID50 through 5x1011 TCID50, given as a single IV dose. The primary objective is safety and tolerability; secondary objectives include monitoring the PK of viral replication through SPECT/CT imaging with NIS gene, viremia, virus shedding, preliminary efficacy, changes in the immune profile of peripheral blood leukocytes, and immunohistochemistry for immune cell infiltrates in tumors. Results. Nine patients have received IV VSV-IFNβ-NIS to date; three with EC and six with hematologic malignancies. The highest dose administered to date is 1.7x1010 TCID50 and dose escalation is ongoing. No DLTs have been observed. Patients experienced the expected infusion related AEs including rigors, chills, nausea, fever, hypotension, and hot flashes. Multiple cytokines increased at 4h post infusion of virus, but most returned to baseline levels by 24h. Viremia was detectable in all patients at the end of infusion, and to varying levels at 30 mins, 1, 2, 4, 24, 48h or 72 hours post virus infusion. No persistent viremia was observed. No infectious virus was recovered in buccal swabs or urine and neutralizing anti-VSV antibodies were present by day 29. Extensive immune phenotyping for T cells, NK, MSDC, myeloid cells performed on peripheral blood cells collected at baseline and at day 3, 8, 15 and 29 post virus infusion showed a trend towards increased PD-1 expression on CD8+ cells. Early IHC data suggests an increase in CD3+ and CD8+ cells in tumor biopsies at day 29 and 3 months in patients with EC treated at the first dose level. Elispot assays for shared EC antigens are pending. Conclusions. IV administration of VSV-IFNβ-NIS up to doses of 1.7x1010 is safe and well tolerated. There is evidence of T cell activation with increased PD-1 expression in CD8+ T cells in the peripheral blood and increased in CD3+ and CD8+ cells in tumor biopsies. Updated results for the EC study will be reported. Citation Format: Jamie Bakkum-Gamez, Matthew S. Block, Nanda Packiriswamy, Bethany A. Brunton, Upreti Deepak, Jonathan M. Mitchell, Lukkana Suksanpaisan, Pamela Atherton, Amylou Dueck, Stephen J. Russell, Martha Q. Lacy, Kah-Whye Peng. First in human (FIH) dose escalation studies of intravenous administration of VSV-IFNβ-NIS (Voyager-V1™) in Stage IV or recurrent endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT072.

Herpes Simplex Virus 1 Inhibits TANK-Binding Kinase 1 through Formation of the Us11-Hsp90 Complex.

The Us11 protein of herpes simplex virus 1 (HSV-1) is an accessory factor with multiple functions. In virus-infected cells, it inhibits double-stranded RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase, RIG-I, and MDA-5. However, its precise role is incompletely defined. By screening a human cDNA library, we showed that the Us11 protein targets heat shock protein 90 (Hsp90), which inactivates TANK binding kinase 1 (TBK1) and antiviral immunity. When ectopically expressed, HSV-1 Us11 precludes TBK1 from access to Hsp90 and interferon (IFN) promoter activation. Consistently, the Us11 protein, upon HSV infection, suppresses the expression of beta interferon (IFN-β), RANTES, and interferon-stimulated genes. This is mirrored by a blockade in the phosphorylation of interferon regulatory factor 3. Mechanistically, the Us11 protein associates with endogenous Hsp90 to disrupt the Hsp90-TBK1 complex. Furthermore, Us11 induces destabilization of TBK1 through a proteasome-dependent pathway. Accordingly, Us11 expression facilitates HSV growth. In contrast, TBK1 expression restricts viral replication. These results suggest that control of TBK1 by Us11 promotes HSV-1 infection.IMPORTANCE TANK binding kinase 1 plays a key role in antiviral immunity. Although multiple factors are thought to participate in this process, the picture is obscure in herpes simplex virus infection. We demonstrated that the Us11 protein of HSV-1 forms a complex with heat shock protein 90, which inactivates TANK binding kinase 1 and IFN induction. As a result, expression of the Us11 protein promotes HSV replication. These experimental data provide a new insight into the molecular network of virus-host interactions.

Perspectives for cancer immunotherapy mediated by p19Arf plus interferon-beta gene transfer

While cancer immunotherapy has gained much deserved attention in recent years, many areas regarding the optimization of such modalities remain unexplored, including the development of novel approaches and the strategic combination of therapies that target multiple aspects of the cancer-immunity cycle. Our own work involves the use of gene transfer technology to promote cell death and immune stimulation. Such immunogenic cell death, mediated by the combined transfer of the alternate reading frame (p14ARF in humans and p19Arf in mice) and the interferon-β cDNA in our case, was shown to promote an antitumor immune response in mouse models of melanoma and lung carcinoma. With these encouraging results, we are now setting out on the road toward translational and preclinical development of our novel immunotherapeutic approach. Here, we outline the perspectives and challenges that we face, including the use of human tumor and immune cells to verify the response seen in mouse models and the incorporation of clinically relevant models, such as patient-derived xenografts and spontaneous tumors in animals. In addition, we seek to combine our immunotherapeutic approach with other treatments, such as chemotherapy or checkpoint blockade, with the goal of reducing dosage and increasing efficacy. The success of any translational research requires the cooperation of a multidisciplinary team of professionals involved in laboratory and clinical research, a relationship that is fostered at the Cancer Institute of Sao Paulo.

Abstract B31: Death of endothelial cells induced by paracrine exposure to factors derived from murine melanoma cells upon interferon-beta gene therapy

Abstract Tumor vasculature plays a central role in tumor progression, making it a therapeutic target. Inhibition of angiogenesis has the potential to slow down tumor progression and inhibit metastasis. Interferon-beta (IFN) is a cytokine that shows antitumor effects, including immune activation. In addition, IFN inhibits angiogenesis by reducing the production of proangiogenic factors and by inducing death of endothelial cells. Gene therapy is showing significant results in cancer treatment including when recombinant adenoviral vectors are used for the delivery of therapeutic genes. In our previous studies, we developed a specialized adenoviral vector for the delivery of IFN to p53-positive cells. We propose that the impact of our cancer gene therapy approach may include the inhibition of angiogenesis. Here we explore the response of endothelial cells exposed to IFN through distinct routes of delivery. The efficient transduction of tEnd cells, a murine endothelial cell line, was confirmed by detection of GFP encoded by the control vector (Ad-GFP). In addition, tEnd cells readily produced IFN, as measured by ELISA, after treatment with Ad-IFN. Unexpectedly, IFN gene transfer did not alter angiogenic characteristics, such as migration or tube formation as evidenced in wound healing and tubulogenesis assays, respectively. Our results also indicate that the gene transfer of IFN does not impact cell viability since treatment did not provoke the accumulation of hypodiploid cells, was not associated with AnnexinV staining, and did not impact clonogenesis or MTT staining. These data indicate that expression of IFN by the transduced endothelial cells may not have any effect, a surprise given IFN's well-known ability to inhibit tumor angiogenesis. We next sought to investigate the impact of IFN treatment in a model that mimics certain aspects of the tumor microenvironment. In the first set of assays, B16 (mouse melanoma, p53wt) tumor cells were transduced, washed to remove excess virus particles, and only after were they cocultivated with tEnd cells, permiting the direct contact of adenoviral transduced tumor cells and non-treated endothelial cells. In order to differentiate the two cell lines, we used tEnd cells marked with the dTomato fluorescent protein (which we call tEndTO). Strikingly, the number of tEndTO cells was drastically reduced in the presence of B16 cells treated with Ad-IFN, suggesting that treated tumor cells promote a paracrine effect that inhibits tEnd cells. This point was further explored by exposing tEnd cells to conditioned medium derived from B16 cells that had been transduced with the recombinant adenoviral vectors before analysis of the viability of the endothelial cells. We observed significantly reduced colony formation and MTT staining accompanied by greatly increased accumulation of hypodiploid cells as well as AnnexinV staining when the tEnd cells were cultivated in conditioned medium derived from Ad-IFN transduced tumor cells, but not Ad-GFP. These data suggest that the exposure of tEnd to the secretome of Ad-IFN tranduced B16 cells results in the death of the endothelial cells in a paracrine fashion. In the context of gene therapy for melanoma, the use of modified adenoviral vectors carrying IFN as the therapeutic gene may be of great interest. Our results show a remarkable paracrine effect on endothelial cells when they are exposed to factors produced by melanoma cells transduced with Ad-IFN. Currently, we are continuing our evaluation of the use of these vectors to inhibit melanoma angiogenesis to cause retardation of tumor growth, though special attention will be given to the differential response associated with the route of IFN delivery and the possibility that additional factors are required to bring about endothelial cell death. Citation Format: Igor de Luna Vieira, Rodrigo Esaki Tamura, Bryan Eric Strauss. Death of endothelial cells induced by paracrine exposure to factors derived from murine melanoma cells upon interferon-beta gene therapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B31.