Human IFN-beta Research Articles

70. Safety, Toxicity and Efficacy of Systemic Recombinant VSV Therapy in Pet Dogs With Spontaneous Cancer

Clinical translation of oncolytic therapy to benefit cancer patients is dependent upon extensive evaluation of viral safety, toxicity and therapeutic efficacy in disease bearing animals. Comparative oncology, the study of naturally occurring cancer in animals, allows evaluation of novel cancer therapies in pet dogs that are similar in size, genetic make-up, physiology and exposure to environmental risk factors to humans. We have previously shown single shot systemic therapy with recombinant Vesicular stomatitis virus (VSV) engineered to express Interferon-beta (IFN) and the sodium-iodide symporter (NIS) in tumor bearing mice results in tumor-specific virus replication that can be monitored noninvasively by SPECT/CT imaging using NIS specific radio-tracer, induces tumor remission and confers significant survival benefit. A comparative oncology approach was utilized to investigate safety, clinical toxicities and therapeutic efficacy of systemic VSV therapy in dogs with spontaneous cancer. A pre-clinical rapid dose-escalation study was performed in healthy lab beagles defined a safe systemic dose range. We initiated evaluation of systemic VSV administration in dogs with spontaneous cancer. 8 dogs with various cancers were treated with VSV expressing either human or canine IFN-beta and NIS. We demonstrate that systemic VSV therapy at a dose of 10(10) TCID is well tolerated in dogs with spontaneous disease. We detected transient hepatotoxicity that resolved spontaneously in 2/8 dogs following systemic VSV-IFN-NIS treatment. No infectious virus was detected in urine or buccal swab samples. Monitoring of disease response indicated that 2/2 dogs with T-cell lymphoma had dramatic disease remission following systemic VSV-hIFN-NIS treatment resulting in partial response, though remission was transient and disease eventually relapsed. Virus pharmacokinetics (PK) studies indicate that dogs with robust responses also had highest viral PK in blood, suggesting therapeutic response is PK dependent. A dose optimization protocol was adopted where gradual dose escalation and multi-dosing protocols are being evaluated. Initial findings indicate two-dose systemic VSV-IFN-NIS therapy in a dog with metastatic Osteosarcoma (OSA) is well tolerated, delays viral decay in blood, and has resulted in improved health of the treated dog with no major disease progression to date. RNAseq analysis of tumor biopsies from treated animals reveals changes in tumor gene expression following systemic VSV therapy. Overall the data demonstrate that recombinant VSV can be safely administered systemically in cancer bearing dogs. Our data suggest that therapeutic response is PK dependent, prompting dose optimization studies that will directly inform and support planned clinical studies to evaluate systemic VSV therapy in patients with relapsed or metastatic cancer.

MONOPEGYLATED HUMAN INTERFERON BE- TA-1A PREPARATION: OPTIMIZATION OF CONDITIONS FOR N-TERMINAL PEGYLATION

A modification of recombinant human IFN beta- 1a by polyethylene glycol at a-amino group of N-terminal methionine has been performed. The PEGylation was conducted using activated butyraldehyde PEG derivative with molecular mass of 30000 Da. As a result of the multifactorial experiment, the dependence of monoPEGylated protein yield on the reaction conditions was demonstrated, and optimal PEGylation conditions were established. We developed a one-step chromatographic purification scheme allowing to obtain the monoPEG-IFN beta-1a conjugate with the above 98% purity . Mass-spectrometric studies showed that the molecular mass of the conjugate is 54130 Da, wherein the N-terminal methionine is associated with one PEG molecule. The obtained PEG-IFN beta-1a has specific antiviral activity, and it can be considered as a perspective candidate to the design of long-acting medicine for the prolonged treatment of multiple sclerosis.

46. Safety, Biodistribution, and Initial Efficacy of ART-I02, an AAV-5 Vector Encoding Human IFN-beta, Delivered Via Intra-Articular Injection in Rhesus Monkeys With Collagen-Induced Arthritis

46. Safety, Biodistribution, and Initial Efficacy of ART-I02, an AAV-5 Vector Encoding Human IFN-beta, Delivered Via Intra-Articular Injection in Rhesus Monkeys With Collagen-Induced Arthritis

Ernest (Pete) Knight Jr. (1932–2013) A Remembrance to a Great Interferon Scientist and Colleague

We lost a great colleague and friend on January 21, 2013, Ernest (Pete) Knight Jr. at the age of 80 years old. We all knew him as Pete, a quiet-spoken, intelligent, and trustworthy man who was also a true gentleman. Pete was born (1932 in Fordyce, Arkansas) and grew up in Arkansas. For those of you who were wondering how he got his nickname “Pete,” it was actually bestowed upon him by a county judge in Arkansas, a colleague of his dad. He attended the University of Arkansas and there he met the love of his life, Patsy. They married in 1955 and went to Illinois where Pete did his graduate work at the University of Illinois (1957–1960). This is where he first developed his interest in protein chemistry and got valuable experience in purifying bacterial proteins. Also during this time he and Patsy had a son named Peter (b. 1960). It was certainly a very busy time for Pete. Following receiving his Ph.D., he was granted funds from the National Institutes of Health (NIH) to continue his protein purification studies as a postdoctoral fellow in Paris, France (1961–1963). It was a wonderful experience for his scientific growth and his family. After his postdoc, Pete joined the DuPont Company in their Central Research Department in Wilmington, Delaware, where he continued to work on bacterial protein purification until 1967. Wanting to move from bacterial to animal proteins, Pete went to Jim Darnell's lab at Albert Einstein Medical School in New York, where he studied animal cells and viruses. This was an important turning point in Pete's scientific journey. Being on the cutting edge of a new field and facing a challenging problem are always exhilarating, and Pete was working at the forefront with interferon (IFN) in his crosshairs. As a protein chemist who had a great deal of experience in protein purification, he took on the challenge of purifying mouse IFN and human IFN-beta and did it successfully (Knight, 1975, 1976a). If you asked Pete what were his major contributions he would say they were “The determination of 2×108 U/mg as the ultimate specific activity for murine and human IFN-beta” (Knight, 1975, 1976a); “The demonstration that murine IFN is a family of at least eight glycoproteins, all having IFN activity” (Knight, 1975); “The demonstration that human IFN-beta is one glycoprotein of molecular weight 20,000” (Knight, 1976a); “The demonstration that pure IFN-beta contains both antiviral and the antiproliferative activity” (Knight, 1976b, 2008). He and his colleagues then went on to sequence the first human IFN-beta with the help of Leroy Hood and Mike Hunkapiller (Knight and others, 1980). This was a true intellectual collaboration. We were proud that our lab at NIH, which was involved in the first sequencing of a human IFN-alpha, and Peter Lengyel's lab, which was involved in sequencing the first mouse IFNs, could participate in such a successful collaboration with Pete and L. Hood and M. Hunkapiller at the California Institute of Technology (Taira and others 1980; Zoon and others 1980). Pete's interest in IFN continued to grow. He discovered and studied a number of IFN-induced genes and proteins (Knight and Korant 1979; Korant and others 1984; Knight and others 1985; Blomstrom and others 1986). Pete shepherded the finances of the International Society for Interferon and Cytokine Research (ISICR) as Treasurer from 1985 to 1998. He was also very active reviewing manuscripts for the Journal of Interferon & Cytokine Research. In 1987 Pete and I co-organized the ISICR Meeting in Washington, DC, to commemorate the 30th anniversary of the discovery of IFN. Pete loved to travel and often that was in conjunction with the interferon meetings. We all have very fond memories of Pete at the meetings and the events surrounding it. Patsy often accompanied him and helped us all enjoy the beauties of the meeting sites. Pete retired from DuPont in 1990 and then went on to work with a new company, Cephalon, that same year, where he continued to provide expert advice for an additional 7 years. In 1997 he and Patsy decided to retire (really), and they went south to Hilton Head, South Carolina. There they had many wonderful years in the sun until they decided to join their son Peter and his family in Kittery, Maine, in 2005. Being together with his family and enjoying his granddaughter Caroline were of great importance to him. He and Patsy enjoyed his last years in Maine very much. Pete died with his family around him, peacefully and with dignity, the way we know he would have wanted it. Goodbye, Pete, we will miss you.

Kinetics of Soluble Mesothelin in Patients with Malignant Pleural Mesothelioma during Treatment

Previous data suggested that serum levels of soluble mesothelin (SM) are related to tumor size and may have prognostic significance in malignant pleural mesothelioma (MPM). We tested the hypothesis that this marker could also be useful for monitoring response to treatment. Serial measurements of SM were determined in 40 patients diagnosed with MPM and subjected to gene-transfer therapy using intrapleural infusion of an adenoviral vector expressing human IFN-beta or conventional treatment (mainly chemotherapy). In patients with baseline SM levels greater than 1 nM/L and disease progression after therapy, SM levels increased by 2.1 nM/L at two, 5.2 nM/L at four and 1.3 nM/L at 6 months. Patients with initial SM below 1 nM/L had a similar but more moderate increase of SM over time. Patients who responded to treatment or were considered stable had an initial small decrease of SM followed by a return to baseline values after 6 months of follow-up. In patients with baseline SM levels greater than 1 nM/L, increasing levels were associated with a significantly shorter median survival than in patients with stable or decreasing SM levels (4.4 vs. 27.7 months; P = 0.012). Increasing serum levels of SM were associated with disease progression and worse outcome, whereas stable or decreasing values suggested response to treatment. If confirmed in larger series, SM could be used to monitor patients with malignant pleural mesothelioma under treatment.

The efficacy of combination therapy using adeno-associated virus—interferon β and trichostatin A in vitro and in a murine model of neuroblastoma

Trichostatin A (TSA) is a potent histone deacetylase inhibitor and has demonstrated significant antitumor activity against a variety of cancer cell lines. Type I interferons have also shown significant antitumor as well as antiangiogenic activity. In this study, we examined the effectiveness of combination therapy of TSA and interferon beta (IFN-beta) on human neuroblastoma cells in vitro and in vivo using a murine model of retroperitoneal neuroblastoma. For in vitro experiments, plated human neuroblastoma cells (NB-1643 and NB-1691) were treated with vehicle or with IFN-beta, TSA, or both for 24 hours. Cytotoxicity was assessed by counting cells and expressing the results as a percentage of controls. Expression of the tumor suppressor p21(Waf1) was assessed by Western blot. For in vivo experiments, retroperitoneal neuroblastomas were established in severe combined immune deficiency (SCID) mice. Interferon beta was given using a gene therapy approach, administering 1.5 x 10(10) particles of an adeno-associated virus vector encoding human IFN-beta (AAV hIFN-beta) via tail vein as a single dose per mouse. Trichostatin A was given at a dose of 5 mg/kg every 48 hours subcutaneously. Treatment groups included controls, AAV hIFN-beta alone, TSA alone, and AAV hIFN-beta together with TSA. Tumor volume was assessed 2 weeks after the treatment began. After 24 hours, treatment with IFN-beta, TSA, and a combination of both resulted in a 45.3%, 68.1%, and 75% reduction in cell count relative to controls in the NB-1691 cell line. In the NB-1643 line, cell counts were reduced by 23%, 58%, and 62.3% respectively. In addition, NB-1691 cells treated with TSA showed increased expression of p21(Waf1) on Western blot. For in vivo experiments, control-, AAV hIFN-beta-, TSA-, and combination-treated tumors had the following final volumes: 1577.7 +/- 264.2 mm(3) (n = 3); 128.5 +/- 74.4 mm(3) (n = 4; P = .0001); 1248.7 +/- 673.9 mm(3) (n = 4; P = .48); and 127.5 +/- 36.8 mm(3) (n = 4; P = .0007), respectively. Neuroblastoma, because of its unique biology, continues to be a challenging tumor to treat, and many times these tumors are refractory to standard chemotherapeutic regimens. These data show that both TSA and IFN-beta inhibit neuroblastoma growth and that the combination may potentially provide a unique way to treat this difficult disease.

Measured Neutralizing Titers of IFN-β Neutralizing Antibodies (NAbs) Can Depend on the Preparations of IFN-β Used in the Assay

An immune response to recombinant human protein therapeutics, including type I interferons (IFNs), has the potential to have a serious negative impact on safety and efficacy. Monitoring of patients for neutralizing antibodies (NAbs) often is advisable. In the case of IFN-beta therapy for multiple sclerosis (MS), we obtained reproducible quantitative titers of NAbs using an improved and well-characterized assay based on a 10-fold reduction of a challenge dose of IFN-beta. However, the observed titer was significantly affected by the preparation of IFN-beta used as the assay challenge. NAb titers obtained using IFN-beta1b averaged 3-5-fold lower than titers of the same sample assayed using either IFN-beta1a or human fibroblast-derived IFN-beta. This was the case whether neutralizing serum was obtained from patients on therapy with IFN-beta1a or IFN-beta1b. The reason for this apparent titer difference is not fully understood but appears to be related to protein folding or other structural properties that differentiate the IFN-beta1b both from commercial IFN-beta1a preparations and from human fibroblast-derived IFN-beta.

Structure of IRF-3 Bound to the PRDIII-I Regulatory Element of the Human Interferon-β Enhancer

Interferon regulatory factor 3 (IRF-3) is a key transcription factor in the assembly of the mammalian interferon-beta (IFN-beta) enhanceosome. We present here the structure of IRF-3 DNA binding domain in complex with the complete PRDIII-I regulatory element of the human IFN-beta enhancer. We show that four IRF-3 molecules bind in tandem to, variably spaced, consensus and nonconsensus IRF sites on the composite element. The ability of IRF-3 to bind these variable sites derives in part from two nonconserved arginines (Arg78 and Arg86) that partake in alternate protein-DNA contacts. We also show that the protein-DNA contacts are highly overlapped and that all four IRF sites are required for gene activation in vivo. In addition, we show that changing the nonconsensus IRF sites to consensus sites creates a more efficient enhancer in vivo. Together, the structure and accompanying biological data provide insights into the assembly of the IFN-beta enhanceosome in mammals.

Adenovirus-mediated interferon-β gene transfer inhibits angiogenesis in and progression of orthotopic tumors of human prostate cancer cells in nude mice

Interferon (IFN)-beta is a multifunctional cytokine. Our previous studies revealed that intratumoral transfer of the murine interferon (IFN)-beta gene inhibited the growth of human and mouse prostate cancer cells in mice. Since IFN-beta activity is species-restricted, we investigated the efficacy and mechanisms of forced expression of human IFN-beta in suppressing the growth of human prostate cancer cells in mice. Orthotopic tumors of PC-3MM2 human prostate cancer cells were forced to express human IFN-beta by intratumoral injection of an adenoviral vector (AdhIFN-beta). Tumor growth and survival of tumor-bearing mice were determined. Cell proliferation and apoptosis were evaluated by immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Angiogenesis and angiogenic molecule expression were evaluated by IHC and quantitative real-time reverse-transcriptional PCR (qRT-PCR). We found that forced expression of human IFN-beta inhibited tumor growth in a dose-dependent manner. An injection of 2 x 10(9) PFU (plaque-forming units) of AdhIFN-beta retarded tumor growth by 90% and prolonged the survival of tumor-bearing mice. Control tumors contained more proliferating cells (PCNA(+)) and fewer apoptotic cells (TUNEL(+)) than did AdhIFN-beta treated-tumors. Treatment with AdhIFN-beta downregulated the expression of interleukin-8 and vascular endothelial cell growth factor-A. Taken together, our data indicated that forced expression of human IFN-beta in human prostate cancer cells significantly inhibited their prostatic growth, which correlated with downregulation of angiogenic molecules and suggested that adenoviral vector-mediated IFN-beta gene therapy could be an effective approach for the management of human prostate cancer.

A possible role of autogenous IFN‐β for cytokine productions in human fibroblasts

It has been already known that human diploid fibroblasts are able to produce not only high levels of IFN-beta but also various kinds of cytokines by poly rI: poly rC, and some inflammatory cytokines are induced by IFN-beta gene activation. We also obtained similar results. However, in our system, cytokine productions were extremely enhanced by treating the cells with a low dose of type 1 IFN and the priming effects on cytokine productions were blocked by cycloheximide similar to those on IFN-beta productions. Most of cytokines were produced later than IFN-beta and synthesis patterns of their mRNA showed the same phenomena. We made clear that cytokine productions by poly rI: poly rC are mediated by secreted IFN-beta at a protein level using a monoclonal antibody against human IFN-beta. Further, it was shown that intra-cellular IFN-beta which is not secreted might also participate in cytokine productions. Meanwhile, IL-1beta induced various kinds of cytokines in human fibroblasts and production time courses of these cytokines were similar to those of poly rI: poly rC induced cytokines. Although secreted IFN-beta was not detected in IL-1beta stimulated culture, expression of IFN-beta mRNA was augmented. These results showed that priming effects of type 1 IFN on cytokine productions by poly rI: poly rC might not be the direct action, but successive IFN-beta production might be essential in the production processes of other cytokines. Further, it was suggested that inducible IFN-beta might also take part in IL-1beta-induced cytokine productions.

N-Glycosylation of Murine IFN-βin a Putative Receptor-Binding Region

Human and mouse genomes contain more than 20 related genes encoding diverse type I interferons (IFNs- alpha/beta), cytokines that are crucial for resistance of organisms against viral infections. Although the amino acid sequences of various IFN-alpha/beta subtypes differ markedly, they are all considered to share a common three-dimensional structure and to bind the same heterodimeric receptor, composed of the IFNAR-1 and IFNAR-2 subunits. Analysis of available mammalian IFN-beta sequences showed that they all carry 1 to 5 predicted N-glycosylation sites. Murine IFN-beta contains three predicted N-glycosylation sites (Asn29, Asn69, Asn76), one of which (Asn29) is located in the AB loop, in a region predicted to interact with the type I IFN receptor. The aim of this work was to test if this site is indeed N-glycosylated and if this glycosylation would affect IFN antiviral activity. We showed that all three N-glycosylation sites predicted from the sequence, including Asn29, carry N-linked sugars. Mutation of individual N-glycosylation sites had a weak negative influence on IFN antiviral activity. In contrast, the complete loss of glycosylation dramatically decreased activity. Our data suggest that interaction of murine IFN-beta with the IFNAR could locally differ from that of human IFN-alpha2 and human IFN-beta.

Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNbeta-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNbeta-related adverse effects in multiple sclerosis.

BackgroundA substantial proportion of multiple sclerosis (MS) patients discontinue interferon-beta (IFNβ) treatment due to various adverse effects, most of which emerge at the early phase after initiation of the treatment and then diminish with time. At present, the molecular mechanism underlying IFNβ-related adverse effects remains largely unknown. The aim of this study is to identify a comprehensive list of early IFNβ-responsive genes (IRGs) in peripheral blood mononuclear cells (PBMC) that may play a key role in induction of adverse effects.MethodsTotal RNA of PBMC exposed to 50 ng/ml recombinant human IFNβ for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis.ResultsAmong 1,258 genes on the array, IFNβ elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNβ markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils.ConclusionIFNβ immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNβ-related early adverse effects in MS patients in vivo.

Diversity and Relatedness Among the Type I Interferons

Type I interferons (IFNs) include the IFN-alpha family of subtypes, IFN-beta, IFN-omega, IFN-tau, IFN-kappa, IFN-lambda, and IFN-zeta. IFN genes lack introns and encode secretory signal peptide sequences that are proteolytically cleaved prior to secretion from the cell. In contrast to the approximately 50% amino acid sequence identity among the human IFN-alpha subtypes, human IFN-alphas share approximately 22% identity with human IFN-beta and 37% identity with human IFN-omega. Many of the conserved residues among the type I IFNs are implicated in receptor recognition and structural integrity. This report provides an update on the gene annotations for the mouse and human IFN gene clusters on chromosome 4 and 9, respectively, with accompanying amino acid sequence alignments. Based on sequence identities, a phylogenic tree analysis for the different mammalian Type I IFNs is also presented, showing the high degree of relatedness among these IFNs. Notably, sequence alignment of the different human and mouse IFN promoter regions reveals different signature patterns for transcription factor binding sites, implying different inducers might differentially activate the transcription of the different IFNs.

Mesenchymal Stem Cells: Potential Precursors for Tumor Stroma and Targeted-Delivery Vehicles for Anticancer Agents

High concentrations of interferon beta (IFN-beta) inhibit malignant cell growth in vitro. However, the therapeutic utility of IFN-beta in vivo is limited by its excessive toxicity when administered systemically at high doses. Mesenchymal stem cells (MSC) can be used to target delivery of agents to tumor cells. We tested whether MSC can deliver IFN-beta to tumors, reducing toxicity. Human MSC were transduced with an adenoviral expression vector carrying the human IFN-beta gene (MSC-IFN-beta cells). Flow cytometry was used to measure tumor cell proliferation among in vitro co-cultures of MSC-IFN-beta cells and human MDA 231 breast carcinoma cells or A375SM melanoma cells. We used a severe combined immunodeficiency mouse xenograft model (4-10 mice per group) to examine the effects of injected MSC-IFN-beta cells and human recombinant IFN-beta on the growth of MDA 231- and A375SM-derived pulmonary metastases in vivo and on survival. All statistical tests were two-sided. Co-culture of MSC-IFN-beta cells with A375SM cells or MDA 231 cells inhibited tumor cell growth as compared with growth of the tumor cells cultured alone (differences in mean percentage of control cell growth: -94.0% [95% confidence interval [CI] = -81.2% to -106.8%; P<.001] and -104.8% [95% CI = -82.1% to -127.5%; P<.001], respectively). Intravenous injection of MSC-IFN-beta cells into mice with established MDA 231 or A375SM pulmonary metastases led to incorporation of MSC in the tumor architecture and, compared with untreated control mice, to prolonged mouse survival (median survival for MDA 231-injected mice: 60 and 37 days for MSC-injected and control mice, respectively [difference = 23.0 days (95% CI = 14.5 to 34.0 days; P<.001]; median survival for A375SM-injected mice: 73.5 and 30.0 days for MSC-injected and control mice, respectively [difference = 43.5 days (95% CI = 37.0 to 57.5 days; P<.001]). By contrast, intravenous injection of recombinant IFN-beta did not prolong survival in the same models (median survival for MDA 231-injected mice: 41.0 and 37.0 days for IFN-beta-injected and control mice, respectively [difference = 4 days, 95% CI = -5 to 10 days; P = .308]; median survival for A375SM-injected mice: 32.0 and 30.0 days for IFN-beta-injected and control mice, respectively [difference = 2 days, 95% CI = 0 to 4.5 days; P = .059]). Injected MSC-IFN-beta cells suppressed the growth of pulmonary metastases, presumably through the local production of IFN-beta in the tumor microenvironment. MSC may be an effective platform for the targeted delivery of therapeutic proteins to cancer sites.

Simultaneous detection of DsRed2-tagged and EGFP-tagged human beta-interferons in the same single cells.

The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon-beta (IFN-beta). Expression plasmids for human IFN-beta tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin-Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20 degrees C) allowed DsRed2-tagged human IFN-beta to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocal microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins.

Epitope specificity of neutralizing antibodies against IFN-beta.

Neutralizing antibodies (NAb), a subset of antibodies against interferon-beta (IFN-beta) that inhibit activation of the IFN-beta receptor, are presumed to bind to the receptor-binding site of IFN-beta. The aim of this study was to identify specific epitopes for human NAb and nonneutralizing antibodies (NNAb) on the IFN-beta molecule. Thirty-one 12-mer peptides and one 11-mer peptide representing the amino acid sequence of the human IFN-beta molecule were used as antigens in an ELISA antibody assay. Significant antibody binding was observed at residues 1-12, 121-132, and 151-162. NNAb and NAb recognized residues 121-132 with equal frequency, but NAb had higher titers. NAb bound significantly more frequently to residues 1-12 and 151-162. There was a significant positive correlation between NAb titers and titers against residues 1-12. Binding to residues 1-12 was more pronounced in patients treated with IFN-beta1a than in patients treated with IFN-beta1b. Our results indicate a qualitative and quantitative difference between NAb and NNAb, with special emphasis on the recognition of different epitopes.

Subcellular trafficking of exogenously expressed interferon-beta in Madin-Darby canine kidney cells.

We have recently demonstrated that when IFN-beta was exogenously expressed in epithelial cells, transiently expressed IFN-beta was predominantly secreted from the cell side to which the transfection was performed, while stably expressed one was almost equally secreted to the apical and basolateral sides. In the present study, we analyzed the subcellular transport of IFN-beta using confocal imaging with green fluorescent protein (GFP)-tagged IFN-beta in Madin-Darby canine kidney (MDCK) cells. Stably expressed and transiently expressed human IFN-beta (HuIFN-beta)-GFPs were seen in upper regions of the nucleus. In stable HuIFN beta-GFP-producing transformants, transiently expressed mouse IFN-beta (MuIFN-beta) was apparently co-localized with the bulk of the constitutive HuIFN beta-GFP proteins at TGN, and a significant quantity of them then appeared to pass into distinct post-TGN vesicles, accepting either type of IFN. Meanwhile, when cells were co-transfected with both expression vectors, transiently expressed both IFNs tended to co-localize not only at TGN but in post-TGN vesicles. These results suggest that stably and transiently expressed IFN-betas, albeit co-localized at TGN, were transported through apparently discriminated post-TGN routes.

Development of recombinant vesicular stomatitis viruses that exploit defects in host defense to augment specific oncolytic activity.

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus normally sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta). Recently, we reported that VSV replicates to high levels in many transformed cells due, in part, to susceptible cells harboring defects in the IFN system. These observations were exploited to demonstrate that VSV can be used as a viral oncolytic agent to eradicate malignant cells in vivo while leaving normal tissue relatively unaffected. To attempt to improve the specificity and efficacy of this system as a potential tool in gene therapy and against malignant disease, we have genetically engineered VSV that expresses the murine IFN-beta gene. The resultant virus (VSV-IFNbeta) was successfully propagated in cells not receptive to murine IFN-alpha/beta and expressed high levels of functional heterologous IFN-beta. In normal murine embryonic fibroblasts (MEFs), the growth of VSV-IFNbeta was greatly reduced and diminished cytopathic effect was observed due to the production of recombinant IFN-beta, which by functioning in a manner involving autocrine and paracrine mechanisms induced an antiviral effect, preventing virus growth. However, VSV-IFNbeta grew to high levels and induced the rapid apoptosis of transformed cells due to defective IFN pathways being prevalent and thus unable to initiate proficient IFN-mediated host defense. Importantly, VSV expressing the human IFN-beta gene (VSV-hIFNbeta) behaved comparably and, while nonlytic to normal human cells, readily killed their malignant counterparts. Similar to our in vitro observations, following intravenous and intranasal inoculation in mice, recombinant VSV (rVSV)-IFNbeta was also significantly attenuated compared to wild-type VSV or rVSV expressing green fluorescent protein. However, VSV-IFNbeta retained propitious oncolytic activity against metastatic lung disease in immunocompetent animals and was able to generate robust antitumor T-cell responses. Our data indicate that rVSV designed to exploit defects in mechanisms of host defense can provide the basis for new generations of effective, specific, and safer viral vectors for the treatment of malignant and other disease.

Deadenylation of interferon-beta mRNA is mediated by both the AU-rich element in the 3'-untranslated region and an instability sequence in the coding region.

Viral infection of fibroblastic and endothelial cells leads to the transient synthesis of interferon-beta (IFN-beta). The down-regulation of IFN-beta synthesis after infection results both from transcriptional repression of the IFN-beta gene and rapid degradation of mRNA. As with many cytokine mRNAs, IFN-beta mRNA contains an AU-rich element (ARE) in its 3'-untranslated region (UTR). AREs are known to mediate mRNA deadenylation and destabilization. Depending on the class of ARE, deadenylation was shown to occur through synchronous or asynchronous mechanisms. In this study, we analysed IFN-beta mRNA deadenylation in natural conditions of IFN-beta synthesis, e.g., after viral infection. We show that human IFN-beta mRNA follows an asynchronous deadenylation pathway typical of a mRNA containing a class II ARE. A deletion analysis of the IFN-beta natural transcript demonstrates that poly(A) shortening can be mediated by the ARE but also by a 32 nucleotide-sequence located in the coding region, that was identified previously as an instability determinant. In fact, these elements are able to act independently as both of them have to be removed to abrogate mRNA deadenylation. Our data also indicate that deadenylation occurs independently of mRNA translation. Moreover, we show that deadenylation of IFN-beta mRNA is not under the control of viral infection as IFN-beta mRNA derived from a constitutively expressed gene cassette is deadenylated in absence of viral infection. Finally, an unidentified nuclear event appears to be a prerequisite for IFN-beta mRNA deadenylation as IFN-beta mRNA introduced directly into the cytoplasm does not undergo deadenylation. In conclusion, our study demonstrates that IFN-beta mRNA poly(A) shortening is under the control of two cis-acting elements recruiting a deadenylating machinery whose activity is independent of translation and viral infection but might require a nuclear event.

Molecular cloning of the pufferfish (Takifugu rubripes) Mx gene and functional characterization of its promoter.

Mx proteins are members of a family of interferon-inducible genes that are expressed by cells in response to viral infection. They are important determinants of innate immunity against viral infection in vertebrates. We cloned the pufferfish ( Takifugu rubripes) Mx gene and sequenced 80 kb from the Mx locus. The Fugu Mx gene spans 3.4 kb from the transcription start site to the polyadenylation signal, and is made up of 12 exons and 11 introns. The protein sequence encoded by the Fugu Mx gene is 77%, 48%, and 51% identical to that of trout Mx1, chicken Mx, and mouse Mx1 genes, respectively. The Fugu Mx gene is expressed in a variety of tissues, with high expression detected in the heart, gill, kidney, intestine, and brain. Analysis of the 5'-flanking sequence of the gene showed the presence of two interferon-stimulated response elements (ISRE) at positions -51 to 38 and -97 to 85, relative to the transcription start site. The Fugu Mx promoter was inducible by human IFN-beta in the human hepatoma (Huh7) cells and by polyinosinic: polycytidilic acid in the top minnow hepatoma (PLHC-1) cells. Deletion analysis of the promoter showed that both ISREs contributed to inducibility. These results demonstrate that the molecular mechanisms involved in Mx gene regulation are conserved between fish and mammals.