Mouse Interferon Research Articles

Adaptive Immune Responses to Zika Virus Are Important for Controlling Virus Infection and Preventing Infection in Brain and Testes.

The recent association between Zika virus (ZIKV) and neurologic complications, including Guillain-Barré syndrome in adults and CNS abnormalities in fetuses, highlights the importance in understanding the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response, suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show in this article, although wild-type C57BL/6 mice mount cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by Abs in Rag1-/- mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Ig-treated Rag1-/- mice or wild-type mice treated with anti-type I IFNR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared with nonpregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in the control of ZIKV infection and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.

Adaptive immune responses to Zika virus are important for controlling virus infection and preventing infection in brain and testes

Abstract The recent association between Zika virus (ZIKV) and neurological complications including Guillain-Barré Syndrome (GBS) in adults and CNS abnormalities in fetuses highlights the urgency to understand the immunological mechanisms controlling this emerging infection. Studies have indicated that ZIKV evades the human type I IFN response suggesting a role for the adaptive immune response in resolving infection. However, the inability of ZIKV to antagonize the mouse IFN response renders the virus highly susceptible to circulating IFN in murine models. Thus, as we show here, although wild type C57BL/6 mice mount both cell-mediated and humoral adaptive immune responses to ZIKV, these responses were not required to prevent disease. However, when the type I IFN response of mice was suppressed, then the adaptive immune responses became critical. For example, when type I IFN signaling was blocked by antibodies in Rag1−/− immunodeficient mice, the mice showed dramatic weight loss and ZIKV infection in the brain and testes. This phenotype was not observed in Rag1−/− mice or mice treated with anti-IFNAR alone. Furthermore, we found that the CD8+ T cell responses of pregnant mice to ZIKV infection were diminished compared to non-pregnant mice. It is possible that diminished cell-mediated immunity during pregnancy could increase virus spread to the fetus. These results demonstrate an important role for the adaptive immune response in control of ZIKV infection, and imply that vaccination may prevent ZIKV-related disease, particularly when the type I IFN response is suppressed as it is in humans.

The Conserved Coronavirus Macrodomain Promotes Virulence and Suppresses the Innate Immune Response during Severe Acute Respiratory Syndrome Coronavirus Infection.

ABSTRACTADP-ribosylation is a common posttranslational modification that may have antiviral properties and impact innate immunity. To regulate this activity, macrodomain proteins enzymatically remove covalently attached ADP-ribose from protein targets. All members of the Coronavirinae, a subfamily of positive-sense RNA viruses, contain a highly conserved macrodomain within nonstructural protein 3 (nsp3). However, its function or targets during infection remain unknown. We identified several macrodomain mutations that greatly reduced nsp3’s de-ADP-ribosylation activity in vitro. Next, we created recombinant severe acute respiratory syndrome coronavirus (SARS-CoV) strains with these mutations. These mutations led to virus attenuation and a modest reduction of viral loads in infected mice, despite normal replication in cell culture. Further, macrodomain mutant virus elicited an early, enhanced interferon (IFN), interferon-stimulated gene (ISG), and proinflammatory cytokine response in mice and in a human bronchial epithelial cell line. Using a coinfection assay, we found that inclusion of mutant virus in the inoculum protected mice from an otherwise lethal SARS-CoV infection without reducing virus loads, indicating that the changes in innate immune response were physiologically significant. In conclusion, we have established a novel function for the SARS-CoV macrodomain that implicates ADP-ribose in the regulation of the innate immune response and helps to demonstrate why this domain is conserved in CoVs.

Chemical constituents and nitric oxide inhibitory activity of supercritical carbon dioxide extracts from Mitragyna speciosa leaves

Mitragyna speciosa Korth., is a controversial plant that possesses various medicinal values and has been used from time immemorial in folk medicine. Recently, it has been widely abused as a narcotic source. The aim of the present study was to characterize the in vitro nitric oxide (NO) inhibition property of the supercritical CO2 fluid extracts from M. speciosa leaves and to identify the chemical constituents of the extract that exhibited the most inhibition without toxicity effect. Samples were extracted using the green technology, supercritical fluid extraction (SFE) technique via CO2 as the mobile phase. The NO inhibitory activity was evaluated by Griess assay, which measures the formation of nitrite ion (NO2−) in recombinant mouse interferon gamma/lipopolysaccharide (IFN-γ/LPS) stimulated RAW 264.7 cells. Matrix 5 Step-1 (M5S1) that was extracted with pure CO2 at 3000psi and 60°C exhibited the highest NO inhibitory activity (60.08%) without cytotoxicity (cell viability, 91.98%) at a concentration of 100μg/mL. GC and GC–MS analysis revealed palmitic acid as the major constituent (34.90%) of M5S1. This study provides first evidence that M5S1, the non-alkaloidal extract obtained by supercritical fluid extraction of M. speciosa leaves possesses potential property in preventing inflammatory diseases mediated by excessive production of NO.

Intrinsic Viral Factors Are the Dominant Determinants of the Hepatitis C Virus Response to Interferon Alpha Treatment in Chimeric Mice.

BackgroundHepatitis C virus infection is a global health problem. New direct-acting antiviral agents have been recently approved. However, due to their high cost and some genotypes remaining difficult to treat, interferon-based therapy with pegylated interferon and ribavirin likely may remain a component of hepatitis C virus treatment for some patients. Unfortunately, pegylated interferon / ribavirin treatment achieved favorable outcomes in less than 50% of patients. Factors determining the outcome to pegylated interferon / ribavirin include both host and viral factors. It has been a major challenge to separate the host and viral factors in most in vivo systems.Aims & MethodsWe used two hepatitis C virus strains from patients with different interferon-sensitivities and three hepatocyte donors, each with distinct interleukin 28B and interferon lambda 4 single nucleotide polymorphisms to investigate the contributions of viral and host factors to the response of hepatitis C virus to interferon treatment in chimeric mice.Results and ConclusionsWe found that viral factors were the dominant factors in determining the interferon treatment outcomes in chimeric mice. Host factors, such as pre-treatment liver interferon-stimulated gene expression and single nucleotide polymorphisms near interleukin 28B and interferon lambda 4 coding regions, were less important determinants of the response to interferon in the chimeric mice than they were in patients. Our results also suggest that a complete immune system as seen in patients may be required for host factors such as single nucleotide polymorphisms near interleukin 28B / interferon lambda 4 and pre-treatment liver interferon-stimulated gene upregulation to have an effect on the interferon response.

Structural Characterization and Immunostimulatory Activity of a Homogeneous Polysaccharide from Sinonovacula constricta.

Sinonovacula constricta has been widely used as a health food and medicine in China, Japan, and Korea. In the present study, a water-soluble polysaccharide fraction (SCP-1) was prepared from S. constricta by enzyme-assisted extraction and purification of chromatography with DEAE-52 cellulose anion-exchange column and Sephadex G-100 size exclusion column. On the basis of the analytical results of high-performance liquid chromatography, Fourier transform-infrared spectroscopy, methylation analysis, and NMR spectroscopy, SCP-1 was found to have an average molecular weight of 15.63 kDa and a linear backbone of (1→4)-linked α-D-Glcp residue with one branch, α-D-Glcp, attached to the main chain by a (1→6) glycosidic bond at every five α-D-Glcp units. Furthermore, it was found that SCP-1 could significantly increase the viability of macrophages, enhance the capability of macrophage phagocytosis, increase the activity of acid phosphatase, and promote the production of nitric oxide, mouse tumor necrosis factor (TNF)-α, mouse interferon (IFN)-γ, and mouse interleukin (IL)-1β. The results suggest that SCP-1 possesses potent immunomodulating effect and may be explored as a potential biological response modifier.

Abstract B37: Interferon-beta gene transfer to human melanoma cell lines using a specialized adenoviral vector induces high levels of cell death

Abstract Melanoma represents only 4% of all skin cancers, yet its prognosis is especially grim once the metastatic disease state has been reached. However, the genotype of melanomas may provide an opportunity for therapeutic intervention. For example, 90% of cases retain wild-type p53 and a considerable number of these present deletions in the chromosome region that encodes interferon-beta; (IFN). In our previous work, we developed adenoviral vectors where transgene expression is controlled by p53 and propose that such vectors would be especially interesting for the treatment of melanoma. We have used these vectors to transfer the mouse IFN cDNA in murine models, including melanoma, and have observed a significant ability of IFN to inhibit cell proliferation in vitro and in vivo (Merkel et al, 2013). In addition, we have shown that the application of IFN contributes to an anti-tumor immune response (unpublished data). Here we present our recent efforts to adapt this strategy to a model of human melanoma. To this end, the human IFN cDNA was cloned into an adenoviral vector that offers CAR-independent transduction due to the incorporation of the RGD tripeptide in the fiber as well as expression of the transgene under the control of p53. We expect that the AdRGDPGhIbeta; vector will provide expression of human IFN in response to cellular p53 activity. Reliable vector expression was confirmed by ELISA. Then SK-MEL-05 and SK-MEL-147 (human melanoma cell lines harboring wild-type p53) were transduced, incubated for 48, 72 or 96 hours of incubation and then cell cycle alterations were assessed by PI/FACS analysis. We observed a time-dependent accumulation of hypodiploid cells in either cell line when treated with the AdRGDPGhIbeta; vector, yet non-treated or treatment with a GFP-expressing virus did not alter the cell cycle profile. At the final time point, more than 90% of cells treated with IFN were hypodiploid. This result suggests that endogenous p53 reliably induced vector expression and that IFN was an efficient inducer of cell death. We observed the paracrine effects of this treatment when we mixed non-transduced cells with transduced cells and obtained a dose-dependent accumulation of hypodiploid cells. In this assay, the presence of only 5% transduced cells was sufficient to kill 40% of the total cell population in both the SK-MEL-05 and SK-MEL-147 cell lines. We are especially encouraged by these results since our mouse models using Ad5 to transfer murine IFN were less efficient in inducing cell death than seen in the human model. At this time we are determining the mechanism of cell death as well as initiating a model of in situ gene therapy with human IFN. Grants #2010/15025-0 and 2013/25167-5, São Paulo Research Foundation (FAPESP). Citation Format: Taynah David, Bryan Eric Strauss. Interferon-beta gene transfer to human melanoma cell lines using a specialized adenoviral vector induces high levels of cell death. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B37.

Poly(I:C) treatment leads to interferon-dependent clearance of hepatitis B virus in a hydrodynamic injection mouse model.

We have previously shown that poly(I:C) activates murine hepatic cells to produce interferon (IFN) and suppresses hepatitis B virus (HBV) replication in vitro. Therefore, we addressed whether poly(I:C) is able to induce the clearance of HBV in vivo. The chronic HBV replication mouse model was established by the hydrodynamic injection (HI) of pAAV-HBV1.2 into the tail veins of wild-type and IFN-α/βR-, IFN-γ-, and CXCR3-deficient C57BL/6 mice. Fourteen days post-HI of pAAV-HBV1.2, mice were administered poly(I:C) by intraperitoneal injection, intramuscular injection, or HI. Only treatment of poly(I:C) by HI led to HBV clearance in wild-type C57BL/6 mice. Serum HBsAg disappeared within 40 days postinfection (dpi) in mice that received poly(I:C) by HI, and this was accompanied by the appearance of anti-HBs antibodies. HBV-specific T-cell and antibody responses were significantly enhanced by HI of poly(I:C). HBV replication intermediates and HBcAg-positive hepatocytes were eliminated in the liver. HI of poly(I:C) induced the production of IFNs in mice and enhanced the levels of cytokines, IFN-stimulated genes, and T-cell markers in the liver. Importantly, poly(I:C)-induced HBV clearance was impaired in IFN-α/βR-, IFN-γ-, and CXCR3-deficient mice, indicating that the induction of type I IFN and the stimulation and recruitment of T cells into the liver are essential for HBV clearance in this model. Taken together, the application of poly(I:C) by HI into the liver enhances innate and adaptive immune responses and leads to HBV clearance in an HBV mouse model, implicating the potential of intrahepatic Toll-like receptor 3 (TLR3) activation for the treatment of chronic hepatitis B patients. It has become well accepted that immunomodulation is a potentially useful approach to treat chronic viral infection. Recently, combinations of antiviral treatment and therapeutic vaccinations were evaluated for therapies of chronic hepatitis B virus (HBV) infection. Activation of the innate immune branch may also be important for viral control and contributes to HBV clearance. Our present study demonstrated that hepatic TLR3 activation led to clearance of hepatitis B virus in an HBV mouse model. For the first time, we showed that HBV clearance in this model is dependent not only on type I interferon (IFN) but also on type II IFN, indicating a coordinated action of innate and adaptive immune responses. T-cell recruitment appeared to be critical for the success of TLR3-mediated antiviral action. These findings implicate the potential of intrahepatic TLR3 activation for the treatment of chronic HBV infection.

Protection against RNA-induced liver damage by myeloid cells requires type I interferon and IL-1 receptor antagonist in mice

Cell types and mechanisms involved in type I interferon (IFN)-mediated anti-inflammatory effects are poorly understood. Upon injection of artificial double-stranded RNA (poly(I:C)), we observed severe liver damage in type I IFN-receptor (IFNAR) chain 1-deficient mice, but not in wild-type (WT) controls. Studying mice with conditional IFNAR ablations revealed that IFNAR triggering of myeloid cells is essential to protect mice from poly(I:C)-induced liver damage. Accordingly, in poly(I:C)-treated WT, but not IFNAR-deficient mice, monocytic myeloid-derived suppressor cells (MDSCs) were recruited to the liver. Comparing WT and IFNAR-deficient mice with animals deficient for the IFNAR on myeloid cells only revealed a direct IFNAR-dependent production of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RA) that could be assigned to liver-infiltrating cells. Upon poly(I:C) treatment, IFNAR-deficient mice displayed both a severe lack of IL-1RA production and an increased production of proinflammatory IL-1β, indicating a severely imbalanced cytokine milieu in the liver in absence of a functional type I IFN system. Depletion of IL-1β or treatment with recombinant IL-1RA both rescued IFNAR-deficient mice from poly(I:C)-induced liver damage, directly linking the deregulated IL-1β and IL-1RA production to liver pathology. Type I IFN signaling protects from severe liver damage by recruitment of monocytic MDSCs and maintaining a balance between IL-1β and IL-1RA production.

Preclinical safety and activity of recombinant VSV‐IFN‐β in an immunocompetent model of squamous cell carcinoma of the head and neck

Recombinant vesicular stomatitis virus expressing interferon-β (VSV-IFN-β) has demonstrated antitumor activity in vitro and in vivo. In preparation for clinical testing in human squamous cell carcinoma (SCC) of the head and neck, we conducted preclinical studies of VSV-IFN-β in syngeneic SCC models. In vitro, VSV-IFN-β (expressing rat or mouse interferon [IFN]-β)-induced cytotoxicity and propagated in rat (FAT-7) or mouse (SCC-VII) SCC cells during normoxia and hypoxia. In vivo, intratumoral administration of VSV-rat-IFN-β or VSV-human-IFN-β in FAT-7 bearing or non-tumor bearing immunocompetent rats did not result in acute organ toxicity or death. VSV-r-IFN-β replicated predominantly in tumors and a dose dependent anti-VSV antibody response was observed. Intratumoral or intravenous administration of VSV-IFN-β resulted in growth delay and improved survival compared with controls. The above data confirm safety and feasibility of VSV-IFN-β administration in immunocompetent animals and support its clinical evaluation in advanced human head and neck cancer.

Bridging the Species Divide: Transgenic Mice Humanized for Type-I Interferon Response

We have generated transgenic mice that harbor humanized type I interferon receptors (IFNARs) enabling the study of type I human interferons (Hu-IFN-Is) in mice. These “HyBNAR” (Hybrid IFNAR) mice encode transgenic variants of IFNAR1 and IFNAR2 with the human extracellular domains being fused to transmembrane and cytoplasmic segments of mouse sequence. B16F1 mouse melanoma cells harboring the HyBNAR construct specifically bound Hu-IFN-Is and were rendered sensitive to Hu-IFN-I stimulated anti-proliferation, STAT1 activation and activation of a prototypical IFN-I response gene (MX2). HyBNAR mice were crossed with a transgenic strain expressing the luciferase reporter gene under the control of the IFN-responsive MX2 promoter (MX2-Luciferase). Both the HyBNAR and HyBNAR/MX2-Luciferase mice were responsive to all Hu-IFN-Is tested, inclusive of IFNα2A, IFNβ, and a human superagonist termed YNSα8. The mice displayed dose-dependent pharmacodynamic responses to Hu-IFN-I injection, as assessed by measuring the expression of IFN-responsive genes. Our studies also demonstrated a weak activation of endogenous mouse interferon response, especially after high dose administration of Hu-IFNs. In sharp contrast to data published for humans, our pharmacodynamic readouts demonstrate a very short-lived IFN-I response in mice, which is not enhanced by sub-cutaneous (SC) injections in comparison to other administration routes. With algometric differences between humans and mice taken into account, the HyBNAR mice provides a convenient non-primate pre-clinical model to advance the study of human IFN-Is.

Gene delivery of albumin binding peptide-interferon-gamma fusion protein with improved pharmacokinetic properties and sustained biological activity

We have demonstrated that gene delivery of a fusion protein of mouse interferon (IFN) γ with mouse serum albumin (IFNγ–MSA) was effective in prolonging the circulation half-life of IFNγ in mice. However, the fusion to MSA greatly reduced the biological activity of IFNγ to less than 1%. In this study, we designed IFNγ fusion proteins with a 20 amino-acid long albumin-binding peptide (ABP) to prolong the in vivo half-life of IFNγ without reducing its biological activity. IFNγ–ABP and ABP–IFNγ, two fusion proteins with the ABP being fused to the C- or N-terminal of IFNγ, retained 40%–50% biological activities determined using a gamma-activated sequence-dependent luciferase assay. These fusion proteins exhibited the ability to bind to MSA. Gene delivery of IFNγ–ABP or ABP–IFNγ to mice using the hydrodynamic injection method resulted in a sustained concentration of IFNγ in the serum compared with gene delivery of IFNγ. In addition, the growth of mouse colon carcinoma CT-26 cells in the lung was efficiently inhibited by gene delivery of the IFNγ fusion proteins. These results indicate that the fusion of ABP is a useful approach to achieving prolonged retention in the blood circulation through binding to serum albumin and retaining biological activity.

257 : Development of a hyperglycosylated interferon ALFACON1: Towards monthly dosing for antiviral therapies in diseases such as chronic hepatitis C

Type 1 interferons (IFN) have been shown to be efficacious against many viruses. However, a significant drawback is the short duration of exposure. We have improved the PK of IFN ALFACON1 (CIFN) by glycoengineering. ALS-624 was generated by introducing four glycosylation sites, which demonstrated a significantly increased Molecular Weight of 30–45 kD compared to 19 kD for CIFN. ALS-624 was purified and fractionated to yield 4-Gly, 3-Gly and 2-Gly fractions by size exclusion chromatography. In a rat PK study, four groups were subcutaneously dosed with the three glycosylated ALS-624 fractions plus a non-glycosylated CIFN control. The high MW fraction of 4-Gly exhibited a 6-fold increase of AUC and over an 11-fold increase in T 1/2 over CIFN suggesting the potential for QW dosing. In a Yellow Fever Virus study, hamsters were injected intraperitoneally with virus. All compounds were administered i.p. at a dose of 1.25 × 10 6 IU/kg/dose, given QW (4 Gly and Peg-IFN-α-2a), BIW (3 Gly), or QD (IFN alfacon-1 and placebo) beginning at −4 h and ending 14 days post infection. Animals were observed for mortality for 21 days. QW dosing of 4-Gly achieved similar efficacy to QD CIFN and QW Peg-IFN-α-2a in overall survival rate and reduction of ALT level. Further refinement resulted in development of ALS-683 which has a MW in the 60–90 kD range and maintains undiminished biological potency. In mouse PK/PD study, both ALS-683 mouse homolog and unglycosylated mouse IFNα1 were dosed at 1000 IU/g. Animals ( N = 3) were sacrificed at pre-dose and various time points post-dose. Mouse plasma and liver were analyzed for PK and PD markers. ALS-683 homolog has ∼37-fold improvement in T 1/2 and ∼33-fold improvement in AUC when compared with unglycosylated mouse IFN α 1. Significantly improved PD responses were also observed. ALS-683 can be further developed for possible once a month dosing.

Effects of Nasal or Pulmonary Delivered Treatments with an Adenovirus Vectored Interferon (mDEF201) on Respiratory and Systemic Infections in Mice Caused by Cowpox and Vaccinia Viruses

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal cowpox (Brighton strain) and vaccinia (WR strain) virus respiratory and systemic infections in mice. Two routes of mDEF201 administration were used, nasal sinus (5-µl) and pulmonary (50-µl), to compare differences in efficacy, since the preferred treatment of humans would be in a relatively small volume delivered intranasally. Lower respiratory infections (LRI), upper respiratory infections (URI), and systemic infections were induced by 50-µl intranasal, 10-µl intranasal, and 100-µl intraperitoneal virus challenges, respectively. mDEF201 treatments were given prophylactically either 24 h (short term) or 56d (long-term) prior to virus challenge. Single nasal sinus treatments of 106 and 107 PFU/mouse of mDEF201 protected all mice from vaccinia-induced LRI mortality (comparable to published studies with pulmonary delivered mDEF201). Systemic vaccinia infections responded significantly better to nasal sinus delivered mDEF201 than to pulmonary treatments. Cowpox LRI infections responded to 107 mDEF201 treatments, but a 106 dose was only weakly protective. Cowpox URI infections were equally treatable by nasal sinus and pulmonary delivered mDEF201 at 107 PFU/mouse. Dose-responsive prophylaxis with mDEF201, given one time only 56 d prior to initiating a vaccinia virus LRI infection, was 100% protective from 105 to 107 PFU/mouse. Improvements in lung hemorrhage score and lung weight were evident, as were decreases in liver, lung, and spleen virus titers. Thus, mDEF201 was able to treat different vaccinia and cowpox virus infections using both nasal sinus and pulmonary treatment regimens, supporting its development for humans.

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.

Targeting TLR3 with no RIG-I/MDA5 activation is effective in immunotherapy for cancer

Introduction: Many forms of RNA duplexes with agonistic activity for pattern-recognition receptors have been reported, some of which are candidates for adjuvant immunotherapy for cancer. These RNA duplexes induce cytokines, interferons (IFNs) and cellular effectors mainly via two distinct pathways, TLR3/TICAM-1 and MDA5/MAVS.Areas covered: We determined which pathway of innate immunity predominantly participates in evoking tumor immunity in response to RNA adjuvants.Expert opinion: In knockout (KO) mouse studies, robust cytokine or IFN production is dependent on systemic activation of the MAVS pathway, whereas maturation of dendritic cells (DCs) to drive cellular effectors (i.e., NK and CTL) depends on the TICAM-1 pathway in DCs. MAVS activation often causes endotoxin-like cytokinemia, while the TICAM-1 activation does not. Unlike the TLR/MyD88 pathway, this TICAM-1 pathway barely accelerates tumor progression. Although the therapeutic effect in human patients of MAVS-activating or TICAM-1-activating RNA duplexes remains undetermined, the design of a TLR3 agonist with optimized toxicity and dose is an important goal for human immunotherapy. Here we summarize current knowledge on available RNA duplex formulations, and offer a possible approach to developing a promising RNA duplex for clinical tests.

Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma

BackgroundInterleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer.MethodsSolid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD50 assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated.ResultsSingle intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect.ConclusionsSingle or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation.

897 CONSENSUS RECOMMENDATIONS FOR VIROLOGIC NOMENCLATURE IN DAA CLINICAL TRIALS

development, hippocampus and prefrontal cortex, through the use of primary cultured mouse neurons. Methods: Hippocampal and prefrontal cortex neurons were isolated from 18-day-old embryos of C57 BL/6 mice. After disaggregation with trypsin, they were cultured at 1.2×105 cells/well in DMEM and 10% FCS, and later in Neurobasal medium. At day 9 of culture, stimulation was performed using 100, 500 and 1000 IU of mouse IFN-a2a, and cells were collected after 12, 24 and 48 hours. The expression of interferon stimulated genes (ISGs) and 10 of the depression-related interferon-inducible genes (DRIIs) was assessed using real time PCR. Results: The stimulation with mouse IFN promoted an increased expression of the studied ISGs (Isg15, Mx2 and Ifit1) in a doseresponse manner, and, in general terms, they reached similar rates at the concentration and time points in both studied areas. Regarding to the DRIIs, differential responses to the treatment were observed, ranging from a nonor mild response in one or both studied areas (i.e. Dynlt1, Mef2A and Glrx showed a light increase in prefrontal cortex but not in hippocampus), to a strong upregulation, as found in Stat1, Rtp4, Mef2A and Ube2L6. Conclusions: IFN-a treatment promotes a response of ISGs in the cultured neurons of hippocampus and prefrontal cortex, and is also associated to the modulation of the expression of most of the studied DRIIs, meanwhile others might be under the regulation of factor(s) different than IFN-a. Linked to our previous findings, the upregulation of some DRIIs may be involved in the pathophysiological mechanisms underlying IFN-a therapyassociated depression.

Direct Upregulation of Iron Regulatory Hormone Hepcidin by Interferon Alpha

Abstract 2103 Introduction:Interferon (IFN) activates various immune systems in vivo and is administered for the patients with various diseases such as viral hepatitis B,C and malignant tumors. During the treatment with IFN, iron metabolism has been reported to be important. For instances, iron overload has reported to contribute to the poor response rate of IFN treatment and poor prognosis in the patients with hepatitis C. However, it has been unclear whether IFN itself effects on the iron metabolism. Therefore, we aimed to determine the effect of IFN itself on iron metabolism. Materials and Methods:The mice were subcutaneously administered the mouse IFN alpha (104 and 105 IU/mouse/day) and sacrificed after 3, 5 and 7 days. Then the serum, the bone marrow, the liver, the spleen, and the duodenum tissues were collected. To analyze serum hepcidin levels, liquid chromatography-tandem mass spectrometry was performed. The changes of mRNA and protein expressions involved in iron metabolism such as transferrin receptor 1, hepcidin, and ferroportin, were also analyzed by quantitative real time RT-PCR and western blotting. Some parts of tissues were processed for the formalin-fixed paraffin embedding blocks, and Hematoxylin-Eosin and immunofluorescence staining for ferroportin. To evaluate the direct effects of IFN alpha on the expression of hepcidin by hepatocytes, primary cultured hepatocytes obtained from the mouse liver and human hepatoma-derived cultured cells were treated with human IFN alpha, and then RNA and the protein were extracted after 24 hours of treatment, followed by the analysis of hepcidin mRNA and transcription factors, such as STAT3. Results:Among the analyzed genes and proteins that are involved in iron metabolism, the hepcidin expression was highly modulated by IFN alpha. Both the serum hepcidin level and hepcidin mRNA expression in the liver were increased in IFN alpha-treated mice. And the expression of ferroportin, target molecule of hepcidin, was decreased in the duodenum of IFN alpha-treated mice. The analysis in vitro showed the upregulation of hepcidin mRNA by the IFNa treatment both in primary hepatocytes of the mouse liver and human hepatoma-derived cells. Interestingly, the upregulation of activated STAT3 was also observed in those cells, and that was cancelled by the addition of neutralizing antibody (IFNAR1) for IFN receptors. Discussion and Conclusion:Our results showed that IFN alpha directly upregulated the hepcidin expression by the liver. Concerning the mechanism of the upregulation of hepcidin, the upregulation of activated STAT3 by IFN should be involved. Commonly, IFN alpha accelerated the heterodimer formation of transcription of STAT1 and STAT2 via the activation of JAK by the binding of IFN alpha with IFN receptor on cell surface. The upstream lesion of the hepcidin gene does not have the binding site for STAT1/STAT2 heterodimer, but have the binding site for STAT3 homodimer. In addition, IFNa has been reported to activate STAT3 in human primary hepatocytes (Radaeva, S. et al. Gastroenterology 2002; 122:1020–34). Combined these, presumably IFN alpha directly effected on hepatocytes to enhance the transcription of hepcidin gene via activated STAT3 homodimer. In addition to the upregulation of hepcidin, our results showed the decreased duodenum expression of ferroportin in IFN alpha-treated mice. Ferroportin is expressed on the basolateral cell membrane of enterocytes and has function of iron transport from the enterocytes to blood. Ferroportin has also been reported to internalize and be degraded when hepcidin binds to ferroportin on the cell surface, resulting in the decrease of iron absorption from the gastrointestinal tract. Therefore, the upregulation of hepcidin by IFN alpha treatment observed in the present study should be physiologically functional, and this indicated that iron absorption should be decreased during IFN treatment. This effect is considered to be favorable because this would inhibit iron overload during IFN treatment and may increase the effect of IFN. Disclosures:Sasaki:Novartis Pharma K.K.: Research Funding. Kohgo:Novartis: Research Funding, Speakers Bureau; Chugai Roche: Research Funding; Asahikasei Kurare Medical: Research Funding; Sapporo Brewery: Research Funding; Kyorin Pharma: Research Funding.

Therapy and Long-Term Prophylaxis of Vaccinia Virus Respiratory Infections in Mice with an Adenovirus-Vectored Interferon Alpha (mDEF201)

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal vaccinia virus (WR strain) respiratory infections in mice. mDEF201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 106 to 108 plaque forming units/mouse. When the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1–49 days pre-infection), with minimal weight loss occurring during infection. Surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mDEF201 did not compromise the immune response against the initial infection. Post-exposure therapy was given between 6–24 h after vaccinia virus exposure and protection was afforded by a 108 dose of mDEF201 given at 24 h, whereas a 107 dose was effective up to 12 h. Comparisons were made of the ability of mDEF201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. Lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). Lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mDEF201 treated mice were nearly at baseline. In contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. These results demonstrate the long-acting prophylactic and treatment capacity of mDEF201 to combat vaccinia virus infections.