Human Interferon-alpha Research Articles

Development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, Yarrowia lipolytica

BackgroundAs an oleaginous yeast, Yarrowia lipolytica is able to assimilate hydrophobic substrates. This led to the isolation of several promoters of key enzymes of this catabolic pathway. Less is known about the behavior of Y. lipolytica in large bioreactors using these substrates. There is therefore a lack of established know-how concerning high cell density culture protocols of this yeast. Consequently, the establishment of suitable induction conditions is required, to maximize recombinant protein production under the control of these promoters.ResultsHuman interferon α2b (huIFN α2b) production in Yarrowia lipolytica was used as a model for the enhancement of recombinant protein production under the control of the oleic acid (OA)-inducible promoter POX2. Cell viability and heterologous protein production were enhanced by exponential glucose feeding, to generate biomass before OA induction. The optimal biomass level before induction was determined (73 g L-1), and glucose was added with oleic acid during the induction phase. Several oleic acid feeding strategies were assessed. Continuous feeding with OA at a ratio of 0.02 g OA per g dry cell weight increased huIFNα2b production by a factor of 1.88 (425 mg L-1) and decreased the induction time (by a factor of 2.6, 21 h). huIFN α2b degradation by an aspartic protease secreted by Y. lipolytica was prevented by adding pepstatin (10 μM), leading to produce a 19-fold more active huIFN α2b (26.2 × 107 IU mg-1).ConclusionY. lipolytica, a generally regarded as safe (GRAS) microorganism is one of the most promising non conventional yeasts for the production of biologically active therapeutic proteins under the control of hydrophobic substrate-inducible promoter.

PS2-050. The use of human recombinant interferon alpha 2b for the treatment of influenza caused by the pandemic influenza virus A /H1N1/ in pregnant women

PS2-050. The use of human recombinant interferon alpha 2b for the treatment of influenza caused by the pandemic influenza virus A /H1N1/ in pregnant women

Binding and activity of all human alpha interferon subtypes

Vertebrates have multiple genes encoding Type I interferons (IFN), for reasons that are not fully understood. The Type I IFN appear to bind to the same heterodimeric receptor and the subtypes have been shown to have different potencies in various experimental systems. To put this concept on a quantitative basis, we have determined the binding affinities and rate constants of 12 human Alpha-IFN subtypes to isolated interferon receptor chains 1 and 2. Alpha-IFNs bind IFNAR1 and IFNAR2 at affinities of 0.5–5μM and 0.4–5nM respectively (except for IFN-alpha1 – 220nM). Additionally we have examined the biological activity of these molecules in several antiviral and antiproliferative models. Particularly for antiproliferative potency, the binding affinity and activity correlate. However, the EC50 values differ significantly (1.5nM versus 0.1nM for IFN-alpha2 in WISH versus OVCAR cells). For antiviral potency, there are several instances where the relationship appears to be more complicated than simple binding. These results will serve as a point of reference for further understanding of this multiple ligand/receptor system.

The anti-cancer activity of human consensus interferon-alpha synthesized in cell-free system

The cell-free method is suitable for rapid and economical production of therapeutic proteins, since it is an open system, which allows us to control the reaction microenvironment to promote folding, solubility of proteins and maximize the protein yield. Consensus interferon is a newly developed type I interferon, a rapid-acting version of interferon that appears more potent than the currently approved pegylated version. Our work aimed to synthesize human consensus interferon-alpha (cIFN-α) in cell-free protein expression system of Escherichia coli cells origin. The cloned cIFN-α gene in pET101/D-TOPO expression system was used in cell-free IFN production. The system was tested by using a standard construct, GFP (green fluorescent protein) gene was cloned into pIVEX2.3 vector; this gene and our gene, both are under the T7 promoter transcriptional control. The synthesis of active cIFN-α gradually increased from 2 to 6 h of the reaction, also reducing the temperature of incubation to ⩽30 °C maximized its solubility. After purification on nickel–nitrilotriacetate acid (Ni–NTA) resin, the yield of cIFN-α was 400 μg/ml cell-free reaction solution. The resultant cIFN-α was fully biologically active as demonstrated by its anti-cancer effect and immunoassay signals.

Knockdown of USP18 Increases Alpha 2a Interferon Signaling and Induction of Interferon-Stimulating Genes but Does Not Increase Antiviral Activity in Huh7 Cells

The current standard of care for hepatitis C virus (HCV) patients is cotreatment with human alpha interferon (IFN-α) and ribavirin. The host factor USP18 functions to regulate the interferon signaling pathway by acting as an off-switch. In order to understand whether the inhibition of USP18 represents a valid target for the enhancement of interferon treatment for chronic viral diseases, we have used a wide range of RNA interference (RNAi) reagents to suppress USP18 gene expression in Huh7 cell lines. We demonstrate that a USP18 knockdown results in IFN-α2a signaling (measured by increased IFN-stimulated response element [ISRE] reporter gene activity, 2',5'-oligoadenylate synthetase [2-5 OAS] expression, and ISG15 induction) that is increased by ∼100-fold, whereas the antiviral (AV) potency in both the Huh7 HCV subgenomic replicon assay and the Huh7.5 HCV infectious virus assay increased by ∼3-fold. While the degree of the USP18 knockdown of USP18 elicited by the different RNAi reagents correlated with the enhancement of IFN-α2a signaling, it did not correlate with the enhancement of AV activity. The failure of increased IFN-α2a signaling to fully translate into increased AV potency was also observed for encephalomyocarditis virus (EMCV) assays using Huh7.5 cells. These data suggest that the IFN-mediated AV response in Huh7.5 cells has only a limited dependence on USP18 activity.

Use of recombinant interferon omega in feline retrovirosis: From theory to practice

Type-I interferons (IFNs) are cytokines that have non-specific antiviral activity, participating mostly in innate defense mechanisms. Their administration has been proposed to treat several viral and immunomediated diseases as an immunomodulatory therapy. Due to its availability, recombinant human interferon-alpha (rHuIFN-α) has been studied in relation to feline retrovirosis, both in vitro and in vivo. However, IFNs are species-specific and antibodies have been shown to develop in response to the high rHuIFN-α doses necessary for an effective therapy. A recombinant feline IFN has been developed, which has been characterized as interferon-omega (rFeIFN-ω), designed to overcome these problems. Nonetheless, very few studies have been undertaken to evaluate its efficacy in cats naturally infected with FIV or FeLV. In an initial study, we here demonstrated that rFeIFN-ω can dramatically improve the clinical condition of infected cats, and induce improvement of hematologic parameters. Minor changes or no change was observed for hypergammaglobulinemia, CD4/CD8 ratio, proviral load, viremia and RT activity, suggesting that the overall effect of IFN was on innate immunity. More studies are needed in order to better understand its in vivo mechanisms.

Human INF-α therapy in desmoid tumors: A retrospective study.

e20505 Background: Aggressive fibromatosis (desmoid tumors) are locally invasive, nonmetastatic and have a high local recurrence rate after primary resection. For patients (pts) where surgery is not an option, effective medical treatments are needed. Positive responses with INF-α therapy are reported and supported by in vitro results, was our reason to treat pts with human leukocyte interferon-alpha (Multiferon [M]). Methods: M is an INF-α product obtained from the leukocyte fraction of human blood. The product is highly purified with multiple subtypes including α1, α2, α8, α10, α14 and α21. Pts from 2 sites were included in this retrospective study. Pts had progressive symptoms, were inoperable without considerable morbidity and for whom RT, chemotherapy or hormone therapy also were unwanted. Results: 11 patients were included and 5 are still on treatment. Average age was 38 years and average follow-up time is currently 344 days. Pts N1 to N8 were INF-α naïve, whereas S1 to S3 were changed from peginterferon treatment to M treatment due to suspected anti-interferon antibodies with progression of disease (S1 and S3) or livertoxicity (S2). Of the 8 naïve pts, 2 had PR, 5 had SD, 1 no follow-up MRI scan yet. Six of 8 pts experienced clinical improvement such as regression of pain, increased mobility or other relevant parameters. For N2 and N5 the MRI sequences revealed lower intensity signal, reflecting a greater amount of less active fibrotic tissue. N5 experienced 13% shrinkage in second largest tumor diameter. N1 reached max response of 60% decrease in largest diameter, after 1½ years treatment. Treatment was stopped after 31 mth and tumor still stable 5 mth later. For pts S1 to S3 all had SD, 2 had clinical improvement, 1 had clinical worsening. Conclusions: Treatment response emerges slowly. M may be a new promising treatment option for desmoid tumors. The presentation will present latest follow-up data. Pt Sex Age Tumor location Response Clinical improvement yes/no % change from baseline Follow-up time days N1 M 44 Abd PR Y -60 929 N2 F 28 Ex SD Y +6 661 N3 F 24 Ex SD N +11 191 N4 M 68 Head and Neck PR Y -38 620 N5 F 29 Gluteal SD Y 0 238 N6 M 29 Abd SD Y -7 137 N7 F 35 Tru SD N -6 57 N8 M 63 Ex - Y - - S1 F 29 Ex SD N +4 84 S2 F 17 Ex SD Y -3 558 S3 F 49 Tru SD Y -2 176 Abbreviations: Abd, abdominal; Tru, superficial trunk; Ex, extremity.

Design of an efficient medium for heterologous protein production in Yarrowia lipolytica: case of human interferon alpha 2b

BackgroundThe non conventional yeast Yarrowia lipolytica has aroused a strong industrial interest for heterologous protein production. However most of the studies describing recombinant protein production by this yeast rely on the use of complex media, such media are not convenient for large scale production particularly for products intended for pharmaceutical applications. In addition medium composition can also affect the production yield. Hence it is necessary to design an efficient medium for therapeutic protein expression by this host.ResultsFive different media, including four minimal media and a complex medium, were assessed in shake flasks for the production of human interferon alpha 2b (hIFN α2b) by Y. lipolytica under the control of POX2 promoter inducible with oleic acid. The chemically defined medium SM4 formulated by Invitrogen for Pichia pastoris growth was the most suitable. Using statistical experimental design this medium was further optimized. The selected minimal medium consisting in SM4 supplemented with 10 mg/l FeCl3, 1 g/l glutamate, 5 ml/l PTM1 (Pichia Trace Metals) solution and a vitamin solution composed of myo-inositol, thiamin and biotin was called GNY medium. Compared to shake flask, bioreactor culture in GNY medium resulted in 416-fold increase of hIFN α2b production and 2-fold increase of the biological activity.Furthermore, SM4 enrichment with 5 ml/l PTM1 solution contributed to protect hIFN α2b against the degradation by the 28 kDa protease identified by zymography gel in culture supernatant. The screening of the inhibitory effect of the trace elements present in PTM1 solution on the activity of this protease was achieved using a Box-Behnken design. Statistical data analysis showed that FeCl3 and MnSO4 had the most inhibitory effect.ConclusionWe have designed an efficient medium for large scale production of heterologous proteins by Y. lipolytica. The optimized medium GNY is suitable for the production of hIFN α2b with the advantage that no complex nitrogen sources with non-defined composition were required.

AAV2-Mediated Subretinal Gene Transfer of hIFN-α Attenuates Experimental Autoimmune Uveoretinitis in Mice

BackgroundRecent reports show that gene therapy may provide a long-term, safe and effective intervention for human diseases. In this study, we investigated the effectiveness of adeno-associated virus 2 (AAV2) based human interferon-alpha (hIFN-α) gene therapy in experimental autoimmune uveoretinitis (EAU), a classic model for human uveitis.Methodology/Principal FindingsAn AAV2 vector harboring the hIFN-α gene (AAV2.hIFN-α) was subretinally injected into B10RIII mice at two doses (1.5×106 vg, 1.5×108 vg). AAV2 vector encoding green fluorescent protein (AAV2.GFP) was used as a control (5×108 vg). The expression of hIFN-α in homogenized eyes and serum was detected by ELISA three weeks after injection. The biodistribution of vector DNA in the injected eyes, contralateral eyes and distant organs was determined by PCR. EAU was induced by immunization with IRBP161–180 three weeks following vector injections, and evaluated clinically and pathologically. IRBP-specific proliferation and IL-17 expression of lymphocytes from the spleen and lymph nodes were assayed to test the influence of the subretinal delivery of AAV2.hIFN-α on the systemic immune response. hIFN-α was effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2.hIFN-α vector. DNA of AAV2.GFP was observed only in the injected eyes, but not in the distant organs or contralateral eyes. Subretinal injection of both doses significantly attenuated EAU activity clinically and histologically. For the lower dose, there was no difference concerning lymphocyte proliferation and IL-17 production among the AAV2.hIFN-α, AAV2.GFP and PBS injected mice. However, the higher dose of AAV2.hIFN-α significantly suppressed lymphocyte proliferation and IL-17 production.Conclusions/SignificanceSubretinal delivery of AAV2.hIFN-α lead to an effective expression within the eye for at least three months and significantly attenuated EAU activity. AAV2.hIFN-α was shown to inhibit the systemic IRBP-specific immune response.

Development of a murine animal model of depression for repeated dosing with human interferon alpha

Background:Neuropsychiatric adverse effects of interferon (IFN) alpha are well known. There is little clinically relevant research on animal models of depression with recombinant human IFN alpha 2b (rhIFN-α2b).Aim:To identify an appropriate dose and duration of administration of recombinant human interferon alpha-2b (rhIFN-α2b) to establish a convenient and clinically relevant murine model of chronic rhIFN-α2b-induced depression using the forced swim test (FST).Materials and Methods:Using a 4×3 factorial design, rhIFN-α2b was administered subcutaneously to mice (n=180) in the dose range of 400, 800, and 1600 IU/g/day for 5, 10, and 15 days; saline-treated mice formed the control groups. In each group, 1 day after the last dose, the mice were assessed for immobility in the FST. In another experiment, at these same doses and time points, the effect of rhIFN-α2b on murine motility was assessed in the small open field.Results:We found that rhIFN-α2b significantly increased immobility in the FST. The immobility was detectable by day 5 and did not increase with duration of IFN treatment. The immobility was apparent with the 400 IU/g/day dose and was not greater at higher IFN doses. At no dose or time point did rhIFN-α2b alter murine motility in the small open field.Conclusion:We conclude that rhIFN-α2b-induced behavioral despair, represented by immobility in the FST, is not due to reduced basal motility. The FST may therefore be used as a convenient Swiss albino mouse model of chronic rhIFN-α2b-induced depression with a 400–1600 IU/g/day dose administered subcutaneously for 5–15 days. The most economical model is 400 IU/g/day administered for 5 days

Human αIFN co-formulated with milk derived E2-CSFV protein induce early full protection in vaccinated pigs

Subunit vaccines are a suitable alternative for the control of classical swine fever. However, such vaccines have as the main drawback the relatively long period of time required to induce a protective response, which hampers their use under outbreak conditions. In this work, type I interferon is used as an immunostimulating molecule in order to increase the immunogenicity of a vaccine candidate based on the E2-CSFV antigen produced in goat milk. Pigs vaccinated with E2-CSFV antigen co-formulated with recombinant human alpha interferon were protected against clinical signs and viremia as early as 7 days post-vaccination. It was also demonstrated that interferon stimulates a response of specific anti-CSFV neutralizing antibodies. The present work constitutes the first report of a subunit vaccine able to confer complete protection by the end of the first week after vaccination. These results suggest that the E2-CSFV antigen combined with type I interferons could be potentially used under outbreak conditions to stop CSFV spread and for eradication programs in CSF enzootic areas.

Production of human interferon ALPHA 2b in plants of Nicotiana excelsior by Agrobacterium-mediated transient expression

Human interferon alpha2b gene was transiently expressed in Nicotiana excelsior plants. Fusion with N. plumbaginifolia calreticulin signal peptide for improved apoplast targeting and carrying out the expression under optimized conditions resulted in maximal interferon activity of 3.2 x 10(3) IU/g fresh weight (FW) with an average of 2.1 +/- 0.8 x 10(3) IU/g FW. It proves that N. excelsior is a suitable host for Agrobacterium-mediated transient expression of genes encoding physiologically active human proteins. The transient expression conditions optimized for GFP marker protein were confirmed to be preferable for hIFN alpha2b.

A molecular approach to optimize hIFN α2b expression and secretion in Yarrowia lipolytica

In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): pre-proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre-proLIP2 signal secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular secretion of small heterologous proteins in Yarrowia lipolytica.

PS3-39 Neutralization activity of the vaccinia virus B18R protein against the human interferon alpha protein family

PS3-39 Neutralization activity of the vaccinia virus B18R protein against the human interferon alpha protein family

PS3-69 Binding and neutralization of monoclonal antibodies to human interferon alpha subtypes

PS3-69 Binding and neutralization of monoclonal antibodies to human interferon alpha subtypes

PS2-71 Differential effects of human interferon-alpha subtypes on CD4 Tcell function

PS2-71 Differential effects of human interferon-alpha subtypes on CD4 Tcell function

Pegylated human interferon alpha 2a does not induce depression-associated changes in mice

Interferon (IFN) alpha proteins are proinflammatory cytokines having immunomodulating and antiviral properties. States during which cytokine systems are activated (e.g., during viral infection or during treatment of chronic hepatitis C and various malignancies with IFN alpha, etc.) can be associated with depression-like syndromes or even full-blown depressive episodes. Therefore, the role of IFN alpha and other cytokines in the pathogenesis of depressive disorder (“cytokine hypothesis of depression”) has been assessed for many years with contradictory results. We have investigated whether intraperitoneal administration of high doses (up to 600 µg/kg body weight) of pegylated, recombinant human IFN alpha 2a in mice induces changes known to be associated with depression using three different readouts: behavior in a model of despair (Porsolt swim test), presence of anhedonia (sucrose preference test), and sensitivity of the hypothalamic–pituitary–adrenal system (dexamethasone suppression test). We also assessed potential IFN-induced changes in gene expression in the liver. In none of the performed experiments, depression-associated effects could be found despite very high serum levels of IFN-induced antiviral activity compared to levels measured in hepatitis C virus (HCV) patients treated routinely with pegylated recombinant human IFN alpha 2a. The lack of such expected effects is probably due to the fact that pegylated human recombinant IFN alpha 2a does not activate the murine class I IFN receptor. Our results do not support the hypothesis that administration of recombinant pegylated human IFN alpha to mice produces a robust model of depression.

Improvement of human interferon alpha secretion by Lactococcus lactis

To improve the secretion and expression of human interferon alpha 2b (IFN) in Lactococcus lactis, a synthetic pro-peptide, LEISSTCDA (LEISS), was fused to the N-terminus of IFN. This gave a higher secretion efficiency (12% vs. 5%) and yield (approximately 2.8-fold) of IFN. The signal peptide, SP(SlpA) (SlpA, an S-layer protein of Lactobacillus brevis), was also tested to secrete IFN instead of SP(Usp45) (Usp45, the main secreted protein in L. lactis). This gave increased IFN secretion (approximately 3-fold) but lower total production. All the recombinant IFN had appropriate bioactivities in an antiviral assay.

Influence of carbohydrates on the stability and structure of a hyperglycosylated human interferon alpha mutein

Protein physical and chemical instability is one of the major challenges in the development of biopharmaceuticals during every step of the process, ranging from production to final delivery. This is particularly applicable to human recombinant interferon alpha-2b (rhIFN-α2b), a pleiotropic cytokine currently used worldwide for the treatment of various cancer and chronic viral diseases, which presents a poor stability in solution. In previous studies, we have demonstrated that the introduction of four N-glycosylation sites in order to construct a heavily glycosylated IFN variant (4N-IFN) resulted in a markedly prolonged plasma half-life which was reflected in an enhanced therapeutic activity in mice in comparison with the commercial non-glycosylated rhIFN-α2b (NG-IFN). Herein, we evaluated the influence of glycosylation on the in vitro stability of 4N-IFN towards different environmental conditions. Interestingly, the hyperglycosylated cytokine showed enhanced stability against thermal stress, acid pH and repetitive freeze-thawing cycles in comparison with NG-IFN. Besides, microcalorimetric analysis indicated a much higher melting temperature of 4N-IFN, also demonstrating a higher solubility of this variant as denoted by the absence of precipitation at the end of the experiment, in contrast with the NG-IFN behaviour. Furthermore, far-UV circular dichroism (CD) spectrum of 4N-IFN was virtually superimposed with that of NG-IFN, indicating that the IFN structure was not altered by the addition of carbohydrate moieties. The same conclusion could be inferred from limited proteolysis studies. Our results suggest that glycoengineering could be a useful strategy for protecting rhIFN-α2b from inactivation by various external factors and for overcoming aggregation problems during the production process and storage.

Expression patterns of Human Interferon-alpha and Interferon-lambda subtypes by monocytes, dendritic cells and B cells. (136.14)

Abstract Human interferon-alpha comprises a family of thirteen highly homologous genes and twelve subtypes (coding sequences of IFN-alpha1 and -13 are identical). We employed two modifications of fluorescent-labeled probe technology--molecular beacons (MB) and locked-nucleic acids (LNA) to develop a highly efficient, sensitive, and specific quantitative RT-PCR assay for each human IFN-alpha subtype as well as the three subtypes of IFN-lambda, and for completion, IFN-beta and -gamma. The assay is formatted to a 384-well plate that measures 16 samples/plate and includes four-point standards for each IFN. Using this assay, we determined expression patterns of these IFN by monocytes, monocyte-derived macrophages and dendritic cells (MDM and MDDC), primary mDC, pDC and B cells in response to ligands for TLR 3, 4, and 9. All the myeloid cells, including mDC expressed high levels of IFN-beta and -lambda1, and moderate levels of IFN-alpha. pDC expressed high levels of almost all IFN, although with quantitative and in one case, a qualitative difference in between the response to CpG (TLR9) vs. imiquod (TLR7). B cells also expressed IFN—more subtypes than myeloid cells, but and fewer than pDC. Type I IFN expression was always accompanied by type III IFN expression. We conclude that IFN expression patterns are both ligand and cell-type specific. Subtypes alone or in combination may have unique roles in the innate immune response.