[The methods of detection of binding and neutralizing antibodies to preparations of interferon-beta].

The human recombinant ß-interferon is most frequently applied for treatment of remittent recurrent form of multiple sclerosis using pharmaceuticals. The clinical response to applied therapy is absent in some of patients that can be conditioned by development of antibodies too preparations. Depending on possibility of blocking binding of human recombinant ß-interferon with its receptor, all antibodies are divided on binding and neutralizing ones. The purpose of study is to investigate analytical and clinical diagnostic parameters of tests using for detection of different types of antibodies synthesized against human recombinant ß-interferon. The study sampling consisted of 33 patients with remittent recurrent form of multiple sclerosis receiving therapy with human recombinant ß-interferon and also of 40 donors and 15 patients with multiple sclerosis without therapy with human recombinant ß-interferon. The concentration of binding antibodies was measured by enzyme-linked immunosorbent assay. Also immune blotting assay was applied. The titer of neutralizing antibodies was determined using cell line HL-116 sensitive to human recombinant ß-interferon. The binding and neutralizing antibodies were not detected in donors and patients without human recombinant ß-interferon therapy. The prevalence of binding antibodies to human recombinant ß-interferon amounted to 57.6% when analysis of samples using immune blotting assay was used and 60.6% when commercial testing system was applied. The statistical analysis of results demonstrated high convergence and correlation of values of concentrations of binding antibodies obtained using immune blotting assay and enzyme-linked immunosorbent assay (r=0.9159, p<0.0001). The clinically significant titers of neutralizing antibodies were detected in 21.21°% of patients. All patients with clinically significant titer of neutralizing antibodies were positive in relation to binding antibodies measured by immune blotting assay and enzyme-linked immunosorbent assay. The high correlation between values of titers of neutralizing antibodies and concentration of binding antibodies measured by immune blotting assay (r=0.7909, p=0.0055). The application in clinical practice of data concerning presence of binding and neutralizing antibodies to human recombinant ß-interferon can input into optimization of therapy with expensive biologic preparations in patients with multiple sclerosis and other autoimmune diseases.

A novel function of human Pumilio proteins in cytoplasmic sensing of viral infection.

RIG-I-like receptor (RLR) plays a pivotal role in the detection of invading pathogens to initiate type I interferon (IFN) gene transcription. Since aberrant IFN production is harmful, RLR signaling is strictly regulated. However, the regulatory mechanisms are not fully understood. By expression cloning, we identified Pumilio proteins, PUM1 and PUM2, as candidate positive regulators of RIG-I signaling. Overexpression of Pumilio proteins and their knockdown augmented and diminished IFN-ß promoter activity induced by Newcastle disease virus (NDV), respectively. Both proteins showed a specific association with LGP2, but not with RIG-I or MDA5. Furthermore, all of these components were recruited to NDV-induced antiviral stress granules. Interestingly, biochemical analyses revealed that Pumilio increased double-stranded (ds) RNA binding affinity of LGP2; however, Pumilio was absent in the dsRNA-LGP2 complex, suggesting that Pumilio facilitates viral RNA recognition by LGP2 through its chaperon-like function. Collectively, our results demonstrate an unknown function of Pumilio in viral recognition by LGP2.

Purification and biological characterization of soluble, recombinant mouse IFNß expressed in insect cells.

Interferon ß (IFNß) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNß utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNß. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNß purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNß than any other reported eukaryotic-based expression system. Recombinant murine IFNß produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNß activity in vivo. Recombinant IFNß also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNß.

Comparison of site-specific PEGylations of the N-terminus of interferon beta-1b: selectivity, efficiency, and in vivo/vitro activity.

PEGylation, including nonspecific and site-directed approaches, is a well-established and validated strategy to increase the stability, in vivo plasma retention time, and efficacy of protein pharmaceutics together with a reduction in immunogenicity and hydrophobicity. Site-directed conjugation by PEG-aldehyde is the most widely used method for N-terminal modification; however, the generation of multimodified products is inevitable because of lysine chemistry, which always leads to difficulties in purification and quantification. In this study, we developed a specific PEGylation strategy through the periodation of the N-terminus of interferon beta-1b (IFN-ß-1b) followed by the coupling of PEG-hydrazide. The prolonged elimination half-life and significantly diminished immunogenicity of the PEG-hydrazide-modified protein indicated the development of an effective process for improving the pharmacology and immunogenicity of IFN-ß-1b. We further conducted comparisons on the selectivity, velocity, yield, and pharmacokinetics of the two methods. The results demonstrated that the hydrazide-based conjugation was a highly specific coupling reaction that only produced homogeneous N-terminal mono-PEGylated conjugate but also generated heterogeneous multimodified products in the aldehyde-based process. In addition, a better PEGylation yield was found for the hydrazide conjugation compared with that of the aldehyde strategy. These investigations supply a practical approach for the site-specific modification of proteins with an N-terminal serine or threonine to achieve improved homogeneity of the conjugates as well as enhanced pharmacological properties.

Expression and purification of chicken beta interferon and its antivirus immunological activity.

Interferon beta (IFN-ß) belongs to a class of natural proteins that can inhibit virus replication. In this paper, nucleic acid sequence encoding chicken interferon beta (chIFN-ß) mature protein was cloned and highly expressed in Escherichia coli (E. coli) by 1mM IPTG induction. The expressed chIFN-ß was about 20% of total bacterial protein. The recombinant protein in form of inclusion body was then solubilised, refolded and purified to a purity of greater than 95% by immobilized metal ion affinity chromatography (IMAC). This recombinant chIFN-ß could inhibit vesicular stomatitis Indiana virus (VSV) and infectious bursal disease virus (IBDV) replication in vitro. Animal experiments showed that the recombinant chIFN-ß could decrease pathological lesions caused by the IBDV in bursa of Fabricius. These results will provide useful information for further investigations on veterinary clinical applications and fundamental research.

A tale of two interferon bioassays: how frustration with discrepant results from slightly dissimilar methods can engender discovery.

This introductory article describes an episode that took place in the mid-1980s when the first wave of cytokine discoveries took place. During studies aimed at complete purification of human interferon-γ from crude mitogen-stimulated lymphokine preparations, the use of two different antiviral bioassays for the cytokine yielded disparate results. Analysis revealed the presence of a "contaminant" IFN-like cytokine that was detectable with only one of the two assays. Superficially, the contaminant resembled IFN-ß. However, further analysis showed that it was not an IFN at all but an IFN-inducing cytokine identifiable as interleukin-1.

[Isolation of expressed in E. coli human interferon beta1b (Ser17) by ion-exchange chromatography].

A method for isolation of interferon beta1b (Serl7) from inclusion bodies, comprising the steps of solution and reduction of protein from the inclusion bodies, refolding, chromatography on DEAE-Sepharose, chromatography on SP-Sepharose, concentrating, desalting and addition of stabilizers. The solution of reduced protein was diluted with pH 8.0 buffer of 50 mM Tris-HCl, 25 microM CuCl2 and 0.5% Twin 20 for refolding. We used gradient of pH (from 9.3 upto 11.3) for elution of interferon-beta from cation-exchange column. We concentrated of eluate and then desalted on the Sephadex G-50 column with 1 mM NaOH. Then the protein solution was neutralized with mannitol and Na-phosphate. Obtained preparation of interferon-beta was pure by gel-electrophoresis and by HPLC analysis, and had practically indentical level of antiproliferative activity with well-known preparation of Betaferone. Thus we show the possibility of isolation and obtaining of pure and active interferone-beta by ion-exchange chromatography in the presence of non-ion detergent Twin 20. We believe this method for interferon betalb preparation is perspective for scaling and using in the develop of industrial technology for production of this preparation.

High productivity of human recombinant beta-interferon from a low-temperature perfusion culture.

Recombinant human interferon-beta (ß-IFN), used in the therapeutic treatment of multiple sclerosis (MS), can be produced on a large-scale from genetically engineered Chinese hamster ovary (CHO) cells. However, its hydrophobicity causes non-reversible, molecular aggregation in culture. The parameters affecting aggregation were determined to be concentration, culture residence time, temperature and glycosylation. Although the protein can be produced in Escherichia coli in a non-glycosylated form, the addition of glycans confers a reduced rate of aggregation as well as a 10-fold higher bioactivity. We report on the application of a low temperature perfusion culture designed to control the parameters that cause aggregation. In this three-phase culture system there is a transition to a low temperature (32°C) in a batch mode prior to implementing perfusion at 1 volume/day using an acoustic cell separator. Perfusion at the low temperature resulted in a 3.5-fold increase in specific productivity and a 7-fold increase in volumetric productivity compared to the batch culture at 37°C. The percentage aggregation of ß-IFN was reduced from a maximum of 43% in batch culture to a minimum of 5% toward the end of the perfusion phase. The glycosylation profile of all samples showed predominantly sialylated biantennary fucosylated structures. The extent of sialylation, which is important for bioactivity, was enhanced significantly in the perfusion culture, compared to the batch culture.

Cloning, high expression and purification of recombinant human intereferon-beta-1b in Escherichia coli.

Sequential evaluation and process control strategy were employed for impurity profile and high recovery with quality of rhIFN-beta-1b expressed in Escherichia coli. The high-level expression was achieved by using codon substitution (AT content of 52.6% at N-terminal region) and optimization of culture conditions. The addition of rifampicin at a concentration of 200 microg/ml has increased the specific product yield of 66 mg optical density(-1) l(-1) (43.5% of total cellular protein). Eighty-three percent of lipopolysaccharides, 32% of host deoxyribonucleic acid (DNA), and 78% of host cell proteins were removed by 0.75% Triton X-100 and 2 M urea wash. Eleven percent of lipopolysaccharides, 39% of host DNA, and 12% of host cell proteins were removed at the solubilization step. Ninety-two percent of protein refolding was achieved by high-pressure diafiltration method. Refolding by high-pressure diafiltration, bed height, and height equivalent to the theoretical plate value in chromatography column were identified as key parameters for high recovery with purity. Finally, the established process yielded 34% of purified protein with greater than 99% purity and is acceptable for preclinical toxicological studies. The purified rhIFN-beta-1b obtained in this study is the highest that has been reported so far.

[Secreted expression of porcine interferon beta in Pichia pastoris and its inhibition effect on the replication of Pseudorabies virus].

In order to develop recombinant porcine interferon beta with high bioactivity, the rare codes that encoded 3th, 7th and 164th amino acids of porcine interferon beta mature protein were mutant into bias codes of Pichia pastoris and then the modified gene was introduced to yeast secreted expression vector pPICZ alphaA which resulted in pPICZalphaA-PIB. The SacI linearized plasmid pPICZalphaA-PIB was transformed into Pichia pastoris X-33 by electroporation. The transformants were identified by PCR using PoIFN-beta and AOX1 specific primers. The expression of PoIFN-beta was induced with methanol. Several positive clones were obtained and the one namely B1 produced the highest level of PoIFN-beta. The B1 was further fermented in shake-flask in larger volume. The concentration of the secreted PoIFN-beta was about 60 microg/mL and its antiviral activity is about 2.5 x 10(5) U/mL, so the specific activity of porcine interferon beta produced by the Pichia pastoris is approximately 4.17 x 10(6) U/mg. The expressed supernatant was concentrated and identified by SDS-PAGE and Western blot. There are two major proteins with respective molecular mass of approximately 25 kDa and 28 kDa in the supernatant. The results of Western blot indicated that the two proteins were positively reacted and manifested well PoIFN-beta antigenicity. In contrast with the deduced theoretical molecular mass value of PoIFN-beta, the expressed two major proteins were larger which maybe due to the difference of glycosylation. The antiviral effect of recombinant porcine interferon beta (rPoIFN-beta) on Pseudorabies virus (PrV) was studied in the present experiment. The result indicated that rPoIFN-beta could effectively inhibit the replication of PrV in MDBK cells, especially during the early phage of the virus replication.

[Purification and identification of human recombinant IFN-beta expressed in yeast Pichia pastoris].

To find an effective and quick way of purifying and identifying recombinant human IFN-beta (rhIFN-beta) expressed in yeast Pichia pastoris, Blue Sepharose 6 fast flow (Blue S6FF) and immunological affinity chromatography (IAC) were compared in this report. rhIFN-beta was produced in 15 liter bioreactor and purified using the two methods mentioned above. The protein concentrations of rhIFN-beta and residual mouse IgG in purified rhIFN-beta were determined with ELISA. The molecular weight and specificity were demonstrated by PAGE and Western blot. The density of the specific precipitation bands was determined by gel scanning. The relative bioactivities were determined by cyto pathogenic effect inhibition (CPEI). The results showed that 2.65 and 3.03 mg of rhIFN-beta were obtained, respectively, by purifying with Blue S6FF or IAC from 2 liter of fermentation supernatant. The molecular weight was 22 kD. The concentrations of the special precipitation of rhIFN-beta were 95.1% and 96.2% respectively. The relative bioactivity of rhIFN-beta purified by Blue S6FF and IAC were 1.63x10(7) IU/mg and 1.43x10(7) IU/mg, respectively. The residual mouse IgG in purified rhIFN-beta by IAC was less than 50 microg/L. The results indicated that rhIFN-beta could be purified effectively and quickly from fermentation supernatant of yeast Pichia pastoris by IAC. The rhIFN-beta products purified by Blue S6FF and IAC had almost the same purity and bioactivity. The data accumulated from the experiment are useful to the preparation of rhIFN-beta on a larger scale.

Expression and characterization of human interferon-beta1 in the methylotrophic yeast Pichia pastoris.

We describe the heterologous expression of a human interferon-beta1 in the methylotrophic yeast Pichia pastoris. Biologically active recombinant human interferon-beta1 (rHuIFN-beta1) was secreted from shake-flask-grown P. pastoris cells into the medium using the Saccharomyces cerevisiae alpha-mating factor prepro-leader sequence at the level of (1-3) x 10(5) i.u. (international units)/ml (6-12 mg/litre). An rHuIFN-beta1 with an N-terminal sequence identical with that of native HuIFN-beta1 was purified and the specific activity was determined (2-3 x 10(7) i.u./mg). It was found that the secreted recombinant protein was partially N-glycosylated.

Characterization of a soluble ternary complex formed between human interferon-beta-1a and its receptor chains.

The extracellular portions of the chains that comprise the human type I interferon receptor, IFNAR1 and IFNAR2, have been expressed and purified as recombinant soluble His-tagged proteins, and their interactions with each other and with human interferon-beta-1a (IFN-beta-1a) were studied by gel filtration and by cross-linking. By gel filtration, no stable binary complexes between IFN-beta-1a and IFNAR1, or between IFNAR1 and IFNAR2 were detected. However, a stable binary complex formed between IFN-beta-1a and IFNAR2. Analysis of binary complex formation using various molar excesses of IFN-beta-1a and IFNAR2 indicated that the complex had a 1:1 stoichiometry, and reducing SDS-PAGE of the binary complex treated with the cross-linking reagent dissucinimidyl glutarate (DSG) indicated that the major cross-linked species had an apparent Mr consistent with the sum of its two individual components. Gel filtration of a mixture of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the three proteins formed a stable ternary complex. Analysis of ternary complex formation using various molar excesses of IFNAR1 and the IFN-beta-1a/IFNAR2 complex indicated that the ternary complex had a 1:1:1 stoichiometry, and reducing SDS-PAGE of the ternary complex treated with DSG indicated that the major cross-linked species had an apparent Mr consistent with the sum of its three individual components. We conclude that the ternary complex forms by the sequential association of IFN-beta-1a with IFNAR2, followed by the association of IFNAR1 with the preformed binary complex. The ability to produce the IFN-beta-1a/IFNAR2 and IFN-beta-1a/IFNAR1/IFNAR2 complexes make them attractive candidates for X-ray crystallography studies aimed at determining the molecular interactions between IFN-beta-1a and its receptor.

Effects of human trophoblast-induced interferons on the expression of proto-oncogenes c-fms/CSF-1R, EGF-R and c-erbB2 in invasive and non-invasive trophoblast.

The human cytotrophoblast is the first fetal cell type to arise during embryogenesis and differentiate along two pathways to the invasive (extravillous) and non-invasive (villous) populations. The non-invasive villous trophoblast differentiate morphologically and biochemically to form terminally differentiated multinucleated syncytial trophoblast. First trimester invasive and non-invasive trophoblast were isolated from human placentae (5-12 weeks) and were cultured in vitro. The villous trophoblast cells differentiated in vitro to form aggregated syncytial cells which was associated with increased expression of epidermal growth factor receptor (EGF-R). The invasive trophoblast cells expressed colony-stimulating factor receptor (c-fms/CSF-1R) and c-erbB2 proteins but low levels of EGF-R. We studied the effects of human trophoblast-induced interferon (IFN)-alpha/beta on the expression of c-fms/CSF-1R, EGF-R and c-erbB2 whose ligands are reported to be involved in the regulation of growth and differentiation of normal invasive and non-invasive trophoblast cells. Human trophoblast-induced IFN-alpha/beta (100 IU/ml) reduced the expression of EGF-R in both invasive and non-invasive trophoblast cells as determined by quantitative enzyme-linked immunosorbant assay ('ELISA') and western immunoblot methods. The same amount of IFN activity reduced the expression of c-fms/CSF-1R and c-erbB2 proto-oncogene products in invasive trophoblast cells. These results may suggest a possible role of trophoblast-induced IFNs in the regulation of normal trophoblast growth, differentiation and function.

Global regulatory considerations for unified assay specifications.

When developing a biotechnology product for global registration, there are several aspects to evaluate in an effort to unify specifications. These include differences between the United States, Europe and Japan, and Rest-of-World (ROW) countries with regard to the respective regulatory guidelines and pharmacopoeias in force, the state-of-the-art of product testing analytical methods, and the interval between submitting a registration dossier to different countries. In terms of regulatory guidelines, one country may have a monograph or required specifications for particular tests, for example the potency that a product has to meet before clinical trials can be initiated. For pharmacopoeias, different assay methods are required for sterility, general safety, and pyrogen testing, so that one may have to test a specific lot of a product at two or three different times to evaluate the same parameter, because of specific testing differences required for each country's pharmacopoeia. In addition, the state of analytical methods is always evolving and better analytical techniques become available. Sometimes, from starting with one set of tests, and based on the time in development, new tests may be added to the existing list of release specifications, because new analytical techniques have become available. Examining the global registration approval process for Betaseron, (interferon beta-1b) illustrates when specifications were able to be unified and when they were not.

Human trophoblast interferons: production and possible roles in early pregnancy.

Human villous and extravillous trophoblast populations were isolated from first- and third-trimester placentae and were stimulated with viral and non-viral inducers to produce interferons (IFNs). Polyriboinosinic/polyribocytidylic acid [poly(I:C)] induced exclusively IFN-beta in trophoblast cultures, whereas viruses induced mixtures of IFN-alpha subtypes and -beta. The level of IFN production was dependent on the trophoblast population, type of inducer and the stage of differentiation of the trophoblast. First-trimester extravillous trophoblast cultures produced greater than five-fold more IFN than third-trimester villous trophoblast on a per cell basis, whereas term syncytiotrophoblast produced twice as much IFN as term mononuclear villous trophoblast when stimulated with the same inducer. Pretreatment of trophoblast cultures with platelet-derived growth factor and granulocyte/macrophage-colony stimulating factor (GM-CSF) increased the trophoblast IFN production. Tandem high-performance affinity chromatography of the virus-induced trophoblast IFNs resulted in the isolation of trophoblast IFN-alpha and -beta with specific antiviral activities of 0.75-2.73 x 10(8) IU/ml protein. The trophoblast-induced IFNs have antiproliferative and immunosuppressive properties, and, furthermore, activated natural killer cell activity. These data may suggest the possible roles of these IFNs during embryonic development with regard to protection of the fetus against viral infection and maternal immunity.

The use of Zwittergent 3-14 in the purification of recombinant human interferon-beta Ser17 (Betaseron).

A new method for purifying human interferon-beta SER17 from E. coli-derived inclusion bodies has been developed. This procedure eliminates the need for strong denaturants, such as sodium dodecyl sulfate or chaotropes. The procedure makes use of a nondenaturing detergent and a brief incubation at pH 12 to solubilize interferon-beta Ser17 from inclusion bodies. The detergent used was Zwittergent 3-14 (nonionic and pH-insensitive), which is included in the class of sulfobetaines (RN+ (CH3)2(CH2)xSO3-). Zwittergent 3-14 was used in combination with urea to produce a urea/Zwittergent 3-14 washed inclusion body preparation enriched in human interferon-beta Ser17 (Betaseron). Solubilization of inclusion bodies was accomplished by employing a brief (1 minute) shift to pH 12 in the presence of 2.5% Zwittergent 3-14 followed by rapid adjustment to pH 8.0. Solubilization was complete, and the solution could be rapidly adjusted to pH 8 without any observable precipitation of protein. The resultant supernatant could be successfully subjected to a number of chromatographic and analytic procedures, many of which are not compatible with strong anionic detergents, such as SDS. Betaseron was purified from Zwittergent 3-14 solubilized inclusion body lysates using both ion-exchange and size-exclusion chromatography. Purified Betaseron retained bioactivity and could be refolded by simple dialysis against a nonreducing buffer. This method represents a novel procedure for purifying Betaseron from inclusion bodies using a nondenaturing detergent and ion-exchange chromatography.

In vitro cytotoxic activity of cord blood NK cells against herpes simplex virus type-1 infected purified human term villous cytotrophoblast.

Transplacental infection of the fetus with herpes simplex virus (HSV) is associated with high morbidity. The present study was undertaken to shed light on the possible participation of the fetal immune system in the elimination of HSV from placental unit. In a chromium release assay cultured term villous trophoblast cells, regardless of infection with HSV-1, were found resistant to lysis by cord blood natural killer (CBNK) cells. In contrast to this, CBNK cells exhibited a basal level of cytotoxic activity against placental fibroblasts, which was significantly increased by preceding infection of the target cells with HSV-1. Stimulation of CBNK cells with interferon-beta purified from trophoblast (tro-IFN-beta) increased the killing of both HSV-1 infected and uninfected fibroblast, while HSV-1-infected and uninfected term villous trophoblast cells remained resistant to lysis. IL-2-stimulated CBNK cells were able to lyse villous trophoblast cells at a low level, but no significant difference in the susceptibility of the HSV-1-infected and uninfected trophoblast cell was detected.