Human Interferon-alpha Research Articles

Еxternal forms of recombinant interferon alfa-2b - ointment and gel in the treatment of acute respiratory viral infections and influenza in children

The review article presents the results of numerous studies on the effectiveness and safety of the newly developed forms of the human recombinant interferon alpha- 2b drug, ointment and gel, intended for the treatment and prevention of acute respiratory viral infections and influenza in children. Еxternal forms of the drug retain immunomodulatory, antiviral and antiproliferative effects. The dosage form of the preparation of interferon alpha-2b VIFERON® in the form of a gel or ointment allows to achieve a prolonged action due to good absorption, especially the gel, which increases its effectiveness, and also has a moisturizing effect and rarely causes allergies. All of the above properties justify the inclusion of the drug interferon alpha-2b VIFERON® in the form of a gel or ointment in the complex treatment of influenza and acute respiratory viral infections in children.

Site-specific monoPEGylated interferon alpha2a mediated by microbial transglutaminase

PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.

Aspects of a Methodological Approach to Determination of Interferon Alpha Specific Activity

Specific antiviral activity is one of the key indicators characterising pharmaceutical quality and pharmacological efficacy of interferon alpha products (IFN-α). Specific activity is determined using a bioassay measuring antiviral activity in cell culture. The aim of the study was to select the most appropriate conditions for in vitro determination of IFN-α product specific activity. Materials and methods: Vero, MDBK, Hep-2, and A-549 homologous and heterologous cell cultures, as well as vesicular stomatitis Indiana virus (VSV) and murine encephalomyocarditis (EMC) virus at a dose of 100 TCD50 /0.1 mL were used for determination of specific antiviral activity. The international reference standard of recombinant interferon alpha-2b activity (Interferon alpha 2b, human, rDNA, E. coli-derived, 2nd WHO International Standard, 1999, NIBSC Code No. 95/566) and human recombinant interferon alpha 2b in the form of solution (batch No. 040214, Pharmapark LLC, Russia) were used as IFN-α samples. Results: the analysis of the obtained data helped to determine: the combinations of cell lines and the indicator virus most sensitive to IFN-α; the optimal concentration of fetal serum in the medium, and the optimal time parameters; the preferred method of reporting test results. Conclusions: the following test conditions were found to be optimal: the MDBK/VSV and Hep-2/EMC combinations proved to be the most sensitive to IFN-α; the optimal period of interferon and cell culture incubation—24 hours; the optimal concentration of fetal bovine serum in the culture medium used for diluting interferon products—2–5%. The instrumental procedure is preferred for reporting the results of interferon antiviral activity determination, because it is up-to-date, reliable, accurate and time-efficient.

A prospective multicenter randomized controlled study on the efficacy and safety of pharyngeal spraying recombinant human interferon alpha 2b in the treatment of children with herpangina

Objective To evaluate the efficacy and safety of pharyngeal spraying recombinant human interfe-ron alpha 2b (rhIFNα2b) in the treatment of herpangina in children. Method s A prospective, multicenter, rando-mized, opened and controlled study was carried out in 11 hospitals in Anhui province from August 2018 to March 2019.According to the time of admission, 180 patients diagnosed as herpangina were prospectively and randomly divided into rhIFNα2b treatment group and Ribavirin control group.On the basis of giving both groups the heat-clearing, detoxifying and anti-infection treatment, the patients in treatment group received pharyngeal spraying rhIFNα2b 9 g/L saline solution[1 million IU/mL, 0.1 million IU/(0.1 mL·press)], and the patients in control group were treated by pharyngeal spraying Ribavirin (0.5 mg RBV/press, 150 press), 3 presses per time, 4 times per day, continuous administration for 5 days for both groups.Those who recovered in advance were no longer given medication.All patients were observed to fully recover.The clinical efficacy and the disappearing time of symptoms and signs between two groups were compared, and the safety of pharyngeal spraying rhIFNα2b for patients was evaluated. Result s All of the 180 patients completed the study, including 90 cases in the treatment group and 90 cases in the control group.There was no statistically significant difference in terms of gender, age, weight and course of illness before treatment between the two groups (all P>0.05), which had clinical comparability.The apparent efficiency of the treatment group [63.3% (57/90 cases)] was significantly higher than that in the control group [38.9% (35/90 cases)] and the difference was statistically significant(χ2=10.934, P=0.004); no significant difference in the total efficiency between the treatment group [96.7% (87/90 cases)]and the control group [92.2% (83/90 cases)]was observed (χ2=2.924, P=0.169). The duration of fever[(32.59±20.73) h vs.(45.72±26.96) h], hyperemia[(76.48±23.12) h vs.(92.44±24.31) h], herpes[(72.99±25.77) h vs.(85.09± 26.62) h], salivation[(45.44±24.96) h vs.(54.42±31.20) h] and anorexia[(62.70±23.99) h vs.(78.71±30.54) h] in the treatment group were significantly shorter than those in the control group, and the differences were statistically significant(all P 0.05). After treatment, the serum levels of TNF-α and IL-6 were(7.26±1.99) ng/L and (2.42±0.73) ng/L in the treatment group, which were significantly lower than those in the control group [(12.09±6.39) ng/L and (7.32±11.51) ng/L](all P 0.05). The comparison on positive rate of virus in pharyngeal swab between the treatment group [65.3% (32/49 cases) and 40.6% (13/32 cases) respectively] and the control group[66.7%(36/54 cases) and 41.0% (16/39 cases), respectively]before and after therapy showed no significant difference (all P>0.05). During the treatment, no serious adverse reactions were observed in the two groups.The incidence of adverse reactions was 1.1% (1/90 cases) in the treatment group and 5.6% (5/90 cases) in the control group.In addition, the serum hemoglobin level of children in the control group after treatment was significantly lower than that before treatment and that in the treatment group (all P<0.05). Conclusions Compared with pharyngeal spraying ribavirin, pharyngeal spraying rhIFNα2b can greatly improve the clinical efficiency, accelerate the disappearance of clinical symptoms and signs, and shorten the total course of disease, and is more safe and worthy of clinical application. Key words: Herpangina; Recombinant human interferon alpha 2b; Ribavirin; Pharyngeal spraying

Therapeutic responses to interferon-alpha in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP): Differing clinical responses to leukocyte-derived human interferon-alpha versus recombinant interferon-alpha

Introduction: HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive spinal cord syndrome seen in patients infected by human T-lymphotropic virus type 1 (HTLV-1). Initial infection by the virus is usually asymptomatic. As the disease progresses, a se

Identification and characterization of human interferon alpha inhibitors through a WISH cell line-based reporter gene assay.

Interferons (IFNs) are important glycoproteins which can stimulate or inhibit up to three hundred different genes encoding proteins involved in antiviral defense mechanisms, inflammation, adaptive immunity, angiogenesis and among other processes. Nevertheless, different genetic alterations may lead to interferon alpha (IFN-α) overproduction in human autoimmune diseases like systemic lupus erythematosus. As a consequence, IFN-α is a central molecule whose activity must be regulated to block their harmful effect on those disorders where the endogenous cytokine production constitutes the etiology of the illnesses. In this work, we evaluate the biological activity of eighty-eight compounds, from our own chemo-library, to find potential IFN-α inhibitors by using a reporter gene assay (RGA) WISH-Mx2/EGFP. We identified some compounds able to modulate negatively the IFN-α activity. The most active IFN-α inhibitors were further studied achieving promising results. In addition, some combinations of the most active compounds were analyzed accomplishing a stronger effect to decrease the IFN-α activity than each compound alone. Furthermore, the complete inhibition of the cytokine activity was reached with some combinations of compounds.

Safety of aerosol inhalation of recombinant human interferon alpha 2b injection(P.putida)and excipient hydroxyethyl starch 40 to healthy SD rats

Objective To investigate the effects of recombinant human interferon alpha 2b injection(P.putida)and hydroxyethyl starch inhalation on physiological indexes and tissues and organs of SD rats, such as lungs, trachea and bronchus, and thus to explore the safety of the drug and excipients for inhalation. Methods Rats were randomly divided into two groups(hydroxyethyl starch 40 group and recombinant human interferon alpha 2b group).5 million IU/ml recombinant human interferon alpha 2b injection or 6% hydroxyethyl starch 40 injection were given respectively, which were diluted with 0.9% sodium chloride injection in a radio of 1: 2.Both groups were given aerosol inhalation for 30 minutes once a day for 14 days.Within 14 days after administration, the behavior of rats and the degree of toxicity were continuously observed.During the drug administration and recovery period, the clinical signs of the rats in each group were observed daily, and the body weight and food consumption were measured once a week.At the end of drug administration and the end of recovery period, hematology test, blood biochemical test, gross anatomical observation, organ weight determination, and histopathological examination were performed. Results The mass median aerodynamic diameter(MMAD)of the drug mist was 3.56 μm, and the geometric standard deviation(GSD)was 1.84 μm, which met the particle size requirements of arrival and deposition in the lower respiratory tract via atomization inhalation.No abnormality in clinical signs, body weight and food consumption, hematology and blood biochemical indexes were found in hydroxyethyl starch 40 group and recombinant human interferon alpha 2b group.No abnormal histopathological changes were observed in oral mucosa, tongue, nasal cavity(paranasal sinus), larynx, trachea, main bronchial tube, lung, heart, liver, spleen, kidney, reproductive system and other organs or tissues examined. Conclusion Results suggest the drug particle size of recombinant human interferon alpha 2b injection(pseudomonas)can reach the lower respiratory tract and deposit in the lower respiratory tract.Continuous atomization inhalation for 14 days has a good safety, and the excipient hydroxyethyl starch 40 has a high safety. Key words: Recombinant human interferon alpha 2b; Injection; Aerosol inhalation; Hydroxyethyl starch 40; Safety

Follow-Up of Viral Parameters in FeLV- or FIV-Naturally Infected Cats Treated Orally with Low Doses of Human Interferon Alpha.

Specific treatments for the long-life infections by feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are either toxic, expensive or not too effective. Interferon α (IFN-α) is an immunomodulatory molecule which has been shown in vitro to decrease the release of infective particles. The aim of this study was to follow the progress of the clinical score and viral parameters of FeLV- and FIV-naturally infected privately owned cats treated with recombinant human IFN-α (rHuIFN-α, Roferon-A). Twenty-seven FeLV-infected cats (FeLV+) and 31 FIV-infected cats (FIV+) were enrolled in the study. Owners were instructed to orally administer 1 mL/day of 60 IU rHuIFN-α/mL in alternating weeks for four months. Blood samples were taken at the beginning of the study (M0), mid-treatment (M2), end of treatment (M4), and 6–10 months later (M10). Clinical status at these time points improved notably with rHuIFN-α treatment, regardless of the initial severity of the disease, an effect which lasted throughout the study in most animals (15 of the 16 FeLV+ symptomatic cats; 20 of the 22 FIV+ symptomatic cats) improved markedly their clinical situation. In FeLV+ cats plasma antigenemia (p27CA), reverse transcriptase (RT) activity, and proviral load decreased at M2 and M4 but increased again at M10 (“rebound effect”). The level of antigenemia or RT activity was below the detection limits in FIV+ cats, and the effect on proviral load was less marked than in FeLV+ cats. Taken together, these results indicate that rHuIFN-α is a good candidate for treating FeLV+ cats, but the “rebound effect” seen when treatment was discontinued suggests that additional studies should be conducted to clarify its effect on progression of the infection in cats.

Systems of intraganglia and intraneural control over latent alpha-herpesvirus in sensory ganglia of peripheral nervous system

The article presents modern data on interferon‑dependent mechanisms of control of latent alpha herpesvirus in the sensory nerve ganglia of the peripheral nervous system. The data on intraganglionar control mechanisms presented by resident natural killers, natural killer T‑lymphocytes and cytotoxic T‑cells that are part of the satellite cells of the nerve node, and the system of intraneuronal defense contained in the centripetal fibers and presented by cascade system TLR‑3, which leads to the production of pre‑immune interferons‑alpha, ‑beta and ‑lambda with a visorathic action, thus counteracting the centripetal transneural spread of the virus from the periphery to the central nervous system. The joint functioning of the interferon‑dependent systems of intraganglionar and intraneuronal control of alpha herpes viruses ensures the maintenance of the latent state of the pathogen, and the disruption of their work is associated with the risk of reactivation of the virus from latency with the development of clinical symptoms. At the same time, lack of intraganglionar cellular immunity is associated with frequent herpetic eruptions on the skin and mucous membranes, while a deficiency of intraneuronal TLR‑3‑dependent immunity leads to the development of herpes virus lesions of the central nervous system — myelitis, encephalitis, and meningitis. It supports the well‑known clinical fact, that herpetic encephalitis often develops in patients without a history of frequent specific exanthema, and individuals with frequent herpetic eruptions on the skin and mucous membranes may never suffer from viral lesions of the brain and spinal cord. New knowledge about the mechanisms of retaining alpha herpesviruses in the latent state of the sensory ganglia can help in the selection of rational therapy for immunocompromised patients with episodes of herpesvirus neuroinfections with the inclusion of drugs of natural, recombinant and lymphoblastoid human alpha interferons and various inducers of interferonogenesis of chemical or natural origin.The article will be useful, first of all, to neurologists, neurosurgeons, infectious diseases specialists, clinical immunologists, pathologists, physiologists.

Control of specific growth rate for the enhanced production of human interferon α2b in glycoengineered Pichia pastoris: process analytical technology guided approach

AbstractBACKGROUNDProcess variability in bioprocess systems involving genetically engineered strains is a common bottleneck and a real‐time insight of the on‐going process is crucial to achieve the desired product and its quality. In this study, a process analytical technology (PAT) platform was developed for the monitoring and control of specific growth rate during methanol induction phase (μmet) of glycoengineered Pichia pastoris fermentation for human interferon alpha 2b (huIFNα2b) production.RESULTSThe PAT guided approach involves real‐time monitoring of capacitance (ΔC) facilitating online estimation of specific growth rate (μest), which serves as a process input during controller application. Fed‐batch experiments using pulsed‐feeding of methanol at different dosage (20 g and 30 g) did not significantly influence μmet. Exponential methanol feeding was achieved using a modified proportional, integral and derivative (PID) controller for different predefined specific growth rate set point (μsp) values, namely 0.015, 0.03, 0.04 and 0.06 h−1. Exponential feeding strategy during the induction phase resulted in two crucial outcomes: (i) controlled methanol feeding rate (regulated by the developed PID controller) balanced the methanol consumption rate (qs, met) of the P. pastoris; (ii) significant improvement in huIFNα2b titer (1483 mg L−1) and specific productivity(&gt;0.4 mg g−1. h) was achieved by the robust control of μmet at optimal (0.04 h−1) value. The purified huIFNα2b was found to exhibit antiproliferative effect against human breast cancer cell lines.CONCLUSIONSEfficient control of μmet at a very low narrow range was achieved with a long‐term controller stability (&gt;10 h) and the highest titer reported to date for huIFNα2b (at optimal μmet) in the yeast expression platform. © 2019 Society of Chemical Industry

S912 LOCAL INTERFERON‐ALPHA DELIVERY INTO THE MYELOMA MICROENVIRONMENT BY GENE‐ENGINEERED AUTOLOGOUS STEM CELL TRANSPLANTATION

Background:Multiple Myeloma (MM) is an incurable malignancy heralded by an immunosuppressive Tumor Microenvironment (TME). Therapeutic attempts aiming at reprogramming the TME and unleashing innate and adaptive immunity may synergize with current anti‐MM drugs, deepen responses and ultimately prolong long‐term survival.Aims:We developed an ex vivo Gene Therapy (GT) approach that locally delivers Interferon‐alpha (IFNa) into the TME through the Tie2+ myeloid progeny of transplanted gene‐modified Hematopoietic Stem and Progenitor Cells (HSPCs).Methods:Murine Lin‐ or human Cord Blood (huCB) HSPCs were transduced with lentiviral vectors (LV) expressing, respectively, murine or human IFNa under transcriptional control of the Tie2 promoter and post‐transcriptional control by miR‐126–3p thereby targeting IFNa expression to a subset of tumor‐infiltrating monocytes. IFNa‐LV‐transduced or mock‐transduced HSPC were transplanted into Vk∗Myc transgenic mice after they have spontaneously developed a serum M‐spike (immunocompetent mouse model) or into NSGW41 mice that were successively challenged with a luciferase+ MM.1S human myeloma cell line (human xenograft model). Myeloma disease progression was monitored by serum protein electrophoresis (Vk∗Myc), whole body bioluminescence imaging (MM.1S) and bone marrow plasma cell burden at the endpoint. In the MM1.S model, IFNa‐GT was combined with systemic Daratumumab (i.p.), Lenalidomide (i.p.) and Carfilzomib (i.v.) treatment.Results:In the spontaneous Vk∗Myc mouse model of myeloma, Ifna GT halted myeloma progression (4 months PFS of 58.3% vs. 15.8% in control) and reduced mortality by &gt;50%. Treatment response was associated with a higher proportion of CD8+ T cells in the BM and seen at donor cell chimerism as low as 4%. Mechanistic studies on the tumor microenvironment are ongoing. The NSGW41 xenograft model transplanted with huCB HSPCs allowed the development of a larger and more diverse human myeloid compartment compared to the classical NSG model. Even in the absence of adaptive immunity, IFNa‐GT resulted in a significant reduction in BM myeloma burden (13.4+/‐2.2% vs. 26.3+/‐3.7% in mice transplanted with control huCB cells; n = 19 vs. 18), independently confirmed by BLI. Interestingly, the IFNa‐GT group showed an increased content of human CD33+ HLA‐DR+ CD11c+ dendritic cells (including a pro‐inflammatory SLAN+ subset) and increased levels of CD16 and HLA‐DR expression on human myeloid cells within the myeloma‐bearing BM, suggestive of myeloid cell reprogramming towards a more immune‐stimulatory state. Moreover, IFNa‐GT induced an upregulation of CD38 expression (on average 1.4 fold on myeloma cells). This provides a rationale for combining IFNa‐GT with other anti‐MM drugs. Indeed, the anti‐MM effect of Daratumumab, Lenalidomide or Carfilzomib was enhanced by IFNa‐GT, without excessive toxicity on the HSPC graft.Summary/Conclusion:Our studies conducted both in the immunocompetent Vk∗Myc murine model as well as in the hematochimeric xenotransplantation setting demonstrate that IFNa‐GT has robust anti‐MM effect and may subvert the immunosuppressive MM TME, acting both on myeloma cells as well as on the immune infiltrate. A phase I/II dose‐escalation study of IFNa‐GT in patients with early‐relapsed multiple myeloma has recently been approved and is starting accrual in March 2019.

Novel glycosylated human interferon alpha 2b expressed in glycoengineered Pichia pastoris and its biological activity: N-linked glycoengineering approach

Human interferon alpha 2b (IFN α2b) is a type I interferon exhibiting antiviral, anti-proliferative and immunomodulatory activities. The clinical outcome of the approved recombinant human IFN α2b drugs in the market suffers from short plasma half-life, rapid clearance and other side effects. Human IFN α2b expression in mammalian cell lines results in significant heterogeneity in glycan moieties, inconsistent product quality and high production cost. Potential scope exists for the design and development of a successful expression platform for enhanced human IFN α2b production with improved pharmacokinetic property. Glycoengineering strategy was employed to construct IFN α2b with potential N-glycosylation site to evade the drawbacks of approved recombinant human IFN α2b drugs. Heterogeneity of glycosylation and hypermannosylation in the wild-type strains of Pichia pastoris was circumvented by employing glycoengineered strain (SuperMan5) to produce glycosylated IFN α2b with human type N-glycans. Recombinant SuperMan5 strain expressed human type N-glycosylated IFN α2b with greater homogeneity elucidated by glycan analysis (MALDI-TOF/MS). The purified glycosylated IFN α2b was biologically active, inhibiting the viral replication of HCV and HEV at 85% and 66%, respectively. Pharmacokinetic studies in Wistar rats revealed 1.3 fold increase in plasma half-life for glycosylated IFN α2b compared to standard IFN α2b produced by E. coli.

High level extracellular production of recombinant human interferon alpha 2b in glycoengineered Pichia pastoris: culture medium optimization, high cell density cultivation and biological characterization.

The present study was aimed at design of experiments (DoE)- and artificial intelligence-based culture medium optimization for high level extracellular production of a novel recombinant human interferon alpha 2b (huIFNα2b) in glycoengineered Pichia pastoris and its characterization. The artificial neural network-genetic algorithm model exhibited improved huIFNα2b production and better predictability compared to response surface methodology. The optimized medium exhibited a fivefold increase in huIFNα2b titre compared to the complex medium. A maximum titre of huIFNα2b (436mgl-1 ) was achieved using the optimized medium in the bioreactor. Real-time capacitance data from dielectric spectroscopy were utilized to model the growth kinetics with unstructured models. Biological characterization by antiproliferative assay proved that the purified recombinant huIFNα2b was biologically active, exhibiting growth inhibition on breast cancer cell line. Culture medium optimization resulted in enhanced production of huIFNα2b in glycoengineered P. pastoris at both shake flask and bioreactor level. The purified huIFNα2b was found to be N-glycosylated and biologically active. DoE-based medium optimization strategy significantly improved huIFNα2b production. The antiproliferative activity of huIFNα2b substantiates its potential scope for application in cancer therapy.

POLYPHENOLIC WEALTHY MORINGA LEAVES EXTRACTS AS ANTICOXSACKIE B VIRUSES (COX-BV)

The antiviral role of Moringaoleifera leaves extracts ((chloroform (CL), Ethyl acetate (E.A), methanol 80% (M) and n.butanol (n.b) to control Coxsackie B (COX-BV) viral infection was monitored in vitro compared with human interferon alpha (IFNᾰ2a) in order to evaluate the antiviral activity of moringa leaves extracts. The phenolic compound was given higher conc. in E.A. extracts. Cytotoxicity was determined for Moringa leaves extracts on viability of HEP2 cell using MTT assay. The 98μg/ml concentrations for four extracts were considerably non-toxic for HEP2 cell line culture. Antiviral activity in Moringa extracts may be attributed to phenolic compounds and / or as a result of stimulation of COX-BV-sensitive HEP2 cells to express MX protein. Antiviral activity of Moringa leaves extracts compared with IFNᾰ-2a to cell lines against COX-BV viral infection was determined by assessment of the COX-BV virus infectivity titer declining rate and relative residual living cell count using End Point Assay, using three ways pretreatment, co-treatment and post-treatment cell line treated with Moringaextracts. It was found that the extract acetyl acetate was better as an MX gene expression level compared with its level in control cells. It was concluded that Moringaleaves extracts have antiviral activity against COX-BV.

The effect of human interferon alpha on replication of different bovine viral diarrhea virus strains.

Bovine viral diarrhea virus (BVDV) exists in two main biotypes: cytopathic (cp) and noncytopathic (ncp). Although some studies were done on the effect of interferon alpha (IFN-α) on BVDV, the effect of exogenous IFN against BVDV biotypes remains unclear. In the present study, we evaluated the comparative effect of exogenous human IFN-α (HuIFN-α) on different BVDV biotypes and genotypes. The results showed that exogenous HuIFN-α greatly inhibited the growth of different BVDV biotypes and genotypes. However, HuINF-α has a significant inhibitory effect on cp biotype compared to ncp one without significant variation between different genotypes. The effect of HuIFN-α on BVDV reached the maximum level at early stages of infection (0-20 h post infection) and increased in a dose-dependent manner (10-500 U/ml). Quantitative real-time RT-PCR was used to evaluate the effect of exogenous HuIFN-α on RNA synthesis of both BVDV biotypes. HuIFN-α reduced RNA production of cp by 4 logs compared to only 2 logs for ncp strains. Additionally, the antiviral effect of IFN-α against both BVDV biotypes seems to be independent of the RNA-dependent protein kinase (PKR) activation as assayed by direct analysis of in vivo phosphorylation of eIF2-α and by 2-aminopurine (2-AP) treatment. Collectively, these results indicated that the exogenous HuIFN-α treatment has an inhibitory effect not only on cp BVDV biotype but also on the ncp BVDV. The antiviral effect of exogenous HuIFN-α was biotype, time, dose but not genotype dependent. PKR has no role in the inhibitory effect suggesting that other IFN-antiviral pathways were involved. Keywords: BVDV biotypes; HuIFN-α; RNA synthesis; PKR-independent.

Эффективность применения препарата интерферона альфа-2b для профилактики острых респираторных вирусных инфекций и подготовки к ежегодной туберкулинодиагностике у часто болеющих детей

Эффективность применения препарата интерферона альфа-2b для профилактики острых респираторных вирусных инфекций и подготовки к ежегодной туберкулинодиагностике у часто болеющих детей

ARID3a gene profiles are strongly associated with human interferon alpha production

Type I interferons (IFN) causes inflammatory responses to pathogens, and can be elevated in autoimmune diseases such as systemic lupus erythematosus (SLE). We previously reported unexpected associations of increased numbers of B lymphocytes expressing the DNA-binding protein ARID3a with both IFN alpha (IFNα) expression and increased disease activity in SLE. Here, we determined that IFNα producing low density neutrophils (LDNs) and plasmacytoid dendritic cells (pDCs) from SLE patients exhibit strong associations between ARID3a protein expression and IFNα production. Moreover, SLE disease activity indices correlate most strongly with percentages of ARID3a+ LDNs, but were also associated, less significantly, with IFNα expression in LDNs and pDCs. Hierarchical clustering and transcriptome analyses of LDNs and pDCs revealed SLE patients with low ARID3a expression cluster with healthy controls and identified gene profiles associated with increased proportions of ARID3a- and IFNα-expressing cells of each type. These data identify ARID3a as a potential transcription regulator of IFNα-related inflammatory responses and other pathways important for SLE disease activity.

Attach great importance to innovative research and clinical application of recombinant human interferon alpha 1b in pediatrics

As the first class Ⅰ novel genetic engineering medical product in China, recombinant human interferon alpha 1b (IFNα1b) shows unique biological significance and clinical value.Especially in the field of pediatrics, IFNα1b has been used in the treatment of viral pneumonia, viral hepatitis, bronchiolitis, herpetic angina, hand-foot-mouth disease and some tumors in children and shown definite efficacy and good safety.It is also used in the prevention and treatment of virus-related wheezing and major infectious diseases, such as influenza.With a greater application prospect, IFNα1b plays an important role in promoting children′s health in China.Now, IFNα1b′s gene and molecular characteristics, new delivery methods, and the frontier clinical research will be discussed. Key words: Interferon α1b; Child; Virus; Inhalation; Wheezing; Influenza

Cloning of PpKAR2 and PpPDI1 genes promoters from yeast Pichia pastoris, an evaluation of their activity and efficacy for the heterologous genes expression

Background. Yeast Pichia pastoris is successfully used in biotechnology, with their help synthesized various compounds. Promoters are a key factor in the productivity of an expression system, since they determine the expression level of a heterologous gene.&#x0D; The aim of our work was to study the promoter regions of the PpKAR2 and PpPDI1 genes and to evaluate their use for effective expression of heterologous genes.&#x0D; Materials and Methods. To evaluate the activity of promoters, we used a reporter system based on the structural gene of acid phosphatase of yeast Saccharomyces cerevisiae PHO5. To determine the effect of overproduction of native and heterologous protein on the activity of the promoters under study, we used the producer strains of P. pastoris protein disulfide isomerase and maize delta-zein. To evaluate the effectiveness of the use of the promoters under study for the expression of heterologous genes, we have expressed under their control a gene encoding human interferon-alpha16.&#x0D; Results. The promoters of the yeast genes PpKAR2 and PpPDI1 were cloned. Their activity was compared with the promoter of the PpAOX1 gene in the native strains, as well as in strains with overproduction of native and heterologous proteins. Under the control of these promoters, the gene encoding human interferon-alpha 16 is expressed.&#x0D; Conclusion. The promoters studied were weaker than the promoter of the AOX1 gene, but increase their activity in response to the production of heterologous proteins and can be used to express heterologous genes.

Natural deep eutectic solvents as a storage medium for human interferon-α2: a green and improved strategy for room-temperature biologics

The inherent vulnerability of protein biologics to high temperature is the origin of their limited use in clinical settings where proper storage and handling systems are not available. Herein, we demonstrate the first application of natural deep eutectic solvents (NADES) as a temperature-stable storage medium for a real therapeutic protein, human interferon-alpha 2 (IFN-α2). A formulation of IFN-α2 based on a NADES composed of choline chloride and fructose at a 1:1 molar ratio (CCl:Fru) had no influence on the activity of IFN-α2. The NADES provided an appropriate environment for effective protection of IFN-α2 from thermal denaturation at mid (37°C) and high (70°C) temperatures during long-term (90 days) and short-term (2h) storage. Protein stability in the presence of CCl:Fru was demonstrated by orthogonal or complementary methods, including in vitro activity assay, circular dichroism spectroscopy and extrinsic fluorescence spectroscopy. In view of their biocompatibility, customizable properties, and the simplicity of synthesis and use, NADESs are suggested as a green and improved short-term and long-term storage medium that could facilitate the development of room-temperature biologics.