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1
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Uversky VN. How to drug a cloud? Targeting intrinsically disordered proteins. Pharmacol Rev 2024; 77:PHARMREV-AR-2023-001113. [PMID: 39433443 DOI: 10.1124/pharmrev.124.001113] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2024] [Revised: 10/03/2024] [Accepted: 10/15/2024] [Indexed: 10/23/2024] Open
Abstract
Biologically active proteins/regions without stable structure (i.e., intrinsically disordered proteins and regions (IDPs and IDRs)) are commonly found in all proteomes. They have a unique functional repertoire that complements the functionalities of ordered proteins and domains. IDPs/IDRs are multifunctional promiscuous binders capable of folding at interaction with specific binding partners on a template- or context-dependent manner, many of which undergo liquid-liquid phase separation, leading to the formation of membrane-less organelles and biomolecular condensates. Many of them are frequently related to the pathogenesis of various human diseases. All this defines IDPs/IDRs as attractive targets for the development of novel drugs. However, their lack of unique structures, multifunctionality, binding promiscuity, and involvement in unusual modes of action preclude direct use of traditional structure-based drug design approaches for targeting IDPs/IDRs, and make disorder-based drug discovery for these "protein clouds" challenging. Despite all these complexities there is continuing progress in the design of small molecules affecting IDPs/IDRs. This article describes the major structural features of IDPs/IDRs and the peculiarities of the disorder-based functionality. It also discusses the roles of IDPs/IDRs in various pathologies, and shows why the approaches elaborated for finding drugs targeting ordered proteins cannot be directly used for the intrinsic disorder-based drug design, and introduces some novel methodologies suitable for these purposes. Finally, it emphasizes that regardless of their multifunctionality, binding promiscuity, lack of unique structures, and highly dynamic nature, "protein clouds" are principally druggable. Significance Statement Intrinsically disordered proteins and regions are highly abundant in nature, have multiple important biological functions, are commonly involved in the pathogenesis of a multitude of human diseases, and are therefore considered as very attractive drug targets. Although dealing with these unstructured multifunctional protein/regions is a challenging task, multiple innovative approaches have been designed to target them by small molecules.
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2
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Clifton BE, Kozome D, Laurino P. Efficient Exploration of Sequence Space by Sequence-Guided Protein Engineering and Design. Biochemistry 2023; 62:210-220. [PMID: 35245020 DOI: 10.1021/acs.biochem.1c00757] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The rapid growth of sequence databases over the past two decades means that protein engineers faced with optimizing a protein for any given task will often have immediate access to a vast number of related protein sequences. These sequences encode information about the evolutionary history of the protein and the underlying sequence requirements to produce folded, stable, and functional protein variants. Methods that can take advantage of this information are an increasingly important part of the protein engineering tool kit. In this Perspective, we discuss the utility of sequence data in protein engineering and design, focusing on recent advances in three main areas: the use of ancestral sequence reconstruction as an engineering tool to generate thermostable and multifunctional proteins, the use of sequence data to guide engineering of multipoint mutants by structure-based computational protein design, and the use of unlabeled sequence data for unsupervised and semisupervised machine learning, allowing the generation of diverse and functional protein sequences in unexplored regions of sequence space. Altogether, these methods enable the rapid exploration of sequence space within regions enriched with functional proteins and therefore have great potential for accelerating the engineering of stable, functional, and diverse proteins for industrial and biomedical applications.
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Affiliation(s)
- Ben E Clifton
- Protein Engineering and Evolution Unit, Okinawa Institute of Science and Technology, 1919-1 Tancha, Onna, Okinawa 904-0495, Japan
| | - Dan Kozome
- Protein Engineering and Evolution Unit, Okinawa Institute of Science and Technology, 1919-1 Tancha, Onna, Okinawa 904-0495, Japan
| | - Paola Laurino
- Protein Engineering and Evolution Unit, Okinawa Institute of Science and Technology, 1919-1 Tancha, Onna, Okinawa 904-0495, Japan
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3
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Strategies for the Production of Soluble Interferon-Alpha Consensus and Potential Application in Arboviruses and SARS-CoV-2. Life (Basel) 2021; 11:life11060460. [PMID: 34063766 PMCID: PMC8223780 DOI: 10.3390/life11060460] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 05/08/2021] [Accepted: 05/14/2021] [Indexed: 12/18/2022] Open
Abstract
Biopharmaceutical production is currently a multibillion-dollar industry with high growth perspectives. The research and development of biologically sourced pharmaceuticals are extremely important and a reality in our current healthcare system. Interferon alpha consensus (cIFN) is a non-natural synthetic antiviral molecule that comprises all the most prevalent amino acids of IFN-α into one consensus protein sequence. For clinical use, cIFN is produced in E. coli in the form of inclusion bodies. Here, we describe the use of two solubility tags (Fh8 and DsbC) to improve soluble cIFN production. Furthermore, we analyzed cIFN production in different culture media and temperatures in order to improve biopharmaceutical production. Our results demonstrate that Fh8-cIFN yield was improved when bacteria were cultivated in autoinduction culture medium at 30 °C. After hydrolysis, the recovery of soluble untagged cIFN was 58% from purified Fh8-cIFN molecule, fourfold higher when compared to cIFN recovered from the DsbC-cIFN, which achieved 14% recovery. The biological activity of cIFN was tested on in vitro model of antiviral effect against Zika, Mayaro, Chikungunya and SARS-CoV-2 virus infection in susceptible VERO cells. We show, for the first time, that cIFN has a potent activity against these viruses, being very low amounts of the molecule sufficient to inhibit virus multiplication. Thus, this molecule could be used in a clinical approach to treat Arboviruses and SARS-CoV-2.
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4
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Ikegami K, de March CA, Nagai MH, Ghosh S, Do M, Sharma R, Bruguera ES, Lu YE, Fukutani Y, Vaidehi N, Yohda M, Matsunami H. Structural instability and divergence from conserved residues underlie intracellular retention of mammalian odorant receptors. Proc Natl Acad Sci U S A 2020; 117:2957-2967. [PMID: 31974307 PMCID: PMC7022149 DOI: 10.1073/pnas.1915520117] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Mammalian odorant receptors are a diverse and rapidly evolving set of G protein-coupled receptors expressed in olfactory cilia membranes. Most odorant receptors show little to no cell surface expression in nonolfactory cells due to endoplasmic reticulum retention, which has slowed down biochemical studies. Here we provide evidence that structural instability and divergence from conserved residues of individual odorant receptors underlie intracellular retention using a combination of large-scale screening of odorant receptors cell surface expression in heterologous cells, point mutations, structural modeling, and machine learning techniques. We demonstrate the importance of conserved residues by synthesizing consensus odorant receptors that show high levels of cell surface expression similar to conventional G protein-coupled receptors. Furthermore, we associate in silico structural instability with poor cell surface expression using molecular dynamics simulations. We propose an enhanced evolutionary capacitance of olfactory sensory neurons that enable the functional expression of odorant receptors with cryptic mutations.
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Affiliation(s)
- Kentaro Ikegami
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
- Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan
| | - Claire A de March
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
| | - Maira H Nagai
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
- Department of Biochemistry, Universidade de Sao Paulo, Sao Paulo, 05508-000, Brazil
| | - Soumadwip Ghosh
- Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, Duarte, CA 91010
| | - Matthew Do
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
| | - Ruchira Sharma
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
| | - Elise S Bruguera
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
| | - Yueyang Eric Lu
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
| | - Yosuke Fukutani
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710
- Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan
| | - Nagarajan Vaidehi
- Department of Computational and Quantitative Medicine, Beckman Research Institute of the City of Hope, Duarte, CA 91010
| | - Masafumi Yohda
- Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan
| | - Hiroaki Matsunami
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710;
- Department of Neurobiology, Duke Institute for Brain Sciences, Duke University, Durham, NC 27710
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5
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Chandler PG, Broendum SS, Riley BT, Spence MA, Jackson CJ, McGowan S, Buckle AM. Strategies for Increasing Protein Stability. Methods Mol Biol 2020; 2073:163-181. [PMID: 31612442 DOI: 10.1007/978-1-4939-9869-2_10] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
The stability of wild-type proteins is often a hurdle to their practical use in research, industry, and medicine. The route to engineering stability of a protein of interest lies largely with the available data. Where high-resolution structural data is available, rational design, based on fundamental principles of protein chemistry, can improve protein stability. Recent advances in computational biology and the use of nonnatural amino acids have also provided novel rational methods for improving protein stability. Likewise, the explosion of sequence and structural data available in public databases, in combination with improvements in freely available computational tools, has produced accessible phylogenetic approaches. Trawling modern sequence databases can identify the thermostable homologs of a target protein, and evolutionary data can be quickly generated using available phylogenetic tools. Grafting features from those thermostable homologs or ancestors provides stability improvement through a semi-rational approach. Further, molecular techniques such as directed evolution have shown great promise in delivering designer proteins. These strategies are well documented and newly accessible to the molecular biologist, allowing for rapid enhancements of protein stability.
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Affiliation(s)
- Peter G Chandler
- Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
| | - Sebastian S Broendum
- Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
| | - Blake T Riley
- Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
| | - Matthew A Spence
- Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
| | - Colin J Jackson
- Research School of Chemistry, Australian National University, Canberra, ACT, Australia
| | - Sheena McGowan
- Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
| | - Ashley M Buckle
- Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
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6
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Naseem MU, Ahmed N, Khan MA, Tahir S, Zafar AU. Production of potent long-lasting consensus interferon using albumin fusion technology in Pichia pastoris expression system. Protein Expr Purif 2019; 166:105509. [PMID: 31604114 DOI: 10.1016/j.pep.2019.105509] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Revised: 09/14/2019] [Accepted: 10/06/2019] [Indexed: 11/15/2022]
Abstract
Consensus interferon (cIFN) is a wholly synthetic therapeutic protein which is used to treat hepatitis C/B and certain types of malignancies. It has short serum half-life, therefore, to maintain its therapeutic level in the human body it requires thrice-weekly administration. Various strategies like PEGylation and micro-encapsulation have been developed during the last few years to enhance the pharmacokinetics of small therapeutic peptides. This study executed the human albumin-fusion technology, a simple and flexible approach to extend the serum circulating half-life of cIFN, because human serum albumin (HSA) has long circulating half-life (19 days) and very minute immunological activities. We integrated the codon-optimized HSA-cIFN fusion gene into Pichia pastoris genome by homologous recombination. The selection of hyper-resistant P. pastoris clone against Zeocin™ achieved a high-level secretory expression (250 mg/L) of fusion protein. HSA-cIFN fusion protein was purified using one-step purification by affinity chromatography with 34% recovery. The SDS-PAGE and SEC-HPLC analysis confirmed the final purified product has molecular weight of 87 kDa with 98% purity. Western blot analysis using anti-IFN antibodies further verified the purified HSA-cIFN fusion protein. The specific biological activity was 2.1 × 106 IU/mg as assessed by cytopathic inhibition assay, and half-life of fusion protein was estimated by in vitro thermal and proteolytic stability studies. This work concludes that by using albumin fusion technology, codon optimization and one-step purification a high yield of 86 mg/L of biologically active protein with improved serum half-life was obtained.
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Affiliation(s)
- Muhammad Umair Naseem
- National Centre of Excellence in Molecular Biology (NCEMB), University of the Punjab, 87 West Canal Bank Road, Thokar Niazbaig, Lahore 53700, Pakistan; Department of Biophysics and Cell Biology, Doctoral School of Molecular Medicine, University of Debrecen, Egyetem ter 1. Debrecen 4032, Hungary.
| | - Nadeem Ahmed
- National Centre of Excellence in Molecular Biology (NCEMB), University of the Punjab, 87 West Canal Bank Road, Thokar Niazbaig, Lahore 53700, Pakistan
| | - Mohsin Ahmad Khan
- National Centre of Excellence in Molecular Biology (NCEMB), University of the Punjab, 87 West Canal Bank Road, Thokar Niazbaig, Lahore 53700, Pakistan
| | - Saad Tahir
- National Centre of Excellence in Molecular Biology (NCEMB), University of the Punjab, 87 West Canal Bank Road, Thokar Niazbaig, Lahore 53700, Pakistan
| | - Ahmad Usman Zafar
- National Centre of Excellence in Molecular Biology (NCEMB), University of the Punjab, 87 West Canal Bank Road, Thokar Niazbaig, Lahore 53700, Pakistan
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7
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Gugliotta A, Ceaglio N, Kratje R, Oggero M. Effect of ANITVNITV peptide fusion on the bioactivity and pharmacokinetics of human IFN-α2b and a hyper-N-glycosylated variant. J Biotechnol 2019; 303:46-52. [PMID: 31336133 DOI: 10.1016/j.jbiotec.2019.07.006] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2019] [Revised: 07/03/2019] [Accepted: 07/19/2019] [Indexed: 11/17/2022]
Abstract
Different strategies have been developed and successfully applied to biotherapeutics in order to improve their in vivo efficacy. The genetic fusion to natural or synthetic glycosylated peptides constitutes a promising strategy since it conserves the protein sequence and results in the improvement of the pharmacokinetic properties. The ANITVNITV peptide described by Perlmann and coworkers presents 9 amino acids and 2 potential N-glycosylation sites. Its fusion to FSH resulted in the increase of the molecular mass and negative charge of the protein. Consequently, the pharmacokinetics was considerably improved. The aim of the present study was to compare the influence of ANITVNITV peptide fusion on the physicochemical, biological and pharmacokinetic properties of native hIFN-α2b (IFNwt), which contains a single O-glycosylation site, and a hyperglycosylated variant (IFN4N), that bears, in addition, 4 N-linked glycans. The resulting molecules, IFNwtNter and IFN4NNter, evidenced a higher molecular mass and negative charge compared to IFNwt and IFN4N, respectively. Therefore, the pharmacokinetic properties of the new molecules were significantly improved. The molecules obtained by the synthetic peptide fusion strategy evidenced a decrease in their in vitro antiviral specific biological activities (SBA). However, in vitro antiproliferative SBA was differentially modified for IFNwtNter and IFN4NNter in comparison with the parental molecules. For IFNwtNter, a reduction in the antiproliferative SBA was also observed. Remarkably, the addition of the ANITVNITV peptide to the N-terminus of IFN4N had a positive impact on its growth-inhibitory activity. This feature together with its improved pharmacokinetics encourages the development of IFN4NNter as an IFN-α based biobetter.
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Affiliation(s)
- Agustina Gugliotta
- UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA) Santa Fe, Argentina
| | - Natalia Ceaglio
- UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA) Santa Fe, Argentina
| | - Ricardo Kratje
- UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA) Santa Fe, Argentina
| | - Marcos Oggero
- UNL, CONICET, FBCB (School of Biochemistry and Biological Sciences), CBL (Biotechnological Center of Litoral), Ciudad Universitaria, Ruta Nacional 168 - Km 472.4 - C.C. 242 - (S3000ZAA) Santa Fe, Argentina.
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8
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Abstract
Outbreaks of severe virus infections with the potential to cause global pandemics are increasing. In many instances these outbreaks have been newly emerging (SARS coronavirus), re-emerging (Ebola virus, Zika virus) or zoonotic (avian influenza H5N1) virus infections. In the absence of a targeted vaccine or a pathogen-specific antiviral, broad-spectrum antivirals would function to limit virus spread. Given the direct antiviral effects of type I interferons (IFNs) in inhibiting the replication of both DNA and RNA viruses at different stages of their replicative cycles, and the effects of type I IFNs on activating immune cell populations to clear virus infections, IFNs-α/β present as ideal candidate broad-spectrum antivirals.
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Affiliation(s)
- Ben X Wang
- Princess Margaret Cancer Center, Tumor Immunotherapy Program, University Health Network, Toronto, ON M5G 2M9, Canada
| | - Eleanor N Fish
- Toronto General Hospital Research Institute, University Health Network, 67 College Street, Toronto, ON M5G 2M1, Canada; Department of Immunology, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
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9
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Consensus sequence design as a general strategy to create hyperstable, biologically active proteins. Proc Natl Acad Sci U S A 2019; 116:11275-11284. [PMID: 31110018 DOI: 10.1073/pnas.1816707116] [Citation(s) in RCA: 111] [Impact Index Per Article: 18.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Consensus sequence design offers a promising strategy for designing proteins of high stability while retaining biological activity since it draws upon an evolutionary history in which residues important for both stability and function are likely to be conserved. Although there have been several reports of successful consensus design of individual targets, it is unclear from these anecdotal studies how often this approach succeeds and how often it fails. Here, we attempt to assess generality by designing consensus sequences for a set of six protein families with a range of chain lengths, structures, and activities. We characterize the resulting consensus proteins for stability, structure, and biological activities in an unbiased way. We find that all six consensus proteins adopt cooperatively folded structures in solution. Strikingly, four of six of these consensus proteins show increased thermodynamic stability over naturally occurring homologs. Each consensus protein tested for function maintained at least partial biological activity. Although peptide binding affinity by a consensus-designed SH3 is rather low, K m values for consensus enzymes are similar to values from extant homologs. Although consensus enzymes are slower than extant homologs at low temperature, they are faster than some thermophilic enzymes at high temperature. An analysis of sequence properties shows consensus proteins to be enriched in charged residues, and rarified in uncharged polar residues. Sequence differences between consensus and extant homologs are predominantly located at weakly conserved surface residues, highlighting the importance of these residues in the success of the consensus strategy.
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10
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Alqahtani SA, Sulkowski MS. The Role of Interferon for the Treatment of Chronic Hepatitis C Virus Infection. TOPICS IN MEDICINAL CHEMISTRY 2019:97-113. [DOI: 10.1007/7355_2018_59] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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11
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Design, biological activity and signaling pathway of bovine consensus omega interferon expressed in Pichia pastoris. Mol Immunol 2018; 106:46-52. [PMID: 30576951 DOI: 10.1016/j.molimm.2018.12.004] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2018] [Revised: 08/19/2018] [Accepted: 12/05/2018] [Indexed: 11/21/2022]
Abstract
The bovine IFN-ω (BoIFN-ω) multigene family is located on chromosome 8, which has 14 potential functional genes and 10 pseudogenes. After aligning 14 BoIFN-ω subtypes and assigning the most frequently occurring amino acids in each position, one artificial consensus BoIFN-ω (CoBoIFN-ω) gene was designed, optimized and synthesized. Then, CoBoIFN-ω was expressed in Pichia pastoris, which was demonstrated to have 3.94-fold and 14.3-fold higher antiviral activity against VSV on MDBK cells than that of BoIFN-ω24 and BoIFN-ω3, respectively. Besides this, CoBoIFN-ω was confirmed to have antiviral activity against VSV on BL, BT, PK-15 cells, and against BEV, BHV-1, BPIV3 on MDBK cells. Additionally, CoBoIFN-ω could bind with bovine type I IFN receptors, and then activate the promoters of NF-κB, ISRE and BoIFN-β, and induce the transcription of ISGs and expression of Mx1 and NF-κB p65, which suggested CoBoIFN-ω exerts antiviral activity via activation of the JAK-STAT signaling pathway. Overall, this research on CoBoIFN-ω not only extends and improves consensus IFN research, but also reveals that CoBoIFN-ω has the potential to be used in the therapy of bovine viral diseases.
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12
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Zhang X, Yang W, Wang X, Zhang X, Tian H, Deng H, Zhang L, Gao G. Identification of new type I interferon-stimulated genes and investigation of their involvement in IFN-β activation. Protein Cell 2018; 9:799-807. [PMID: 29427062 PMCID: PMC6107486 DOI: 10.1007/s13238-018-0511-1] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2017] [Accepted: 01/09/2018] [Indexed: 12/01/2022] Open
Abstract
Virus infection induces the production of type I interferons (IFNs). IFNs bind to their heterodimeric receptors to initiate downstream cascade of signaling, leading to the up-regulation of interferon-stimulated genes (ISGs). ISGs play very important roles in innate immunity through a variety of mechanisms. Although hundreds of ISGs have been identified, it is commonly recognized that more ISGs await to be discovered. The aim of this study was to identify new ISGs and to probe their roles in regulating virus-induced type I IFN production. We used consensus interferon (Con-IFN), an artificial alpha IFN that was shown to be more potent than naturally existing type I IFN, to treat three human immune cell lines, CEM, U937 and Daudi cells. Microarray analysis was employed to identify those genes whose expressions were up-regulated. Six hundred and seventeen genes were up-regulated more than 3-fold. Out of these 617 genes, 138 were not previously reported as ISGs and thus were further pursued. Validation of these 138 genes using quantitative reverse transcription PCR (qRT-PCR) confirmed 91 genes. We screened 89 genes for those involved in Sendai virus (SeV)-induced IFN-β promoter activation, and PIM1 was identified as one whose expression inhibited SeV-mediated IFN-β activation. We provide evidence indicating that PIM1 specifically inhibits RIG-I- and MDA5-mediated IFN-β signaling. Our results expand the ISG library and identify PIM1 as an ISG that participates in the regulation of virus-induced type I interferon production.
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Affiliation(s)
- Xiaolin Zhang
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Wei Yang
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Xinlu Wang
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Xuyuan Zhang
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Huabin Tian
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Hongyu Deng
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Liguo Zhang
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Guangxia Gao
- CAS Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
- University of Chinese Academy of Sciences, Beijing, 100049, China.
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13
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Miersch S, Kuruganti S, Walter MR, Sidhu SS. A panel of synthetic antibodies that selectively recognize and antagonize members of the interferon alpha family. Protein Eng Des Sel 2017; 30:697-704. [PMID: 28981904 PMCID: PMC5914384 DOI: 10.1093/protein/gzx048] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Revised: 07/25/2017] [Accepted: 08/09/2017] [Indexed: 11/12/2022] Open
Abstract
The 12 distinct subtypes that comprise the interferon alpha (IFNα) family of cytokines possess anti-viral, anti-proliferative and immunomodulatory activities. They are implicated in the etiology and progression of many diseases, and also used as therapeutic agents for viral and oncologic disorders. However, a deeper understanding of their role in disease is limited by a lack of tools to evaluate single subtypes at the protein level. Antibodies that selectively inhibit single IFNα subtypes could enable interrogation of each protein in biological samples and could be used for characterization and treatment of disease. Using phage-displayed synthetic antibody libraries, we have conducted selections against 12 human IFNα subtypes to explore our ability to obtain fine-specificity antibodies that recognize and antagonize the biological signals induced by a single IFNα subtype. For the first time, we have isolated antibodies that specifically recognize individual IFNα subtypes (IFNα2a/b, IFNα6, IFNα8b and IFNα16) with high affinity that antagonize signaling. Our results show that highly specific antibodies capable of distinguishing between closely related cytokines can be isolated from synthetic libraries and can be used to characterize cytokine abundance and function.
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Affiliation(s)
- S Miersch
- The Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada M5G 1L6
| | - S Kuruganti
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - M R Walter
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - S S Sidhu
- The Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada M5G 1L6
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14
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Abstract
A popular and successful strategy in semi-rational design of protein stability is the use of evolutionary information encapsulated in homologous protein sequences. Consensus design is based on the hypothesis that at a given position, the respective consensus amino acid contributes more than average to the stability of the protein than non-conserved amino acids. Here, we review the consensus design approach, its theoretical underpinnings, successes, limitations and challenges, as well as providing a detailed guide to its application in protein engineering.
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Affiliation(s)
- Benjamin T Porebski
- Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Faculty of Medicine, Monash University, Clayton, Victoria 3800, Australia Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK
| | - Ashley M Buckle
- Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Faculty of Medicine, Monash University, Clayton, Victoria 3800, Australia
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15
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Yu D, Zhao M, Dong L, Zhao L, Zou M, Sun H, Zhang M, Liu H, Zou Z. Design and evaluation of novel interferon lambda analogs with enhanced antiviral activity and improved drug attributes. Drug Des Devel Ther 2016; 10:163-82. [PMID: 26792983 PMCID: PMC4708225 DOI: 10.2147/dddt.s91455] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
Type III interferons (IFNs) (also called IFN-λ: IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4) are critical players in the defense against viral infection of mucosal epithelial cells, where the activity of type I IFNs is weak, and unlike type I IFNs that are associated with severe and diverse side effects, type III IFNs cause minimal side effects due to the highly restricted expression of their receptors, and thus appear to be promising agents for the treatment and prevention of respiratory and gastrointestinal viral infection. However, the antiviral potency of natural type III IFNs is weak compared to type I and, although IFN-λ3 possesses the highest bioactivity among the type III IFNs, IFN-λ1, instead of IFN-λ3, is being developed as a therapeutic drug due to the difficulty to express IFN-λ3 in the prokaryotic expression system. Here, to develop optimal IFN-λ molecules with improved drug attributes, we designed a series of IFN-λ analogs by replacing critical amino acids of IFN-λ1 with the IFN-λ3 counterparts, and vice versa. Four of the designed analogs were successfully expressed in Escherichia coli with high yield and were easily purified from inclusion bodies. Interestingly, all four analogs showed potent activity in inducing the expression of the antiviral genes MxA and OAS and two of them, analog-6 and -7, displayed an unexpected high potency that is higher than that of type I IFN (IFN-α2a) in activating the IFN-stimulated response element (ISRE)-luciferase reporter. Importantly, both analog-6 and -7 effectively inhibited replication of hepatitis C virus in Huh-7.5.1 cells, with an IC50 that is comparable to that of IFN-α2a; and consistent with the roles of IFN-λ in mucosal epithelia, both analogs potently inhibited replication of H3N2 influenza A virus in A549 cells. Together, these studies identified two IFN-λ analogs as candidates to be developed as novel antiviral biologics.
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Affiliation(s)
- Debin Yu
- National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, People’s Republic of China
| | - Mingzhi Zhao
- State Key Laboratory of Proteomics, National Engineering Research Center for Protein Drugs, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, People’s Republic of China
| | - Liwei Dong
- National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, People’s Republic of China
| | - Lu Zhao
- National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, People’s Republic of China
| | - Mingwei Zou
- Department of Psychology, College of Liberal Arts and Social Sciences, University of Houston, Houston, TX, USA
| | - Hetong Sun
- Prosit Sole Biotechnology, Co., Ltd., Beijing, People’s Republic of China
| | - Mengying Zhang
- Prosit Sole Biotechnology, Co., Ltd., Beijing, People’s Republic of China
| | - Hongyu Liu
- Prosit Sole Biotechnology, Co., Ltd., Beijing, People’s Republic of China
| | - Zhihua Zou
- National Engineering Laboratory for AIDS Vaccine, Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, People’s Republic of China
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16
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Sobel D, Ahvazi B, Pontzer C. The Role of Type I Interferon Subtypes and Interferon-Gamma in Type I Interferon Diabetes Inhibitory Activity in the NOD Mouse. J Interferon Cytokine Res 2015; 36:238-46. [PMID: 26716812 DOI: 10.1089/jir.2014.0232] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
As in bacterial infections and endotoxin shock, type I interferons (IFNs) also have complex and often opposing effects in various models of autoimmune disease. We have shown that type I IFN paradoxically inhibits autoimmune diabetes in the nonobese diabetic mouse (NOD) and biobreeding (BB) rat. We hypothesize that type I IFN activity differs by IFN subtype and interaction with IFN-gamma. We examined the structure-function relationship of the type I IFN molecule and the mechanism of its diabetes-sparing activity in the NOD mouse. While both recombinant human IFN-alpha A/D (bgl 11) (rHuIFN-alphaA/D) and ovine IFN-tauImod (ovIFN-tau) potently inhibited the development of diabetes (P < 0.01), neither recombinant human IFN-alpha B/D (rHuIFN-alphaB/D) nor recombinant human IFN-alpha consensus (CIFN) were efficacious. The activity of IFN subtypes correlate with their NH3-terminal amino acid sequences. All type I IFN save CIFN, which has no diabetes-sparing activity, inhibited the accessory cell function. IFN-tau administration decreased the expression of Fas and ICAM on total cells, class II MHC expression on B cells, and CD40L expression on T cells by 39%, 45%, 45%, and 60%, respectively. In addition, IFN-tau inhibited the development of diabetes in the NOD.IL4(null) but not the NOD.IFN-gamma(null) mice, suggesting a coordinated interaction between type I and type II IFNs to suppress diabetes development. Thus, the amino terminal portion of the type I IFN molecule influences its ability to inhibit the development of autoimmune diabetes in NOD mice. These data also support the contention that IFN-gamma may have a role in mediating the diabetes-sparing effect of high-dose type I IFNs by the inhibition of the IFN-gamma-inducible immune modulators, class II MHC, Fas, ICAM, and CD40L.
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Affiliation(s)
- Douglas Sobel
- 1 Department of Pediatrics, Georgetown University , Washington, District of Columbia
| | - Behrouz Ahvazi
- 1 Department of Pediatrics, Georgetown University , Washington, District of Columbia
| | - Carol Pontzer
- 2 Department of Molecular Biology, University of Maryland , College Park, Maryland
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17
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Wei L, Bello AM, Majchrzak-Kita B, Salum N, Lewis MM, Kotra LP, Fish EN. Small Molecule Agonists for the Type I Interferon Receptor: An In Silico Approach. J Interferon Cytokine Res 2015; 36:180-91. [PMID: 26700737 DOI: 10.1089/jir.2015.0123] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Type I interferons (IFNs) exhibit broad-spectrum antiviral activity, with potential utility against emerging acute virus infections that pose a threat to global health. Recombinant IFN-αs that have been approved for clinical use require cold storage and are administered through intramuscular or subcutaneous injection, features that are problematic for global distribution, storage, and administration. Cognizant that the biological potency of an IFN-α subtype is determined by its binding affinity to the type I IFN receptor, IFNAR, we identified a panel of small molecule nonpeptide compounds using an in silico screening strategy that incorporated specific structural features of amino acids in the receptor-binding domains of the most potent IFN-α, IFN alfacon-1. Hit compounds were selected based on ease of synthesis and formulation properties. In preliminary biological assays, we provide evidence that these compounds exhibit antiviral activity. This proof-of-concept study validates the strategy of in silico design and development for IFN mimetics.
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Affiliation(s)
- Lianhu Wei
- 1 Center for Molecular Design and Preformulations , Toronto, Canada .,2 Toronto General Research Institute, University Health Network , Toronto, Canada .,3 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Canada
| | - Angelica M Bello
- 1 Center for Molecular Design and Preformulations , Toronto, Canada .,2 Toronto General Research Institute, University Health Network , Toronto, Canada .,3 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Canada
| | - Beata Majchrzak-Kita
- 2 Toronto General Research Institute, University Health Network , Toronto, Canada
| | - Noruê Salum
- 1 Center for Molecular Design and Preformulations , Toronto, Canada .,4 Federal University of Paraná , Paraná, Brazil
| | - Melissa M Lewis
- 1 Center for Molecular Design and Preformulations , Toronto, Canada .,2 Toronto General Research Institute, University Health Network , Toronto, Canada
| | - Lakshmi P Kotra
- 1 Center for Molecular Design and Preformulations , Toronto, Canada .,2 Toronto General Research Institute, University Health Network , Toronto, Canada .,3 Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, Canada
| | - Eleanor N Fish
- 2 Toronto General Research Institute, University Health Network , Toronto, Canada .,5 Department of Immunology, Faculty of Medicine, University of Toronto , Toronto, Canada
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18
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El-Baky NA, Uversky VN, Redwan EM. Human consensus interferons: Bridging the natural and artificial cytokines with intrinsic disorder. Cytokine Growth Factor Rev 2015; 26:637-45. [DOI: 10.1016/j.cytogfr.2015.07.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2015] [Revised: 07/01/2015] [Accepted: 07/02/2015] [Indexed: 12/13/2022]
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19
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Bashir H, Ahmed N, Khan MA, Zafar AU, Tahir S, Khan MI, Khan F, Husnain T. Simple procedure applying lactose induction and one-step purification for high-yield production of rhCIFN. Biotechnol Appl Biochem 2015; 63:708-714. [PMID: 26256695 DOI: 10.1002/bab.1426] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2015] [Accepted: 07/26/2015] [Indexed: 11/11/2022]
Abstract
Recombinant consensus interferon (CIFN) is a therapeutic protein with molecular weight of 19.5 kDa having broad spectrum antiviral activity. Recombinant human CIFN (rhCIFN) has previously been expressed in Escherichia coli using isopropyl-β-d-thiogalactopyranoside (IPTG), a non-metabolizable and expensive compound, as inducer. For economical and commercial-scale recombinant protein production, it is greatly needed to increase the product yield in a limited time frame to reduce the processing cost. To reduce the cost of production of rhCIFN in E. coli, induction was accomplished by using lactose instead of IPTG. Lactose induction (14 g/L) in shake flask experiment resulted in higher yield as compared with 1 mM IPTG. Finally, with single-step purification on DEAE sepharose, 150 mg/L of >98% pure rhCIFN was achieved. In the present study, an attempt was made to develop a low cost process for producing quality product with high purity. Methods devised may be helpful for pilot-scale production of recombinant proteins at low cost.
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Affiliation(s)
- Hamid Bashir
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Nadeem Ahmed
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
| | - Mohsin Ahmad Khan
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Ahmad Usman Zafar
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Saad Tahir
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Muhammad Islam Khan
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Faidad Khan
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Tayyab Husnain
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
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20
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Interferon Beta and Interferon Alpha 2a Differentially Protect Head and Neck Cancer Cells from Vesicular Stomatitis Virus-Induced Oncolysis. J Virol 2015; 89:7944-54. [PMID: 25995245 DOI: 10.1128/jvi.00757-15] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2015] [Accepted: 05/15/2015] [Indexed: 12/21/2022] Open
Abstract
UNLABELLED Oncolytic viruses (OV) preferentially kill cancer cells due in part to defects in their antiviral responses upon exposure to type I interferons (IFNs). However, IFN responsiveness of some tumor cells confers resistance to OV treatment. The human type I IFNs include one IFN-β and multiple IFN-α subtypes that share the same receptor but are capable of differentially inducing biological responses. The role of individual IFN subtypes in promoting tumor cell resistance to OV is addressed here. Two human IFNs which have been produced for clinical use, IFN-α2a and IFN-β, were compared for activity in protecting human head and neck squamous cell carcinoma (HNSCC) lines from oncolysis by vesicular stomatitis virus (VSV). Susceptibility of HNSCC lines to killing by VSV varied. VSV infection induced increased production of IFN-β in resistant HNSCC cells. When added exogenously, IFN-β was significantly more effective at protecting HNSCC cells from VSV oncolysis than was IFN-α2a. In contrast, normal keratinocytes and endothelial cells were protected equivalently by both IFN subtypes. Differential responsiveness of tumor cells to IFN-α and -β was further supported by the finding that autocrine IFN-β but not IFN-α promoted survival of HNSCC cells during persistent VSV infection. Therefore, IFN-α and -β differentially affect VSV oncolysis, justifying the evaluation and comparison of IFN subtypes for use in combination with VSV therapy. Pairing VSV with IFN-α2a may enhance selectivity of oncolytic VSV therapy for HNSCC by inhibiting VSV replication in normal cells without a corresponding inhibition in cancer cells. IMPORTANCE There has been a great deal of progress in the development of oncolytic viruses. However, a major problem is that individual cancers vary in their sensitivity to oncolytic viruses. In many cases this is due to differences in their production and response to interferons (IFNs). The experiments described here compared the responses of head and neck squamous cell carcinoma cell lines to two IFN subtypes, IFN-α2a and IFN-β, in protection from oncolytic vesicular stomatitis virus. We found that IFN-α2a was significantly less protective for cancer cells than was IFN-β, whereas normal cells were equivalently protected by both IFNs. These results suggest that from a therapeutic standpoint, selectivity for cancer versus normal cells may be enhanced by pairing VSV with IFN-α2a.
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21
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El-Baky NA, Redwan EM. Therapeutic alpha-interferons protein: structure, production, and biosimilar. Prep Biochem Biotechnol 2015; 45:109-27. [PMID: 24785737 DOI: 10.1080/10826068.2014.907175] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
In 2007, the world solemnized the golden jubilee of the discovery of interferon (IFN). Interferon is a small protein messenger called a pluripotent cytokine, produced by several cells of the host in response to various biological as well as synthetic stimuli. There are three major classes of interferons in humans: IFN-alpha, IFN-beta, and IFN-gamma. As a treatment option, interferon-alpha (IFN-α) is the most effective one. IFN-α has proved to be effective as an antiviral therapy and tumor-fighting drug in the past two decades. Meanwhile, great progress has been achieved in establishing IFN-α as the first choice of antiviral therapy for chronic hepatitis C virus (HCV) patients. Recently, novel pegylated IFN-α2 products with extended in vivo half-lives and consensus interferon, an artificially engineered type I interferon, have been developed to substantially improve treatment regimes for HCV patients. Undesirable acute and chronic side effects in addition to immunogenicity of therapeutic IFN products remain constraints to conquer for further improvements in clinical applications of IFN. It is certainly expected that more research will be conducted in the future, not only to face these challenges but also to extend the range of IFN products and their clinical targets. The objective herein is to review the current therapeutic alpha-interferons production, formulation technologies, and prospective future for the original entity and its biogeneric version.
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Affiliation(s)
- Nawal Abd El-Baky
- a Biological Sciences Department, Faculty of Science , King Abdulaziz University , Jeddah , Saudi Arabia
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22
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EL-Baky NA, Linjawi MH, Redwan EM. Auto-induction expression of human consensus interferon-alpha in Escherichia coli. BMC Biotechnol 2015; 15:14. [PMID: 25886839 PMCID: PMC4372041 DOI: 10.1186/s12896-015-0128-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2014] [Accepted: 02/18/2015] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Isopropyl-β-D-1-thiolgalactopyranoside (IPTG)-inducible expression of recombinant proteins in E. coli is commonly used and effective. Nevertheless, unintended induction was encountered as a problem when using these bacterial expression systems, generating cultures that give reduced or variable protein yields. Auto-induction allows for production of much higher target protein yield and cell mass than conventional procedures using induction with IPTG without monitoring cell growth then adding IPTG at the appropriate cell density. This method involves special media recipes that promote growth to high density and automatically induce expression of target protein from T7 promoter. Consensus interferon is a synthetic artificially engineered interferon having an amino acid sequence that is a rough average of the sequences of all natural human alpha interferon subtypes and has greater potency than other interferons even the pegylated versions. The purpose of this study was high-level expression of human consensus interferon-alpha (cIFN-α) in E. coli using an auto-induction protocol. The cIFN-α gene was cloned into pET101/D-TOPO expression vector under the T7 promoter transcriptional regulation. Expression was optimized with respect to temperature and length of incubation in shake flask cultures. The antiviral potency and anticancer activity of cIFN-α were evaluated in comparison to IFN-α2a. RESULTS The expressed cIFN-α protein in auto-induction T7 system was found mostly in soluble fraction of the cell lysate (about 70% of yield in total cell lysate) after lowering incubation temperature to 25°C or 30°C. Protein expression was maximal after 24 h incubation at 25°C or 30°C. After purification via single-step chromatography using DEAE-Sepharose, the yield was 270 mg/L in shake flask E. coli cultures which is much higher than IPTG-inducible T7 expression system and other systems according to available data. The synthesized cIFN-α was biologically active as confirmed by its anticancer and antiviral effects and was significantly more potent than IFN-α2a. CONCLUSIONS The auto-induction process was reliable and convenient for production of cIFN-α protein in E. coli, and can be adapted for large-scale therapeutic protein production.
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Affiliation(s)
- Nawal Abd EL-Baky
- Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab, 21934, Alexandria, Egypt.
| | - Mustafa H Linjawi
- College of Applied Medical Sciences, King Abdulaziz University, P.O. Box 80203, Jeddah, 21589, Saudi Arabia.
| | - Elrashdy M Redwan
- Therapeutic and Protective Proteins Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, New Borg EL-Arab, 21934, Alexandria, Egypt. .,Biological Sciences Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah, 21589, Saudi Arabia.
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23
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Ahmed N, Bashir H, Zafar AU, Khan MA, Tahir S, Khan F, Khan MI, Akram M, Husnain T. Optimization of conditions for high-level expression and purification of human recombinant consensus interferon (rh-cIFN) and its characterization. Biotechnol Appl Biochem 2015; 62:699-708. [PMID: 25402716 DOI: 10.1002/bab.1320] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2014] [Accepted: 11/09/2014] [Indexed: 02/04/2023]
Abstract
Recombinant human consensus interferon (rh-cIFN) is an artificially engineered interferon (IFN) developed by recombining and reordering the protein sequences that exist in standard IFN. This recombination resulted into a drug that has the potential to work better than natural, standard IFN. In this study, we described optimized conditions for high-level expression and recovery of biologically active consensus IFN from inclusion bodies (IBs). A synthetic gene coding 166 amino acids of consensus IFN was cloned under the T7 promoter. Escherichia coli strain BL21DE3Plys was used to transform expression construct. For high-level expression, shake-flask fermentation conditions were standardized. For isolation of IBs, the sonication method was optimized. A variety of chaotropic agents including guanidine hydrochloride, urea, SDS, and detergents were studied for solubilization of IBs. For renaturation of solubilized denatured protein by the dilution process, parameters of dilution factor, temperature, and l-arginine were optimized. A one-step chromatography method was developed for high-yield purification of consensus IFN. rh-cIFN was characterized by SDS-PAGE, Western blot, and high-performance liquid chromatography. Purified protein has a molecular weight of 19.5 kDa and specific activity was 2.0 × 10(8) as determined by the cytopathic inhibition assay. This study concludes that by using optimized conditions, we obtained a yield of 100 mg/L of biologically active rh-cIFN, which is highest ever reported according to available data.
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Affiliation(s)
- Nadeem Ahmed
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Hamid Bashir
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Ahmad Usman Zafar
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Mohsin Ahmad Khan
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Saad Tahir
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Faidad Khan
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Muhammad Islam Khan
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
| | - Muhammad Akram
- Centre for Applied Molecular Biology, Ministry of Science and Technology, Lahore, Pakistan
| | - Tayyab Husnain
- National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan
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Lipiäinen T, Peltoniemi M, Sarkhel S, Yrjönen T, Vuorela H, Urtti A, Juppo A. Formulation and stability of cytokine therapeutics. J Pharm Sci 2014; 104:307-26. [PMID: 25492409 DOI: 10.1002/jps.24243] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2014] [Revised: 09/29/2014] [Accepted: 10/13/2014] [Indexed: 12/12/2022]
Abstract
Cytokines are messenger proteins that regulate the proliferation and differentiation of cells and control immune responses. Interferons, interleukins, and growth factors have applications in cancer, autoimmune, and viral disease treatment. The cytokines are susceptible to chemical and physical instability. This article reviews the structure and stability issues of clinically used cytokines, as well as formulation strategies for improved stability. Some general aspects for identifying most probable stability concerns, selecting excipients, and developing stable cytokine formulations are presented. The vast group of cytokines offers possibilities for new biopharmaceuticals. The formulation approaches of the current cytokine products could facilitate development of new biopharmaceuticals.
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Affiliation(s)
- Tiina Lipiäinen
- University of Helsinki, Faculty of Pharmacy, Helsinki, Finland
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25
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Abstract
Interferon was discovered by Alick Isaacs and Jean Lindenmann in 1957. It was originally thought that interferon could be used as a general anti-viral agent and in anti-cancer therapy. There are many different types of interferons, now known as interferons “alpha,” “beta,” “gamma” and “lambda,” with different cellular receptors and modes of action, and there are possibly 24 different types of alpha interferon. Independently and simultaneously, a group of Japanese scientists found an “interfering factor,” which upon subsequent analysis turned out to be interferon, probably of the alpha type. The interferon alpha gene was the first mammalian gene to be cloned in a bacterial system and became the prototype for gene cloning technology. Until the cloning of the interferons in Escherichia coli, and expression of the interferon genes in mammalian cells in culture, it was impossible to obtain enough material for clinical use. Interferon today is predominantly used in the treatment of hepatitis B and C, leukemia and Kaposi’s sarcoma. As an anti-viral agent, interferon has not lived up to its initial promise, since in vitro most viruses block the activity of interferon and clinical trials have given inconclusive results with severe side effects. Interferon induces hundreds of genes in vivo and in vitro, each interferon producing overlapping and distinct gene profiles. The mechanism of both interferon induction and anti-viral response is complicated and involves the interaction of many regulatory molecules. Interferon is now known to be a component of the large family of cytokines or interleukins.
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26
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Targeting the Interferon Response for Antiviral Therapy. Antiviral Res 2014. [DOI: 10.1128/9781555815493.ch18] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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Peciak K, Tommasi R, Choi JW, Brocchini S, Laurine E. Expression of soluble and active interferon consensus in SUMO fusion expression system in E. coli. Protein Expr Purif 2014; 99:18-26. [PMID: 24680730 DOI: 10.1016/j.pep.2014.03.009] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2013] [Revised: 03/15/2014] [Accepted: 03/17/2014] [Indexed: 12/24/2022]
Abstract
Protein production can be improved if methods for soluble protein expression are developed. Interferon consensus (IFN-con) is used to treat hepatitis C. IFN-con has superior activity compared to other clinically used interferon α subtypes. However IFN-con is a challenging protein to produce in a soluble form using an Escherichia coli expression system. Here we describe the expression of soluble and active recombinant IFN-con in E. coli. The IFN-con gene sequence was optimised for expression in E. coli, which was then cloned into the Champion™ pET SUMO expression vector downstream of the SUMO fusion protein and under strong T7lac promoter. The SUMO-IFN-con fusion protein was efficiently expressed using the SHuffle™ E. coli strain and existed in soluble form as 86-88% of the total IFN-con. After removal of the SUMO fusion partner, approximately 50mg of recombinant IFN-con of at least 98% purity (by RP-HPLC) was obtained from a 1L fermentation culture. Using an A549/EMCV antiviral assay, the specific activity of the recombinant IFN-con was determined to be 960×10(6) IU/mg as calculated to NIBSC standard for IFN-con (3×10(5)pfu/mL virus titre). Comparison of the antiviral activity of the produced IFN-con to IFN α-2a showed that IFN-con displays 2.8 times greater activity, which is in good agreement with what has been reported in the literature for pure protein. IFN-con expression in a soluble form from E. coli allowed us to use a simple, two-step purification process to yield highly pure and active IFN-con which is more efficient than obtaining IFN-con from inclusion bodies.
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Affiliation(s)
- Karolina Peciak
- UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK; PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK
| | - Rita Tommasi
- PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK
| | - Ji-won Choi
- PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK
| | - Steve Brocchini
- UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London WC1N 1AX, UK; PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK
| | - Emmanuelle Laurine
- PolyTherics Ltd., The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, UK.
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Alao H, Jake Liang T. Alternative interferons and immunomodulators in the treatment of hepatitis C. Liver Int 2014; 34 Suppl 1:133-8. [PMID: 24373090 DOI: 10.1111/liv.12402] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Interferon-α (IFN-α) has been the mainstay of therapy for hepatitis C and is currently being combined with other drugs to improve the response rate. Newer therapeutic regimens are being developed to spare the use of IFN because of the important side effects associated with IFN-based therapy. However, there may still be a need for the use of IFN in certain populations. In addition, agents that mimic the actions of IFN but with fewer side effects may still be of major value. This review focuses on the development of alternative and new forms of IFNs and other immunomodulatory agents that may supplant IFN-α in combination therapy for hepatitis C.
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Affiliation(s)
- Hawwa Alao
- Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA
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29
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Bello AM, Wei L, Majchrzak-Kita B, Salum N, Purohit MK, Fish EN, Kotra LP. Small molecule mimetics of an interferon-α receptor interacting domain. Bioorg Med Chem 2014; 22:978-85. [DOI: 10.1016/j.bmc.2013.12.049] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2013] [Revised: 12/12/2013] [Accepted: 12/21/2013] [Indexed: 10/25/2022]
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Abbas Z, Tayyab GN, Qureshi M, Memon MS, Subhan A, Shakir T, Jafri W, Hamid S. Consensus interferon plus ribavirin for hepatitis C genotype 3 patients previously treated with pegylated interferon plus ribavirin. HEPATITIS MONTHLY 2013; 13:e14146. [PMID: 24358041 PMCID: PMC3867024 DOI: 10.5812/hepatmon.14146] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/11/2013] [Revised: 10/02/2013] [Accepted: 11/17/2013] [Indexed: 12/11/2022]
Abstract
BACKGROUND Not enough data are available about the effectiveness of consensus interferon (CIFN) among HCV genotype 3 patients who failed to respond to pegylated interferon and ribavirin. OBJECTIVES We aimed to assess the efficacy and safety of CIFN and ribavirin in non-responders and relapsers to pegylated interferon with ribavirin therapy. PATIENTS AND METHODS This open-label investigator-initiated study included 44 patients who received CIFN 15 µg /day plus ribavirin 800-1200 mg daily. In patients with an early virological response (EVR), the dose of CIFN was reduced to 15 µg thrice a week for further 36 weeks. Patients with delayed virological response continued to receive daily CIFN plus ribavirin to complete 48 weeks. The patients were considered "non-responders" if there were less than 2 log reduction in HCV RNA at 12 weeks and detectable HCV RNA at 24 weeks. RESULTS Twenty-four patients (55%) were non-responders and 20 patients were relapsers to the previous treatment with pegylated interferon plus ribavirin (mean age 43.6 ± 9.4 years, males 25 (57%)). Nine patients were clinically cirrhotic (Child A). End of treatment virological response was achieved in 19 (43.1%) patients and sustained virological response (SVR) occurred in 12 (27.3%). Out of these 12 patients, eight were non-responders and four were relapsers to the previous treatment. Advanced fibrosis or clinical cirrhosis was associated with low SVR. Adverse events were fever, myalgia, anorexia, depression, and weight loss. Two patients received granulocyte colony stimulating factor for transient neutropenia. Seven patients were given erythropoietin to improve hemoglobin, and six were treated for mild depression. Two patients developed portosystemic encephalopathy. CONCLUSIONS More than one-quarter of treatment-experienced patients with HCV genotype 3 achieved SVR after re-treatment with consensus interferon plus ribavirin.
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Affiliation(s)
- Zaigham Abbas
- Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan
- Corresponding Author: Zaigham Abbas, Department of Medicine, The Aga Khan University Hospital, Stadium Road, Karachi, Pakistan. Tel/Fax: +92-214930051, E-mail: ,
| | | | - Mustafa Qureshi
- Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan
| | | | - Amna Subhan
- Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan
| | - Tanzila Shakir
- Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan
| | - Wasim Jafri
- Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan
| | - Saeed Hamid
- Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan
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Gibbert K, Schlaak JF, Yang D, Dittmer U. IFN-α subtypes: distinct biological activities in anti-viral therapy. Br J Pharmacol 2013; 168:1048-58. [PMID: 23072338 PMCID: PMC3594665 DOI: 10.1111/bph.12010] [Citation(s) in RCA: 125] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2012] [Revised: 08/15/2012] [Accepted: 09/07/2012] [Indexed: 12/12/2022] Open
Abstract
During most viral infections, the immediate host response is characterized by an induction of type I IFN. These cytokines have various biological activities, including anti-viral, anti-proliferative and immunomodulatory effects. After induction, they bind to their IFN-α/β receptor, which leads to downstream signalling resulting in the expression of numerous different IFN-stimulated genes. These genes encode anti-viral proteins that directly inhibit viral replication as well as modulate immune function. Thus, the induction of type I IFN is a very powerful tool for the host to fight virus infections. Many viruses evade this response by various strategies like the direct suppression of IFN induction or inhibition of the IFN signalling pathway. Therefore, the therapeutic application of exogenous type I IFN or molecules that induce strong IFN responses should be of great potential for future immunotherapies against viral infections. Type I IFN is currently used as a treatment in chronic hepatitis B and C virus infection, but as yet is not widely utilized for other viral infections. One reason for this restricted clinical use is that type I IFN belongs to a multigene family that includes 13 different IFN-α subtypes and IFN-β, whose individual anti-viral and immunomodulatory properties have so far not been investigated in detail to improve IFN therapy against viral infections in humans. In this review, we summarize the recent achievements in defining the distinct biological functions of type I IFN subtypes in cell culture and in animal models of viral infection as well as their clinical usage in chronic hepatitis virus infections.
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Affiliation(s)
- K Gibbert
- Department of Virology, University Hospital Essen, Essen, Germany.
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Mohammed Y, El-Baky NA, Redwan EM. Expression, purification, and characterization of recombinant human consensus interferon-alpha in Escherichia coli under λP(L) promoter. Prep Biochem Biotechnol 2012; 42:426-47. [PMID: 22897766 DOI: 10.1080/10826068.2011.637600] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2022]
Abstract
Recombinant human consensus interferon-alpha (cIFN-α) was obtained by synthesizing a codon-optimized gene composed of the consensus nucleotides at each position in the human alpha interferon family and expressing it in Escherichia coli. The full cIFN-α gene was synthesized in two steps of assembly and amplification by polymerase chain reaction (PCR) using long (45-50 nucleotides) overlapped primers. The two-step PCR resulted in a DNA band of 504 base pairs (bp) corresponding to the calculated size of the cIFN-α gene. The synthetic gene was cloned into temperature-regulated Power3 expression vector. The ligated Power3-cIFN-α (Power3-cIFNα) plasmid carried the cIFN-α gene under transcriptional regulation of the heat-inducible λP(L) promoter. This expression system was optimized with respect to heat-shock temperature and time of induction in shake flask cultures. The produced cIFN-α protein was characterized by polyacrylamide gel electrophoresis and immunoassays. The majority of the expressed cIFN-α protein of about 19 kD in size accumulated in the form of inclusion bodies. After refolding and purification utilizing single-step ion-exchange chromatography on DEAE-Sepharose, the yield was 70 mg/L. cIFN-α anti-cancer activity was assayed and compared with the commercially available IFN-α 2a.
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Affiliation(s)
- Y Mohammed
- Antibody Laboratory, Protein Research Department, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technology Applications, Alexandria, Egypt
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Wang BX, Fish EN. The yin and yang of viruses and interferons. Trends Immunol 2012; 33:190-7. [PMID: 22321608 PMCID: PMC7106503 DOI: 10.1016/j.it.2012.01.004] [Citation(s) in RCA: 79] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2011] [Revised: 12/21/2011] [Accepted: 01/04/2012] [Indexed: 12/15/2022]
Abstract
Interferons (IFNs)-α/β are critical effectors of the innate immune response to virus infections. Through activation of the IFN-α/β receptor (IFNAR), they induce expression of IFN-stimulated genes (ISGs) that encode antiviral proteins capable of suppressing viral replication and promoting viral clearance. Many highly pathogenic viruses have evolved mechanisms to evade an IFN response and the balance between the robustness of the host immune response and viral antagonistic mechanisms determines whether or not the virus is cleared. Here, we discuss IFNs as broad-spectrum antivirals for treatment of acute virus infections. In particular, they are useful for treatment of re-emerging virus infections, where direct-acting antivirals (DAAs) have limited utility due to DAA-resistant mutations, and for newly emerging virus strains in which the time to vaccine availability precludes vaccination at the onset of an outbreak.
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Affiliation(s)
- Ben X Wang
- University Health Network, Toronto, Ontario, Canada
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Koehler JW, Dupuy LC, Garrison AR, Beitzel BF, Richards MJ, Ripoll DR, Wallqvist A, Teh SY, Vaewhongs AA, Vojdani FS, Padgett HS, Schmaljohn CS. Novel plant-derived recombinant human interferons with broad spectrum antiviral activity. Antiviral Res 2011; 92:461-9. [PMID: 22020161 PMCID: PMC9628711 DOI: 10.1016/j.antiviral.2011.10.008] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2011] [Revised: 09/27/2011] [Accepted: 10/07/2011] [Indexed: 02/03/2023]
Abstract
Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.
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Key Words
- ifns, interferons
- ifnar, interferon-α receptor
- veev, venezuelan equine encephalitis virus
- rvfv, rift valley fever virus
- ebov, ebola virus
- mpxv, monkeypox virus
- grammr™, genetic reassortment by mismatch resolution
- bsa, bovine serum albumin
- gfp, green fluorescent protein
- pspp, protein structure prediction pipeline
- pdb, protein data bank
- type i interferons
- ebola virus
- rift valley fever virus
- venezuelan equine encephalitis virus
- monkeypox virus
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Affiliation(s)
- Jeffrey W Koehler
- U.S. Army Medical Research Institute of Infectious Diseases, Virology Division, Fort Detrick, MD 21702, USA
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Cai Y, Zhang Z, Fan K, Zhang J, Shen W, Li M, Si D, Luo H, Zeng Y, Fu P, Liu C. Pharmacokinetics, tissue distribution, excretion, and antiviral activity of pegylated recombinant human consensus interferon-α variant in monkeys, rats and guinea pigs. ACTA ACUST UNITED AC 2011; 173:74-81. [PMID: 21985916 DOI: 10.1016/j.regpep.2011.09.008] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2011] [Revised: 08/28/2011] [Accepted: 09/16/2011] [Indexed: 11/25/2022]
Abstract
The study aims to characterize the pharmacokinetic, tissue distribution, excretion, and antiviral activity properties of a novel pegylated recombinant human consensus interferon-α variant (PEG-IFN-SA) following a single subcutaneous administration to monkeys, rats and guinea pigs. Studies included: (1) pharmacokinetic properties of PEG-IFN-SA and comparison with those of non-pegylated IFN-SA in rhesus monkeys and rats; (2) tissue distribution and urinary, fecal, and biliary excretion patterns of (125)I-PEG-IFN-SA in guinea pigs; and (3) antiviral activity assessment of PEG-IFN-SA in cynomolgus monkeys. The pegylated protein exhibited improved pharmacokinetic properties compared to IFN-SA in both monkeys and rats, with a 12-fold and 15-fold increase in elimination half-life, and a 100-fold and 10-fold decrease in serum clearance, as well as a 2.5-fold and 10-fold increase in the time to reach peak serum concentration, respectively. (125)I-PEG-IFN-SA was found to be distributed to most of the tissues examined and has character of targeting special distribution, and urinary appeared to be a major route for the excretion of PEG-IFN-SA in guinea pigs. Serum sample analysis from PEG-IFN-SA-treated monkeys showed dose-dependent antiviral activity for one week. These findings demonstrate that pegylation of IFN-SA results in more desirable pharmacokinetic properties, enhanced drug exposure and sustained-efficacy of in vivo antiviral action.
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Affiliation(s)
- Yongming Cai
- Department of Pharmaceutical Engineering, School of Chemical and Technology, Tianjin University, 92 Weijin Road, Tianjin 300072, China
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Consensus interferon used to treat prior partial-responders to pegylated interferon plus ribavirin. Dig Dis Sci 2011; 56:3032-7. [PMID: 21879283 DOI: 10.1007/s10620-011-1869-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/01/2011] [Accepted: 08/12/2011] [Indexed: 01/08/2023]
Abstract
BACKGROUND The response to pegylated interferon (peg-IFN) plus ribavirin therapy remains less than ideal with 40-50% of treated subjects failing to clear the virus. Moreover, retreatment is only minimally effective. Consensus interferon (c-IFN) has been shown to be efficacious in HCV genotype 1 patients who have failed therapy with peg-IFN. AIM To evaluated the response to re-treatment of peg-IFN plus ribavirin partial-responders with c-IFN plus ribavirin. METHODS Forty-two subjects who had previously failed to clear virus after treatment with peg-IFN plus ribavirin were treated with c-IFN (15 μg/day) plus ribavirin (800-1,200 mg/day) until 12 months of therapy or a total of six consecutive months of PCR negativity was achieved. RESULTS The study population consisted predominantly of males (71%), Caucasians (76%), with African Americans comprising the remaining 24%, subjects with HCV genotype 1 infection (81%) and 21% had cirrhosis by liver biopsy. The overall SVR rate was 29%. The only pretreatment variable that distinguished responders from partial-responders was the serum triglyceride level. CONCLUSIONS The use of c-IFN plus ribavirin in the retreatment of prior peg-IFN plus ribavirin partial responders is essentially twice that achieved in prior re-treatment regimens consisting of a second course of peg-IFN plus ribavirin. These results will need to be evaluated against the use of triple therapy consisting of a peg-IFN plus ribavirin and a protease inhibitor. More studies utilizing c-IFN plus ribavirin with either a protease inhibitor or polymerase inhibitor need to be performed as well.
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Wu D, Chu J, Hao YY, Wang YH, Zhuang YP, Zhang SL. Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris. J Biotechnol 2011; 157:107-12. [PMID: 21924302 DOI: 10.1016/j.jbiotec.2011.08.032] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2011] [Revised: 08/30/2011] [Accepted: 08/30/2011] [Indexed: 10/17/2022]
Abstract
Previous report has shown that the expression of recombinant human consensus interferon-α mutant (cIFN) in Pichia pastoris in bioreactor is limited with respect to the incorrectly folded cIFN with incomplete disulfide bond, which lead to the degradation and aggregation of cIFN. In this study, the origin of incorrectly folded cIFN is firstly studied. Fed-batch fermentation in bioreactor shows that the incorrectly folded cIFN is formed intramolecularly and secreted to the extracellular environment. Further chemostat cultures indicate that the specific growth rate is the critical factor for the production of incorrect cIFN. In addition, cell shows reduced expression level of cIFN at high specific growth rate. We also demonstrate that the incorrectly folded cIFN could form aggregates intracellularly and these aggregates are non-covalent forms. Taken together, these results suggest that the efficient heterologous expression of cIFN is limited by high cell growth that is unique from expression limitations seen for soluble proteins. A balance has to be found between the increase for high efficient expression of heterologous proteins and requirement of the high cell growth during the expression of recombinant proteins in P. pastoris.
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Affiliation(s)
- Dan Wu
- State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave, Wuxi, Jiangsu 214122, People's Republic of China
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Kamionka M. Engineering of therapeutic proteins production in Escherichia coli. Curr Pharm Biotechnol 2011; 12:268-74. [PMID: 21050165 PMCID: PMC3179032 DOI: 10.2174/138920111794295693] [Citation(s) in RCA: 103] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2010] [Accepted: 06/04/2010] [Indexed: 11/22/2022]
Abstract
Low cost and simplicity of cultivating bacteria make the E. coli expression system a preferable choice for production of therapeutic proteins both on a lab scale and in industry. In addition straightforward recombinant DNA technology offers engineering tools to produce protein molecules with modified features. The lack of posttranslational modification mechanisms in bacterial cells such as glycosylation, proteolytic protein maturation or limited capacity for formation of disulfide bridges may, to a certain extent, be overcome with protein engineering. Protein engineering is also often employed to improve protein stability or to modulate its biological action. More sophisticated modifications may be achieved by genetic fusions of two proteins. This article presents a variety of examples of genetic engineering of therapeutic proteins. It emphasizes the importance of designing a construct without any unnecessary amino acid residues.
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Affiliation(s)
- Mariusz Kamionka
- School of Pharmacy and Pharmaceutical Sciences, Trinity College, University of Dublin, Dublin 2, Ireland.
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Chary A, Holodniy M. Interferon combination therapy for HIV/hepatitis C virus coinfection. Immunotherapy 2011; 3:1087-102. [PMID: 21913831 DOI: 10.2217/imt.11.105] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
IFN-α has been the cornerstone of chronic hepatitis C virus (HCV) treatment for over a decade. Yet, rates of sustained virologic response of HCV infection to interferon-based therapy, particularly in difficult-to-treat populations, have been disappointingly low. This is particularly true in HIV/HCV coinfection, in which less than a third of patients typically respond to therapy. New HCV protease inhibitors, most of which will need to be administered with pegylated interferon, are in development, but comprehensive, long-term data for their use in coinfected patients is not yet available. Understanding the basis of this population's poor response to interferon-based therapy is crucial to future exploration of new therapeutic options, immunotherapy and prognosis in HIV/HCV-coinfected population.
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Affiliation(s)
- Aarthi Chary
- VA Palo Alto Health Care System, Palo Alto, CA, USA.
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Vazquez E, Corchero JL, Villaverde A. Post-production protein stability: trouble beyond the cell factory. Microb Cell Fact 2011; 10:60. [PMID: 21806813 PMCID: PMC3162505 DOI: 10.1186/1475-2859-10-60] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2011] [Accepted: 08/01/2011] [Indexed: 12/21/2022] Open
Abstract
Being protein function a conformation-dependent issue, avoiding aggregation during production is a major challenge in biotechnological processes, what is often successfully addressed by convenient upstream, midstream or downstream approaches. Even when obtained in soluble forms, proteins tend to aggregate, especially if stored and manipulated at high concentrations, as is the case of protein drugs for human therapy. Post-production protein aggregation is then a major concern in the pharmaceutical industry, as protein stability, pharmacokinetics, bioavailability, immunogenicity and side effects are largely dependent on the extent of aggregates formation. Apart from acting at the formulation level, the recombinant nature of protein drugs allows intervening at upstream stages through protein engineering, to produce analogue protein versions with higher stability and enhanced therapeutic values.
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Affiliation(s)
- Esther Vazquez
- Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
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Abstract
Twenty-five years have passed since interferon-α was first used in the treatment of chronic hepatitis C infection, and even now it remains an essential part of the standard of care for this condition. At present, the recommended treatment is a combination of pegylated interferon and ribavirin A. There have been enormous advances in our understanding of the mechanisms through which interferon works and in identifying factors related to the response to this treatment. Even with the development of new protease inhibitors, it is likely that interferon will remain an essential component of hepatitis C treatment.
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Affiliation(s)
- Maria Buti
- Liver Unit, Hospital General, Universitario Valle Hebron, Paseo Valle Hebron 119, Barcelona 08035, Spain.
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42
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Yee HS, Currie SL, Tortorice K, Cozen M, Shen H, Chapman S, Cunningham F, Monto A. Retreatment of hepatitis C with consensus interferon and ribavirin after nonresponse or relapse to pegylated interferon and ribavirin: a national VA clinical practice study. Dig Dis Sci 2011; 56:2439-48. [PMID: 21633833 DOI: 10.1007/s10620-011-1746-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/16/2010] [Accepted: 05/03/2011] [Indexed: 12/21/2022]
Abstract
BACKGROUND Studies of the retreatment with consensus interferon (CIFN) and ribavirin (RBV) of hepatitis C virus (HCV)-infected patients who failed prior pegylated interferon alfa/ribavirin (PEG-IFN/RBV) have found quite variable efficacy and tolerability of this therapy. As such, CIFN/RBV use and efficacy in clinical practice were evaluated within the Department of Veterans Affairs (VA), the largest national, integrated system for HCV care. AIMS The purpose of this study was to determine rates of sustained virologic response (SVR) and patterns of CIFN/RBV use in the VA. Methods included retrospective review of national VA data in HCV-infected patients who had previously failed≥12 weeks of PEG-IFN/RBV and were prescribed CIFN/RBV between October 1, 2003 and September 30, 2006. RESULTS A total of 597 patients met the study criteria. CIFN was primarily dosed as 15 mcg subcutaneously daily combined with standard doses of RBV. Mean treatment duration was 21 weeks; CIFN was discontinued within 4 weeks in 24%. Hematological growth factors were used in 49%. Post-treatment viral loads were available in 385 patients. SVR to CIFN/RBV was achieved in 11%, and was significantly higher in prior PEG-IFN/RBV relapsers compared with nonresponders (31% vs. 6%, respectively; P<0.0001). A 2-log10 or greater drop in HCV RNA after 24 weeks of PEG-IFN/RBV was a predictor of subsequent SVR to CIFN/RBV. CONCLUSIONS CIFN/RBV was used frequently in clinical practice for retreatment of PEG-IFN/RBV. In this setting, early treatment discontinuation was common. Overall SVR was low, although response was significantly better in prior PEG-IFN/RBV relapsers and those who had a 2-log(10) or greater decline than in nonresponders.
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Affiliation(s)
- Helen S Yee
- Gastroenterology Section, Department of Veterans Affairs Medical Center (VAMC), 4150 Clement Street (111B), San Francisco, CA 94121, USA
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Wu D, Chu J, Hao YY, Wang YH, Zhuang YP, Zhang SL. High efficient production of recombinant human consensus interferon mutant in high cell density culture of Pichia pastoris using two phases methanol control. Process Biochem 2011. [DOI: 10.1016/j.procbio.2011.05.015] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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Abstract
Consensus interferon (CIFN) is an artificially engineered interferon that reflects most of the human genotype 1 interferons and shows a higher biological and antiviral capacity in vitro. It has been used internationally to treat patients with chronic hepatitis C (HCV) infection before pegylated IFN became available. To mimic the half-life of PEG-IFN it has to be administered on a daily basis. The gold standard in the treatment of hepatitis C is well established and recommended. Today patients are being treated with a combination therapy of pegylated IFN and ribavirin. Length and dosage of therapy depends on the genotype of the virus. Patients with genotype 1 and 4 and high viral load should be treated for 48 weeks; for patients with these genotypes along with either low viral load or early virological response, therapy for 24 weeks is sufficient. Patients with genotype 2 and 3 should be treated for up to 24 weeks. However, daily dosing of IFN-α, eg, CIFN, resulting in a higher cumulative dosage, might be beneficial and more efficacious in some chronic HCV-infected patients. Patients with genotype 1, having initially high viral load (>800,000 IU/mL) and showing advanced liver disease with progressive fibrosis or even cirrhosis comprise the difficult-to-treat in order to overcome the infection. This review summarizes and critically discusses the published data on the treatment of HCV with CIFN.
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Affiliation(s)
- Th Witthöft
- University Hospital Schleswig Holstein, Campus Lübeck, Dept of Medicine I, Division of Gastroenterology, Lübeck, Germany
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Lazear HM, Pinto AK, Vogt MR, Gale M, Diamond MS. Beta interferon controls West Nile virus infection and pathogenesis in mice. J Virol 2011; 85:7186-94. [PMID: 21543483 PMCID: PMC3126609 DOI: 10.1128/jvi.00396-11] [Citation(s) in RCA: 78] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2011] [Accepted: 04/27/2011] [Indexed: 12/21/2022] Open
Abstract
Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR(-)(/)(-)) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β(-)(/)(-) mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β(-)(/)(-) mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR(-)(/)(-) mice. The increased susceptibility of IFN-β(-)(/)(-) mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β(-)(/)(-) mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8(+) T cells, we observed an altered CD4(+) CD25(+) FoxP3(+) regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.
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Affiliation(s)
| | | | | | - Michael Gale
- Department of Immunology, University of Washington School of Medicine, Seattle, Washington 98195-7650
| | - Michael S. Diamond
- Departments of Medicine
- Pathology & Immunology
- Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110
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Ho SB, Aqel B, Dieperink E, Liu S, Tetrick L, Falck-Ytter Y, DeComarmond C, Smith CI, McKee DP, Boyd W, Kulig CC, Bini EJ, Pedrosa MC. U.S. multicenter pilot study of daily consensus interferon (CIFN) plus ribavirin for "difficult-to-treat" HCV genotype 1 patients. Dig Dis Sci 2011; 56:880-8. [PMID: 21221804 PMCID: PMC3041922 DOI: 10.1007/s10620-010-1504-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/09/2010] [Accepted: 11/17/2010] [Indexed: 12/19/2022]
Abstract
BACKGROUND Patients with chronic hepatitis C genotype 1 (HCV-1) and difficult-to-treat characteristics respond poorly to pegylated interferon alfa and ribavirin (RBV), and could benefit from an interferon with increased activity (consensus interferon or CIFN), favorable viral kinetics from daily dosing, and a longer duration of therapy. The purpose of this pilot study was to determine the efficacy and safety of daily CIFN + RBV for initial treatment of patients with HCV-1 infection. METHODS Patients with difficult-to-treat characteristics (92% male, 33% African American, 78% Veterans Affairs [VA]; 67% high viral load, 59% stage 3-4 fibrosis, and mean weight of 204 lbs) were enrolled at seven VA and two community medical centers. They were randomized to daily CIFN (15 mcg/day SQ) and RBV (1-1.2 g/d PO) given for either 52 weeks (group A, n = 33) or 52-72 weeks (from time of viral response +48 weeks) (group B, n = 31). RESULTS Intention to treat analysis for treatment groups A and B demonstrated 33% (11/33) and 32% (10/31) sustained virologic response (SVR), respectively. Only 2/31 patients in group B received more than 52 weeks of treatment. The overall group demonstrated a 31% (20/64) rapid virologic response rate (RVR), 54% (34/64) end of treatment virologic response and a 33% (21/64) SVR. Patients with RVR at 4 weeks, early virologic response from 8-12 weeks, and late virologic response from 16-24 weeks demonstrated SVR of 75% (15/20), 31% (4/13), and 22% (2/9), respectively. Overall early non-protocol discontinuation occurred in 26/64 (40%) patients. CONCLUSION Daily CIFN and ribavirin for initial treatment of HCV-1 patients has potential for achieving a relatively high RVR rate, but discontinuations are frequent and successful use of this regimen is highly dependent on adequate patient support to maintain adherence.
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Affiliation(s)
- Samuel B Ho
- VA San Diego Healthcare System, 3350 La Jolla Village Drive, San Diego, CA 92161, USA.
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Ferenci P. Safety and efficacy of treatment for chronic hepatitis C with a focus on pegylated interferons: the backbone of therapy today and in the future. Expert Opin Drug Saf 2011; 10:529-44. [PMID: 21345149 DOI: 10.1517/14740338.2011.555079] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
INTRODUCTION Approximately 170 million people are infected with HCV. The efficacy of treatment for chronic hepatitis C has increased markedly over the last 2 decades. Optimal patient management requires thorough knowledge of the adverse effect profiles of drugs used for this condition and strategies to mitigate these effects. AREAS COVERED The efficacy, safety and tolerability data associated with IFN-based therapy, with particular attention given to the two licensed pegylated IFNs (peg-IFNs), are identified by focused searches of Medline. Recommendations for the management of adverse events are also given. Focused searches of PubMed are done using the terms peginterferon and chronic hepatitis C. The results of large randomized clinical trials are emphasized. EXPERT OPINION Patients receiving treatment with peg-IFN plus ribavirin for chronic hepatitis C must be monitored closely for adverse events. These events can be effectively managed to maximize patients' adherence and thus the chance of treatment success. Direct-acting antiviral agents are expected to be approved in the near future and will be used in select patients with a peg-IFN plus ribavirin 'backbone'.
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Affiliation(s)
- Peter Ferenci
- Medical University of Vienna, Univ. Klinik für Innere Medizin III, Währinger Gürtel 18-20, A 1090 Vienna , Austria.
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Vezali E, Aghemo A, Colombo M. Interferon in the treatment of chronic hepatitis C: a drug caught between past and future. Expert Opin Biol Ther 2011; 11:301-13. [DOI: 10.1517/14712598.2011.552906] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
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Kumaki Y, Ennis J, Rahbar R, Turner JD, Wandersee MK, Smith AJ, Bailey KW, Vest ZG, Madsen JR, Li JKK, Barnard DL. Single-dose intranasal administration with mDEF201 (adenovirus vectored mouse interferon-alpha) confers protection from mortality in a lethal SARS-CoV BALB/c mouse model. Antiviral Res 2010; 89:75-82. [PMID: 21093489 PMCID: PMC3018546 DOI: 10.1016/j.antiviral.2010.11.007] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2010] [Revised: 11/04/2010] [Accepted: 11/11/2010] [Indexed: 11/21/2022]
Abstract
Interferons (IFNs) are a first line of defense against viral infection. Herein we describe the use of an adenovirus vectored mouse IFN alpha gene (mDEF201) as a prophylactic and treatment countermeasure in a SARS-CoV-infected BALB/c mouse model. Complete survival protection was observed in mice given a single dose of mDEF201 administered intranasally 1, 3, 5, 7, or 14 days prior to lethal SARS-CoV challenge (p < 0.001), and body weights of these treated mice were unaffected by the challenge. In addition, low doses of mDEF201 protected lungs in a dose dependent manner as measured by a reduction in gross pathology. Intranasal treatment with mDEF201 ranging from 106 to 108 PFU significantly protected mice against a lethal SARS-CoV infection in a dose dependent manner up to 12 h post infection (p < 0.001). The data suggest that mDEF201 is a new class of antiviral agent further development as treatment for SARS-CoV infections.
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Affiliation(s)
- Yohichi Kumaki
- Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, 5600 Old Main Hill, Logan, Utah State University, Logan, UT 84322, USA
| | - Jane Ennis
- Defyrus Inc., 2 Bloor Street West, Suite 2602, Toronto, Ontario, Canada M4W 3E2
- Corresponding author. Tel.: +1 416 966 5536.
| | - Ramtin Rahbar
- Defyrus Inc., 2 Bloor Street West, Suite 2602, Toronto, Ontario, Canada M4W 3E2
| | - Jeffrey D. Turner
- Defyrus Inc., 2 Bloor Street West, Suite 2602, Toronto, Ontario, Canada M4W 3E2
| | - Miles K. Wandersee
- Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, 5600 Old Main Hill, Logan, Utah State University, Logan, UT 84322, USA
| | - Aaron J. Smith
- Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, 5600 Old Main Hill, Logan, Utah State University, Logan, UT 84322, USA
| | - Kevin W. Bailey
- Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, 5600 Old Main Hill, Logan, Utah State University, Logan, UT 84322, USA
| | - Zachary G. Vest
- Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, 5600 Old Main Hill, Logan, Utah State University, Logan, UT 84322, USA
| | - Jason R. Madsen
- Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, 5600 Old Main Hill, Logan, Utah State University, Logan, UT 84322, USA
| | - Joseph K.-K. Li
- Department of Biology, 5305 Old Main Hill, Utah State University, Logan, UT 84322, USA
| | - Dale L. Barnard
- Institute for Antiviral Research, Department of Animal, Dairy and Veterinary Science, 5600 Old Main Hill, Logan, Utah State University, Logan, UT 84322, USA
- Corresponding author. Tel.: +1 435 797 2696; fax: +1 435 797 3959.
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