ABSTRACT
The pandemic 2009 H1N1 influenza virus broke out in North America and spread rapidly throughout the world. The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, the protective potential of human exogenous IFN-ω against pandemic 2009 A (H1N1) influenza virus was assessed both in vitro and in guinea pigs. The viral loads of pandemic 2009 A (H1N1) influenza virus strains A/California/04/2009 and A/Beijing/501/2009 were reduced by up to 5000-fold in Caco-2 cells by the addition of human IFN-ω. With daily intranasal treatment with human IFN-ω the viral load of pandemic 2009 A (H1N1) influenza virus strain A/California/04/2009 decreased by 1000-fold in lung tissues of guinea pigs. These results provide strong support for the application of human IFN-ω pretreatment to human influenza control.
Subject(s)
Antiviral Agents/administration & dosage , Influenza A Virus, H1N1 Subtype/drug effects , Interferon Type I/administration & dosage , Orthomyxoviridae Infections/drug therapy , Pandemics , Recombinant Proteins/administration & dosage , Administration, Intranasal , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Caco-2 Cells , Cell Line , Disease Models, Animal , Female , Guinea Pigs , Humans , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H1N1 Subtype/pathogenicity , Interferon Type I/genetics , Interferon Type I/pharmacology , Interferon Type I/therapeutic use , Lung/virology , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/virology , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Virus Replication/drug effectsABSTRACT
Interferon-omega (IFN-ω) may be a useful, promising and alternative antiviral agent, in addition to IFN-α-2a and IFN-α-2b. To improve the pharmacokinetics of IFN-ω for clinical use, the recombinant human IFN-ω-Fc fusion protein (rhIFN-ω-Fc) was expressed in a Chinese hamster ovary cell line (CHO-S), due to the longer serum half-life of rhIFN-ω-Fc compared to the native IFN-ω protein, and purified by affinity chromatography. Physicochemical characterization of the purified fusion protein was performed by SDS-PAGE electrophoresis, dot blot analysis and N-terminal amino acid sequence analysis. The results show that rhIFN-ω-Fc was highly expressed at the predicted size and with the N-terminal amino acid sequence. The antiviral activity was determined by the ability of IFNs to inhibit the cytopathic effects (CPEs) of vesicular stomatitis virus (VSV) on the human amnion WISH cells. The rhIFN-ω-Fc expressed in CHO-S cells has a specific activity of 1.6×10(7) IU/mg compared to rhIFN-ω expressed in yeast, which has a specific activity of 7×10(7) IU/mg. Equimolar concentrations of rhFN-ω and rhIFN-ω-Fc were administered to rabbits for pharmacokinetics comparison. The terminal half-life of rhIFN-ω-Fc was 35 times higher than that of rhIFN-ω. Thus, rhIFN-ω-Fc can be used as a prospective antiviral candidate especially for the treatment of chronic viral disease, such as hepatitis C virus (HCV) infection.
Subject(s)
Antiviral Agents/isolation & purification , Antiviral Agents/metabolism , Biotechnology/methods , Immunoglobulin Fc Fragments/isolation & purification , Immunoglobulin Fc Fragments/metabolism , Interferon Type I/isolation & purification , Interferon Type I/metabolism , Animals , Antiviral Agents/pharmacology , Cell Culture Techniques , Cell Line , Chromatography, Affinity , Cricetinae , Cricetulus , Electrophoresis, Polyacrylamide Gel , Gene Expression , Humans , Immunoblotting , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/pharmacology , Interferon Type I/genetics , Interferon Type I/pharmacology , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Recombinant Fusion Proteins/pharmacology , Sequence Analysis, Protein , Vesiculovirus/drug effectsABSTRACT
The whole encoding sequence for Yersinia pestis LcrV antigen was cloned into pET-32a(+) and expressed in Escherichia coli BL21 (DE3). The LcrV was high level expressed in the E. coli cytoplasm in a completely soluble form. Recombinant LcrV could be purified from the supernatant of the bacteria lysate after chromatography using a combination of Phenyl-Sepharose F F, DEAE-Sepharose F F and Hiload Superdex 75. The final yield of approximately 3 g of purified rLcrV from 42 L bioreactor containing 25 L LB medium was obtained. High-titer IgG directed against rLcrV was detected positive after immunization on the BALB/c mice. The results presented here exhibit the ability to generate multi-gram quantities of non-tagged rLcrV from E. coli that can be used for the development of vaccine for preventing plague.
Subject(s)
Antigens, Bacterial/biosynthesis , Antigens, Bacterial/immunology , Escherichia coli/metabolism , Pore Forming Cytotoxic Proteins/biosynthesis , Pore Forming Cytotoxic Proteins/immunology , Yersinia pestis/metabolism , Animals , Antibodies, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Genetic Vectors , Mice , Mice, Inbred BALB C , Pore Forming Cytotoxic Proteins/isolation & purification , Protein Subunits/biosynthesis , Protein Subunits/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sequence Analysis, ProteinABSTRACT
The entire encoding region for Aspergillus flavus uricase was cloned into pET-32a and expressed in Escherichia coli BL21 (DE3). The uricase was expressed in the E. coli cytoplasm in a completely soluble, biologically active form. A scalable process aimed to produce and purify multi-gram quantities of highly pure, recombinant urate oxidase (rUox) from E. coli was developed. The rUox protein was produced in a 30 L fermentor containing 25 L of 2x YT medium and purified to >99% purity using hydrophobic interaction, anion-exchange, and gel filtered chromatography. The final yield of purified rUox from fermentation resulted in approximately 27 g of highly pure, biologically active rUox per kg of cell paste (approximately 238 mg/8.8 g cell paste/L). The results presented here exhibit the ability to generate multi-gram quantities of rUox from E. coli that may be used for the development of pharmaceutics of reducing the hyperuricemia.
