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1.
Viruses ; 7(1): 154-79, 2015 Jan 14.
Article in English | MEDLINE | ID: mdl-25594836

ABSTRACT

A new anti-influenza remedy that can tolerate the virus antigenic variation is needed. Influenza virus matrix protein-1 (M1) is highly conserved and pivotal for the virus replication cycle: virus uncoating, assembly and budding. An agent that blocks the M1 functions should be an effective anti-influenza agent. In this study, human scFv that bound to recombinant M1 middle domain (MD) and native M1 of A/H5N1 was produced. Phage mimotope search and computerized molecular docking revealed that the scFv bound to the MD conformational epitope formed by juxtaposed helices 7 and 9 of the M1. The scFv was linked molecularly to a cell penetrable peptide, penetratin (PEN). The PEN-scFv (transbody), when used to treat the cells pre-infected with the heterologous clade/subclade A/H5N1 reduced the viral mRNA intracellularly and in the cell culture fluids. The transbody mitigated symptom severity and lung histopathology of the H5N1 infected mice and caused reduction of virus antigen in the tissues as well as extricated the animals from the lethal challenge in a dose dependent manner. The transbody specific to the M1 MD, either alone or in combination with the cognate human scFvs specific to other influenza virus proteins, should be an effective, safe and mutation tolerable anti-influenza agent.


Subject(s)
Antibodies, Viral/therapeutic use , Antiviral Agents/therapeutic use , Carrier Proteins/metabolism , Influenza A Virus, H5N1 Subtype/drug effects , Orthomyxoviridae Infections/drug therapy , Single-Chain Antibodies/therapeutic use , Viral Matrix Proteins/antagonists & inhibitors , Animals , Antibodies, Viral/genetics , Carrier Proteins/genetics , Cell-Penetrating Peptides , Disease Models, Animal , Female , Mice, Inbred BALB C , Molecular Docking Simulation , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/virology , Peptide Library , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic use , Single-Chain Antibodies/genetics , Survival Analysis , Treatment Outcome
2.
Toxins (Basel) ; 6(5): 1526-58, 2014 May 13.
Article in English | MEDLINE | ID: mdl-24828754

ABSTRACT

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.


Subject(s)
Antivenins/metabolism , Elapid Venoms/chemistry , Neurotoxins/pharmacology , Proteome , Single-Chain Antibodies/immunology , Animals , Chromatography, Liquid , Elapid Venoms/metabolism , Elapid Venoms/toxicity , Elapidae , Electrophoresis, Polyacrylamide Gel , Humans , Lethal Dose 50 , Male , Mice , Mice, Inbred ICR , Neurotoxins/immunology , Neutralization Tests , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry
3.
Antiviral Res ; 100(1): 226-37, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23928258

ABSTRACT

Currently, there is a need of new anti-influenza agents that target influenza virus proteins other than ion channel M2 and neuraminidase. Non-structural protein-1 (NS1) is a highly conserved multifunctional protein which is indispensable for the virus replication cycle. In this study, fully human single chain antibody fragments (HuScFv) that bound specifically to recombinant and native NS1 were produced from three huscfv-phagemid transformed Escherichia coli clones (nos. 3, 10 and 11) selected from a human ScFv phage display library. Western blot analysis, mimotope searching/epitope identification, homology modeling/molecular docking and phage mimotope ELISA inhibition indicated that HuScFv of clone no. 3 reacted with NS1 R domain important for host innate immunity suppression; HuScFv of clone nos. 10 and 11 bound to E domain sites necessary for NS1 binding to the host eIF4GI and CPSF30, respectively. The HuScFv of all clones could enter the influenza virus infected cells and interfered with the NS1 activities leading to replication inhibition of viruses belonging to various heterologous A subtypes and type B by 2-64-fold as semi-quantified by hemagglutination assay. Influenza virus infected cells treated with representative HuScFv (clone 10) had up-expression of IRF3 and IFN-ß genes by 14.75 and 4.95-fold, respectively, in comparison with the controls, indicating that the antibodies could restore the host innate immune response. The fully human single chain antibodies have high potential for developing further as a safe (adjunctive) therapeutic agent for mitigating, if not abrogating, severe symptoms of influenza.


Subject(s)
Antibodies, Viral/pharmacology , Down-Regulation/drug effects , Influenza A virus/drug effects , Influenza, Human/virology , Single-Chain Antibodies/pharmacology , Viral Nonstructural Proteins/immunology , Virus Replication/drug effects , Animals , Chick Embryo , Humans , Influenza A virus/classification , Influenza A virus/immunology , Influenza A virus/physiology , Influenza, Human/drug therapy , Viral Nonstructural Proteins/genetics
4.
Virol J ; 10: 148, 2013 May 14.
Article in English | MEDLINE | ID: mdl-23672256

ABSTRACT

BACKGROUND: Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies. METHODS: Fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking. RESULTS: HuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages. CONCLUSIONS: HuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.


Subject(s)
Antibodies, Viral/immunology , Antiviral Agents/isolation & purification , Influenza A Virus, H5N1 Subtype/immunology , Influenza A Virus, H5N1 Subtype/physiology , Single-Chain Antibodies/immunology , Viral Matrix Proteins/immunology , Virus Replication , Animals , Antibodies, Viral/isolation & purification , Antibodies, Viral/metabolism , Antiviral Agents/metabolism , Chick Embryo , Escherichia coli/genetics , Gene Expression , Genetic Vectors , Humans , Peptide Library , Protein Binding , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/metabolism , Viral Matrix Proteins/metabolism
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