A Human Single Chain Transbody Specific to Matrix Protein (M1) Interferes with the Replication of Influenza A Virus

Poungpair, Ornnuthchar; Pootong, Anek; Maneewatch, Santi; Srimanote, Potjanee; Tongtawe, Pongsri; Songserm, Thaweesak; Tapchaisri, Pramuan; Chaicumpa, Wanpen

doi:10.1021/bc900251u

Cited by 34 publications

(46 citation statements)

References 45 publications

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“…Chemical conjugation was employed for Antibodies or antibody fragments linked to PTDs have been used to neutralize tetanus toxin in chromaffin cells and influenza A viral activity. 142,156 Further applications included interfering with the cell cycle, such as inhibition of a G 1 -S-phase arrest by an anti-Cdk (cyclin dependent kinase) inhibitor, 154 or inhibition of cell cycle progression by an anti-cyclin D1 transbody. 151 Transbodies have also been used to target apoptosis in order to promote, as well as to suppress, apoptosis.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Targeting antibodies to the cytoplasm

Marschall

Frenzel

Schirrmann

et al. 2011

mAbs

110

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Section: O N O T D I S T R I B U T Ementioning

confidence: 99%

Targeting antibodies to the cytoplasm

Marschall

Frenzel

Schirrmann

et al. 2011

mAbs

110

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“…Moreover, they can be made as cell penetrable molecules that can reach intracellular targets. 26 The antigen-antibody interaction usually involves many contact residues between the partners; thus, an antibody is relatively more tolerable to target mutation than small molecular drugs/ inhibitors. 27 There is a need for a new remedy for the treatment of HCV infection that is safer than the existing regimens.…”

Section: Discussionmentioning

confidence: 99%

“…26 The scfv sequences of individual E. coli clones that inhibited the NS5BD55 RdRp activity in vitro were subcloned to the recombinant pen-pET23b…”

Section: Production Of Cell Penetrable Human Scfvs Specific To Hcv Ns5bmentioning

confidence: 99%

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Thueng-in

Thanongsaksrikul

Jittavisutthikul

et al. 2014

mAbs

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dodecyl sulfate; SOC, standard-of-care; STAT-C, specifically targeted anti-viral therapy for hepatitis C; SVR, sustained virologic response; VH, variable heavy chain domain of conventional four-chain IgG; V H H, variable heavy chain domain of heavy chain antibody; VL, variable light chain domainA new class of hepatitis C virus (HCV)-targeted therapeutics that is safe, broadly effective and can cope with virus mutations is needed. The HCV's NS5B is highly conserved and different from human protein, and thus it is an attractive target for anti-HCV therapeutics development. In this study, NS5B bound-phage clones selected from a human single chain variable antibody fragment (scFv) phage display library were used to transform appropriate E. coli bacteria. Two scFv inhibiting HCV polymerase activity were selected. The scFvs were linked to a cell penetrating peptide to make cell penetrable scFvs. The transbodies reduced the HCV RNA and infectious virus particles released into the culture medium and inside hepatic cells transfected with a heterologous HCV replicon. They also rescued the innate immune response of the transfected cells. Phage mimotope search and homology modeling/molecular docking revealed the NS5B subdomains and residues bound by the scFvs. The scFv mimotopes matched residues of the NS5B, which are important for nucleolin binding during HCV replication, as well as residues that interconnect the fingers and thumb domains for forming a polymerase active groove. Both scFvs docked on several residues at the thumb armadillo-like fold that could be the polymerase interactive sites of other viral/host proteins for the formation of the replication complex and replication initiation. In conclusion, human transbodies that inhibited HCV RdRp activity and HCV replication and restored the host innate immune response were produced. They are potentially future interferon-free anti-HCV candidates, particularly in combination with other cognates that are specific to NS5B epitopes and other HCV enzymes.

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“…The LDH release assay was performed as described previously (35). Huh7 monolayer in individual wells of a 96-well tissue culture plate (2 × 10 4 cells/well) were washed with sterile PBS, added with 25 μg of R9-HuscFvs in complete DMEM, and incubated for 24 h at 37°C in 5% CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

“…In our laboratory, cell-penetrable antibodies (transbodies) specific to viral proteins have been prepared by linking the antibody molecules to either penetratin or R9. These fusion proteins readily entered mammalian cells and bound to their respective intracellular targets (31–35). …”

Section: Introductionmentioning

confidence: 99%

Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity

et al. 2016

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A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins.

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A Human Single Chain Transbody Specific to Matrix Protein (M1) Interferes with the Replication of Influenza A Virus

Cited by 34 publications

References 45 publications

Targeting antibodies to the cytoplasm

Targeting antibodies to the cytoplasm

Interference of HCV replication by cell penetrable human monoclonal scFv specific to NS5B polymerase

Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity

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