Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes

Yodsheewan, Rungrueang; Maneewatch, Santi; Srimanote, Potjanee; Thueng-in, Kanyarat; Songserm, Thaweesak; Dong-din-on, Fonthip; Bangphoomi, Kunan; Sookrung, Nitat; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

doi:10.1016/j.antiviral.2013.07.019

Cited by 22 publications

(19 citation statements)

References 41 publications

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“…19 Nowadays, recombinant antibodies to any epitope can be rapidly produced in vitro (regardless of the tissue culture facility and immunogenicity and toxicity of the antigen) from E. coli bacteria transfected with the antigen-binding phage selected from an antibody phage display library. [20][21][22][23][24][25] The engineered antibodies can also be made into intact four-chain molecules when the biological activities of the Fc fragment are needed for the therapeutic effect. Alternatively, smaller antibody fragments that are devoid of the Fc fragment or constant domains, e.g., scFv 21,22 or single domain antibodies (nanobodies/VH/ V H H) 13,[23][24][25] can be used to increase the tissue penetrating ability and reduce tissue inflammation.…”

Section: Discussionmentioning

confidence: 99%

“…NM_145725.2, Z56281 and NM_002176, respectively) using Primer3 software. 21 The PCR reaction mixture (12. Determination of phage mimotopes that bound to NS5B specific-human scFvs and validation of the mimotopes Phage mimotopic peptides that bound to the NS5B specifichuman scFvs were determined by a Ph.D. -12 TM phage display peptide library (New England Biolab, USA) as described previously.…”

Section: Foci Assaymentioning

confidence: 99%

“…Determination of phage mimotopes that bound to NS5B specific-human scFvs and validation of the mimotopes Phage mimotopic peptides that bound to the NS5B specifichuman scFvs were determined by a Ph.D. -12 TM phage display peptide library (New England Biolab, USA) as described previously. 20,21 Wells of a 96-well ELISA plate were coated separately with scFv-14 and -34 (1 mg in 100 ml) at 4 C overnight. After washing with Tris buffered saline, pH 7.5 (TBS) containing 0.1% Tween-20 (TBST), each well was blocked with 200 ml of 0.5% BSA in TBS for 1 h and washed.…”

Section: Foci Assaymentioning

confidence: 99%

“…To assess the mechanisms of the scFv-mediated inhibitions of the RdRp activity and the HCV replication, the regions and residues of the NS5B molecule bound by the scFvs were determined by means of 12 mer-peptide phage mimotope search 21,22 and computerized homology modeling and molecular docking. [31][32][33][34][35][36][37][38] The NS5B of HCV genotype 3a's linear sequence (accession number NP751928) was used in the alignment with the scFv mimotope sequences for tentative epitope identification and the 3D structure of PDB 1C2P was used in the molecular docking with the scFv 3D structures.…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Foci Assaymentioning

confidence: 99%

Section: Foci Assaymentioning

confidence: 99%

mentioning

confidence: 99%

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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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“…In particular, the scFv phage display technique has recently been applied to screen antibodies against NS1 (viral nonstructural protein) and M1 (viral matrix protein) of an H5N1 strain [27,28]. However, the emergence of resistant influenza virus strains against drugs targeting neuraminidase (NA), M2, or the receptor-binding domain of HA1 of IAV renders it more urgent to search for other, more-conserved viral protein targets [1,5] such as HA2.…”

Section: Introductionmentioning

confidence: 99%

Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2)

Cheng

Tseng

et al. 2015

Arch Virol

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Influenza A viruses (IAV) are widespread in birds and domestic poultry, occasionally causing severe epidemics in humans and posing health threats. Hence, the need to develop a strategy for prophylaxis or therapy, such as a broadly neutralizing antibody against IAV, is urgent. In this study, single-chain variable fragment (scFv) phage display technology was used to select scFv fragments recognizing influenza envelope proteins. The Tomlinson I and J scFv phage display libraries were screened against the recombinant HA2 protein (rHA2) for three rounds. Only the third-round elution sample of the Tomlinson J library showed high binding affinity to rHA2, from which three clones (3JA18, 3JA62, and 3JA78) were chosen for preparative-scale production as soluble antibody by E. coli. The clone 3JA18 was selected for further tests due to its broad affinity for influenza H1N1, H3N2 and H5N1. Simulations of the scFv 3JA18-HA trimer complex revealed that the complementarity-determining region of the variable heavy chain (VH-CDR2) bound the stem region of HA. Neutralization assays using a peptide derived from VH-CDR2 also supported the simulation model. Both the selected antibody and its derived peptide were shown to suppress infection with H5N1 and H1N1 viruses, but not H3N2 viruses. The results also suggested that the scFvs selected from rHA2 could have neutralizing activity by interfering with the function of the HA stem region during virus entry into target cells.

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Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display

Kühn

Fühner

Unkauf

et al. 2016

Proteomics Clinical Apps

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Antibodies are valuable molecules for the diagnostic and treatment of diseases caused by pathogens and toxins. Traditionally, these antibodies are generated by hybridoma technology. An alternative to hybridoma technology is the use of antibody phage display to generate recombinant antibodies. This in vitro technology circumvents the limitations of the immune system and allows-in theory-the generation of antibodies against all conceivable molecules. Phage display technology enables obtaining human antibodies from naïve antibody gene libraries when either patients are not available or immunization is not ethically feasible. On the other hand, if patients or immunized/infected animals are available, it is common to construct immune phage display libraries to select in vivo affinity-matured antibodies. Because the phage packaged DNA sequence encoding the antibodies is directly available, the antibodies can be smoothly engineered according to the requirements of the final application. In this review, an overview of phage display derived recombinant antibodies against bacterial, viral, and eukaryotic pathogens as well as toxins for diagnostics and therapy is given.

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Human monoclonal ScFv specific to NS1 protein inhibits replication of influenza viruses across types and subtypes

Cited by 22 publications

References 41 publications

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

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

Development of single-chain variable fragments (scFv) against influenza virus targeting hemagglutinin subunit 2 (HA2)

Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display

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