Humanized-VHH Transbodies that Inhibit HCV Protease  and Replication

Jittavisutthikul, Surasak; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Chulanetra, Monrat; Srimanote, Potjanee; Seesuay, Watee; Malik, Aijaz Ahmad; Chaicumpa, Wanpen

doi:10.3390/v7042030

Cited by 36 publications

(41 citation statements)

References 49 publications

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“…This approach is inherently limited by the ability of the virus to shut down host protein synthesis, including that of inhibitory VHHs. While we were able to circumvent these limitations using inducible VHH expression to some extent, cell-penetrating VHHs or other means of permeabilization that allow efficient delivery from extracellular space could also find application (42, 43). Such an approach would allow an evaluation of the importance of the blocked surface at later stages of infection and, importantly, transform VHHs into discovery tools for antiviral agents suitable for therapeutic intervention.…”

Section: Discussionmentioning

confidence: 99%

The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

Hanke

Knockenhauer

Brewer

et al. 2016

mBio

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Alpaca-derived single-domain antibody fragments (VHHs) that target the influenza A virus nucleoprotein (NP) can protect cells from infection when expressed in the cytosol. We found that one such VHH, αNP-VHH1, exhibits antiviral activity similar to that of Mx proteins by blocking nuclear import of incoming viral ribonucleoproteins (vRNPs) and viral transcription and replication in the nucleus. We determined a 3.2-Å crystal structure of αNP-VHH1 in complex with influenza A virus NP. The VHH binds to a nonconserved region on the body domain of NP, which has been associated with binding to host factors and serves as a determinant of host range. Several of the NP/VHH interface residues determine sensitivity of NP to antiviral Mx GTPases. The structure of the NP/αNP-VHH1 complex affords a plausible explanation for the inhibitory properties of the VHH and suggests a rationale for the antiviral properties of Mx proteins. Such knowledge can be leveraged for much-needed novel antiviral strategies.

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Section: Discussionmentioning

confidence: 99%

The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

Hanke

Knockenhauer

Brewer

et al. 2016

mBio

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“…The nanobodies also led to a significant reduction of HCV RNA levels in Huh7 cells transfected with the JFH1 genotype 2a strain, both inside the cells as in the culture medium, when compared to control conditions. Overall, the nanobodies were capable of eliciting responses of a magnitude similar as seen for conventional therapeutic strategies (ribavirin + PEGylated interferon or telaprevir) [28,[31][32][33]. Furthermore, treatment of cells with the nanobodies against NS3/NS4A and NS4B induced the expression of genes involved in the innate immune response (IRF3, IL28-B, and IFN-β).…”

mentioning

confidence: 80%

“…Several nonstructural HCV proteins have been targeted via nanobodies: NS5B, NS3, NS4A, and NS4B [28,[31][32][33]. NS5B functions as an RNA-dependent RNA polymerase.…”

Section: Hepatitismentioning

confidence: 99%

“…The helicase is involved in the replication of the viral genome and is also thought to increase the translational efficiency of the polyprotein by melting highly stable secondary structures in the HCV RNA [34]. The N-terminal serine protease domain is, together with NS4A, involved in downstream processing of the HCV polyprotein with the formation of mature proteins [32]. NS4B plays a major role in HCV replication by inducing an ER membrane web on which HCV replication takes place [31].…”

mentioning

confidence: 99%

“…Furthermore, treatment of cells with the nanobodies against NS3/NS4A and NS4B induced the expression of genes involved in the innate immune response (IRF3, IL28-B, and IFN-β). This is interesting because the innate immune response signaling is interrupted by the virus [31,32]. In general, these studies lack a detailed insight into the molecular mechanisms behind the observed effects.…”

mentioning

confidence: 99%

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Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

Steels¹,

Bertier²,

Gettemans³

2018

Antibody Engineering

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The discovery of heavy-chain-only antibodies (HCAbs) in camelids and sharks led to the rise of a new research field in which single-domain antibodies are used for various applications. Single-domain antibodies are the antigen-binding fragments derived from HCAbs showing several beneficial properties (e.g., small size, specificity, stability under extreme conditions, cost-effective production, and ease of engineering). Importantly, they are stable in reducing cytoplasmic environment, which allows their use as an intrabody to target a wide range of intracellular targets. In this chapter, we discuss both the therapeutic potential of camelid single-domain antibodies (nanobodies) and their use as a research tool with the main focus on its intracellular employment. Targeting intracellular proteins using nanobodies as a therapeutic per se is, up to now, limited due to its incapacity to traverse the cellular membrane. They can however serve as a stepping stone to small compound development, since they directly target a resident, endogenous protein, similar to how a conventional drug acts. In addition, nanobodies are highly adaptable tools and possess interesting properties for more fundamental research objectives like the elucidation of protein function, the tracking and visualization of endogenous proteins in an in vivo setting, and the assessment of protein-protein interactions.Keywords: VHH, single-domain antibody, nanobody, intrabody, therapy, research tool Nanobodies: a concise introductionIn 1993, Hamers-Casterman discovered the presence of heavy-chain-only antibodies in the sera of Camelidae and assessed that these antibodies are still capable of recognizing an extensive repertoire of antigens despite the absence of the light chain. Single-domain antibodies from camels are called nanobodies. They stated that this discovery could be of inestimable © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.value to the development and engineering of soluble V H domains or new immunological molecules for diagnostic, therapeutic, and biochemical purposes [1]. This discovery gave rise to a whole new research field in which single-domain antibodies are used for a wide range of applications. Some of these will be reviewed in the current chapter.The structural properties of conventional IgG antibodies are well known. These consist of two heavy-chain polypeptides and two light-chain polypeptides, each of which is folded into four and two domains, respectively. A variable domain is situated at the N-terminus of both chains (VH and VL) and, as the name suggests, its sequence diverges between IgG antibodies. Paired VH-VL domains make up the variable fragment (Fab) and are responsible for the recognition and binding of the target antigen. The sequence of the other domains is well conse...

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Single domain antibodies for the knockdown of cytosolic and nuclear proteins

Böldicke

2017

Protein Science

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Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, nonaggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain antibody libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic.

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Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

Cited by 36 publications

References 49 publications

The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

The Antiviral Mechanism of an Influenza A Virus Nucleoprotein-Specific Single-Domain Antibody Fragment

Use, Applications and Mechanisms of Intracellular Actions of Camelid VHHs

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

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