In vitro antiviral activity of single domain antibody fragments against poliovirus

Thys, Bert; Schotte, Lise; Wernery, Ulrich; Hassanzadeh-Ghassabeh, Gholamreza; Rombaut, Bartholomeus

doi:10.1016/j.antiviral.2010.05.012

Cited by 40 publications

(39 citation statements)

References 29 publications

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“…Four VHHs with the lowest capturing titer (highest affinity) for H-antigen were selected from this panel. Detailed methods for the selection, production, and purification of the VHHs were reported in previous papers (6,23,26). The amino acid sequences for the four VHHs are shown in Fig.…”

Section: Vhhs Sample Range Of Expanded Poliovirus Conformationsmentioning

confidence: 99%

“…Through the collection of lymphocytes, production of a cDNA library, and phage display panning, several poliovirus binding VHHs were selected. One panel of VHHs was found to bind and neutralize N-antigen, and a second panel selectively bound H-antigen and had no neutralizing activity (23). Four VHHs with the lowest capturing titer (highest affinity) for H-antigen were selected from this panel.…”

Section: Vhhs Sample Range Of Expanded Poliovirus Conformationsmentioning

confidence: 99%

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Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State

Strauss

Schotte

Karunatilaka

et al. 2017

J Virol

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By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion.IMPORTANCE When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productiveinfection events.KEYWORDS 80S, VHH, cryo-electron microscopy, expanded virus, poliovirus, singledomain antibodies, three-dimensional structure P oliovirus (PV) is a small, nonenveloped, positive-sense, single-stranded RNA (ssRNA) virus that is a member of the enterovirus genus of the picornavirus family (1, 2). The virus consists of an RNA genome that is surrounded by a protein shell. The shell is formed by an icosahedrally symmetric arrangement of 60 copies each of 3 large proteins (VP1, VP2, and VP3), each of which has a wedge-shaped beta barrel core (3), and the small myristoylated protein VP4 (4), which is located on the inner surface of the capsid. To infect its natural host (human epithelial cells in the gut), poliovirus must solve the problem of getting its genome through the host's protective cellular membrane. Details of how this is accomplished are now gradually emerging (5, 6).

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Section: Vhhs Sample Range Of Expanded Poliovirus Conformationsmentioning

confidence: 99%

Section: Vhhs Sample Range Of Expanded Poliovirus Conformationsmentioning

confidence: 99%

Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State

Strauss

Schotte

Karunatilaka

et al. 2017

J Virol

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“…Five different recombinant VHHs were used: PVSP6A, PVSS8A, PVSP19B, PVSS21E, and PVSP29F. They all show an in vitro neutralizing activity against poliovirus type 1 at nanomolar concentrations (4).…”

Section: Methodsmentioning

confidence: 99%

“…The selection, production, and purification of the VHHs were previously described in detail (4,5). Originally, the VHHs used in these experiments were obtained from a dromedary immunized against poliovirus type 1.…”

Section: Methodsmentioning

confidence: 99%

“…Motivated by searches for potential antiviral agents, VHHs that specifically bind to and neutralize poliovirus type 1 were recently selected and identified (4) and were further characterized (5,6). In a previous publication (5), we showed that the binding footprints of two VHHs (PVSP6A and PVSP29F) on the poliovirus surface overlap extensively with the footprint of the poliovirus receptor (PVR; also called CD155) and that they produce well-ordered icosahedrally symmetric complexes.…”

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confidence: 99%

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Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

Strauß

Schotte

Thys

et al. 2016

J Virol

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Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. IMPORTANCEWe describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. In addition to conventional antibodies, members of the Camelidae family possess a set of unusual antibodies that consist only of heavy chains in which a constant domain is missing and that are therefore named heavy-chain-only antibodies (1). These antibodies offer a very interesting new tool in scientific research, as they contain variable domains (VHHs) that are fully responsible for and fully capable of recognizing and binding to antigens. The variable domains are also more hydrophilic than their IgG counterparts, as they are not required to bind to a complementary domain of a light chain. Moreover, they are very stable (2), and due to their rather small size (ϳ14 kDa), they are able to bind to epitopes within clefts (3) that are more difficult to reach for larger antibodies. Both their single-domain nature and their lack of glycosylation allow them to be produced at high levels using bacterial expression systems.Motivated by searches for potential antiviral agents, VHHs that specifically bind to and neutralize poliovirus type 1 were recently selected and identified (4) and were further characterized (5, 6). In a previous publication (5), we showed that the binding footprints of two VHHs (PVSP6A and PVSP29F) on the poliovirus surface overlap extensively with the footprint of the poliovirus receptor (PVR; also called CD155) and that they produce well-ordered icosahedrally symmetric complexes. It was also shown that the mechanisms of neutralization operate at multiple stages of the infection process and that all of the neutralizing VHHs stabilize the virus to prevent viral expansion (5), which is an essential step in the infection of cells.As a member of the Picornaviridae family, poliovirus is a naked virus comprised only of a protein capsid surrounding its positively single-stranded RNA. The capsid has a pseudo Tϭ3 icosahedral surface with 60 copies of each of four viral proteins (VPs), VP1 to VP4, of which VP4, the smalles...

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An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell transmission

et al. 2013

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Severe liver disease caused by chronic hepatitis C virus is the major indication for liver transplantation. Despite recent advances in antiviral therapy, drug toxicity and unwanted side effects render effective treatment in liver-transplanted patients a challenging task. Virus-specific therapeutic antibodies are generally safe and well-tolerated, but their potential in preventing and treating hepatitis C virus (HCV) infection has not yet been realized due to a variety of issues, not least high production costs and virus variability. Heavy-chain antibodies or nanobodies, produced by camelids, represent an exciting antiviral approach; they can target novel highly conserved epitopes that are inaccessible to normal antibodies, and they are also easy to manipulate and produce. We isolated four distinct nanobodies from a phage-display library generated from an alpaca immunized with HCV E2 glycoprotein. One of them, nanobody D03, recognized a novel epitope overlapping with the epitopes of several broadly neutralizing human monoclonal antibodies. Its crystal structure revealed a long complementarity determining region (CD3) folding over part of the framework that, in conventional antibodies, forms the interface between heavy and light chain. D03 neutralized a panel of retroviral particles pseudotyped with HCV glycoproteins from six genotypes and authentic cell culture-derived particles by interfering with the E2-CD81 interaction. In contrast to some of the most broadly neutralizing human anti-E2 monoclonal antibodies, D03 efficiently inhibited HCV cell-to-cell transmission. Conclusion: This is the first description of a potent and broadly neutralizing HCV-specific nanobody representing a significant advance that will lead to future development of novel entry inhibitors for the treatment and prevention of HCV infection and help our understanding of HCV cell-to-cell transmission. (HEPATOLOGY 2013;58:932-939) A n estimated 180 million people worldwide are infected with hepatitis C virus (HCV). Chronic infection is frequent and often leads to progressive liver disease, with chronic HCV infection being the leading indication for liver transplantation.1 HCV exhibits significant genetic diversity, and at least six major genotypes, which differ by up to 30% in their nucleotide sequence, have been identified.2 Within an infected individual, the virus exists as a population of genetically related variants or quasispecies. This contributes to viral persistence by facilitating escape from antiviral selection pressures, 3 with significant implications for the design of antiviral therapeutics and vaccines.The standard treatment for chronic HCV infection is a combination of pegylated interferon-a and

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In vitro antiviral activity of single domain antibody fragments against poliovirus

Cited by 40 publications

References 29 publications

Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State

Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State

Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site

An alpaca nanobody inhibits hepatitis C virus entry and cell-to-cell transmission

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