Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

Hinz, Andreas; Hulsik, D. Lutje; Forsman, Anna; Koh, Willie; Belrhali, Hassan; Gorlani, Andrea; Haard, Hans de; Weiss, Robin A.; Verrips, Theo; Weissenhorn, Winfríed

doi:10.1371/journal.pone.0010482

Cited by 36 publications

(41 citation statements)

References 55 publications

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“…One of the most direct mechanisms is blocking the receptor binding sites. Such neutralizing mAbs and Nanobodies were previously identified for influenza virus, HIV, herpes simplex virus, rhinovirus, and others [35][36][37][38][39][40][41]. For example, in the case of HIV, with the aid of an extra long CDR3 loop, the neutralizing Nanobody D7 effectively competed for the CD4 binding site on gp120 protein [42].…”

Section: Discussionmentioning

confidence: 99%

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Koromyslova

Hansman

2018

Biophysical Journal

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Norovirus is the leading cause of gastroenteritis worldwide. Despite recent developments in norovirus propagation in cell culture, these viruses are still challenging to grow routinely. Moreover, little is known on how norovirus infects the host cells, except that histo-blood group antigens (HBGAs) are important binding factors for infection and cell entry. Antibodies that bind at the HBGA pocket and block attachment to HBGAs are believed to neutralize the virus. However, additional neutralization epitopes elsewhere on the capsid likely exist and impeding the intrinsic structural dynamics of the capsid could be equally important. In the current study, we investigated a panel of Nanobodies in order to probe functional epitopes that could trigger capsid rearrangement and/ or interfere with HBGA binding interactions. The precise binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) were identified using X-ray crystallography. We showed that these Nanobodies bound on the top, side, and bottom of the norovirus protruding domain. The impact of Nanobody binding on norovirus capsid morphology was analyzed using electron microscopy and dynamic light scattering. We discovered that distinct Nanobody epitopes were associated with varied changes in particle structural integrity and assembly. Interestingly, certain Nanobody-induced capsid morphological changes lead to the capsid protein degradation and viral RNA exposure. Moreover, Nanobodies employed multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, which included steric obstruction (Nano-14), allosteric interference (Nano-32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only compromised capsid integrity and inhibited VLPs attachment to HBGAs, but also recognized a broad panel of norovirus genotypes with high affinities. Consequently, Nano-26 and Nano-85 have a great potential to function as novel therapeutic agents against human noroviruses. PLOS Author summaryWe determined the binding sites of six novel human norovirus specific Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, Nano-32, and Nano-42) using X-ray crystallography. The unique Nanobody recognition epitopes were correlated with their potential neutralizing capacities. We showed that one Nanobody (Nano-26) bound numerous genogroup II genotypes and interacted with highly conserved capsid residues. Four Nanobodies (Nano-4, Nano-26, Nano-27, and Nano-42) bound to occluded regions on the intact particles and impaired normal capsid morphology and particle integrity. One Nanobody (Nano-14) bound contiguous to the HBGA pocket and interacted with several residues involved in binding HBGAs. We found that the Nanobodies delivered multiple inhibition mechanisms, which included steric obstruction, allosteric interference, and disruption of the capsid stability. Our data suggested that the HBGA pocket might not be an ideal target for drug development, since the surrounding regio...

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Section: Discussionmentioning

confidence: 99%

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Koromyslova

Hansman

2018

Biophysical Journal

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“…They have been used for virus detection of HIV (30,31), vaccinia virus (32), Marburg virus (33), and Dengue virus (34). Nanobodies were shown to block virus attachment to cellular receptors in HIV and influenza virus (35)(36)(37). They also have been evaluated in phase I and II clinical trials in other areas, including oncology, neurology, and immunology (38).…”

Section: Discussionmentioning

confidence: 99%

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

Koromyslova

Hansman

2015

J Virol

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Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ϳ10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability. IMPORTANCEWe characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral.

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“…Other advantages of VHHs include (1) preference for cleft recognition and for binding into active sites [200,201], (2) ease of cloning and expression in bacteria and yeast [202] and (3) excellent stability and solubility [203,204]. These characteristics have led to the development of camelid nanobodies for a number of biological targets [205][206][207][208][209][210][211][212][213], including HIV [199,214,215].…”

Section: Entry/fusion Inhibitorsmentioning

confidence: 99%

“…Furthermore, the A12, D7 and C8 VHHs were found to neutralise a different spectrum of viruses compared to mAb b12 [199,215] and are currently being evaluated for microbicide application.…”

Section: Entry/fusion Inhibitorsmentioning

confidence: 99%

HIV sexual transmission and microbicides

Ariën

Jespers

Vanham

2011

Reviews in Medical Virology

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Pathogens often rely on the contacts between hosts for transmission. Most viruses have adapted their transmission mechanisms to defined behaviours of their host(s) and have learned to exploit these for their own propagation. Some viruses, such as HIV, the human papillomavirus (HPV), HSV-2 and HCV, cause sexually transmitted infections (STIs). Understanding the transmission of particular viral variants and comprehending the early adaptation and evolution is fundamental to eventually inhibiting sexual transmission of HIV. Here, we review the current understanding of the mechanisms of sexual transmission and the biology of the transmitted HIV. Next, we present a timely overview of candidate microbicides, including past, ongoing and future clinical trials of HIV topical microbicides.

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Crystal Structure of the Neutralizing Llama VHH D7 and Its Mode of HIV-1 gp120 Interaction

Cited by 36 publications

References 55 publications

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and Potential Mechanisms of Neutralization

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

HIV sexual transmission and microbicides

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