Characterisation of nanobodies directed against emerging viruses

The emergence of new viral pathogens, such as SARS-CoV-2, or re-emergence of known pathogens, like CHIKV, point out the need for further understanding of the biology behind viruses, as well as the urgent need for the development of therapeutic and diagnostic tools. Nanobodies, small antigen-binding fragments derived from camelid heavy-chain antibodies, have gained attention for their use in viral research due to their wide range of applications: from the study of protein-protein interactions, uncovering of new viral targets, to the generation of new diagnostic tools or therapeutics. In paper I, we isolated a nanobody, Ty1, targeting the receptor binding domain (RBD) of SARS-CoV-2. We showed the ability of Ty1 to neutralise SARS-CoV-2 pseudotyped lentivirus potently (IC50 of 0.77 μg mL-1). The highly neutralising ability of Ty1 was likely due to its ability to bind the RBD in the ‘up’ and ‘down’ conformations, causing direct blocking to the cellular receptor and steric hindrance, respectively. Moreover, staining of SARS-CoV-2-infected cells with Ty1 confirmed its high specificity. In paper II we made use of a novel and rapid strategy to create nanobody multimers. We first functionalised the nanobodies using sortase A ligation to attach click chemistry functional groups. Then, the functionalised nanobodies were used to create C-to-C terminal bi- and tetravalent nanobody constructs by Cu-free strain-promoted azide-alkyne click chemistry (SPAAC). The bivalent and tetrameric nanobody constructs showed an increased potency with respect to the monomeric Ty1 of 150-fold and 4000-fold, respectively. This was true both for SARS-CoV-2 spike pseudotyped lentivirus and infectious SARS-CoV-2. In paper III, we generated nanobodies targeting the spike complex of CHIKV. We used a combinatory immunisation strategy with a cDNA prime followed by a protein boost. The CHIKV spike complex is formed by homotrimers of heterodimers of the E1 and E2 proteins. While E2 binds to the cellular receptor, E1 is responsible for the fusion of viral and cellular membranes, both essential steps of viral entry. We made use of a bivariate mining approach coupled to NGS and calculation of enrichment (fold difference in frequency between basal and enriched libraries) for the quick selection of nanobodies targeting either protein. We identified 12 nanobodies that detected cells infected with the 3 CHIKV lineages (ECSA, Asian and WA). Surprisingly, neutralisation of the ECSA and Asian genotypes was below 50% for all tested nanobodies, while 2 nanobodies, Dy010 and Dy059 could neutralise the WA lineage above 50% with PRNT50 values of 563 and 722 nM, respectively. Fusion to an Fc fragment produced an increase in potency of 130- and 63-fold for Dy010 and Dy059. Moreover, 4 of the nanobodies, Dy009, Dy025, Dy027 and Dy201 cross-reacted with other alphaviruses including ONNV, RRV and SFV, while one nanobody, Dy007, showed great specificity for CHIKV. These nanobodies expand the toolbox for research of this important human pathogen and could form a basis for the development of therapeutic or diagnostic tools

Mutation of CD2AP and SH3KBP1 binding motif in alphavirus nsP3 hypervariable domain results in attenuated virus

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3

A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo

Experiments with replication-competent SARS-CoV-2 were performed in the Biomedicum BSL3 core facility, Karolinska Institutet. We thank Jonas Klingström for providing Calu-3 cells and sharing the Swedish SARS-CoV-2 isolate, and Alex Sigal from the Africa Health Research Institute for providing the beta variant (B.1.351/501Y.V2) isolate. We thank Penny Moore and the NICD (South Africa) for providing the B.1.351/beta variant spike plasmid, which was generated using funding from the South African Medical Research Council. We gratefully acknowledge the G2P-UK National Virology consortium funded by MRC/UKRI (grant ref: MR/W005611/1.) and the Barclay Lab at Imperial College for providing the B.1.617.2 spike plasmid. All cryo-EM data were collected in the Karolinska Institutet’s 3D-EM facility. We thank Agustin Ure for assistance with figure generation and Tomas Nyman (Protein Science Facility at KI) for providing access to SPR instruments. L.H. was supported by the David och Astrid Hageléns stiftelse, the Clas Groschinskys Minnesfond and a Jonas Söderquist’s scholarship. This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 101003653 (CoroNAb), to B.M. and G.M.M. B.M.H. is supported by the Knut and Alice Wallenberg Foundation (KAW 2017.0080 and KAW 2018.0080). The work was supported by project grants from the Swedish Research Council to E.S. (2020-02682), B.M.H. (2017-6702 and 2018-3808), B.M. (2018-02381) and to G.M.M. (2018-03914 and 2018-03843). E.S. is supported by Karolinska Institutet Foundation Grants, National Molecular Medicine Program Grants, and the grants from the SciLifeLab National COVID-19 Research Program, financed by the Knut and Alice Wallenberg Foundation. We thank National Microscopy Infrastructure, NMI (VR-RFI 2016-00968).N

Picomolar SARS-CoV-2 Neutralization Using Multi-Arm PEG Nanobody Constructs

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range

An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

Here, Hanke et al. immunize an alpaca with SARS-CoV-2 spike protein domains and identify a nanobody that binds the receptor binding domain of spike in both the up and down conformations and sterically hinders ACE2 engagement

High-content imaging, immunoblot and immunofluorescence data related to: Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH

G3BP is the central node within stress-induced protein–RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.See README file for full details. general notes on deposited file formats:*.TIF or *.tif image files can be opened using standard software for images (ImageJ, Photoshop,...)*.xlsx can be opened using Microsoft Excel*.cpproj project files can be opened in Cellprofiler (https://cellprofiler.org/) High-throughput (HT) imaging datasets can be analysed by opening the deposited Cellprofiler pipelines in Cellprofiler (https://cellprofiler.org/).TIF files can also be viewed using any image viewing software (https://imagej.nih.gov/ij/download.html). Scripts to analyse the Cellprofiler output, and additional information is found on Github: https://github.com/derpaule/RSOB-22-0369 Funding provided by: Swedish Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004359Award Number: 2018-03843Funding provided by: Swedish Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004359Award Number: 2018-03914Funding provided by: Swedish Cancer FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100012538Award Number: CAN 2018/829Funding provided by: Swedish Cancer FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100012538Award Number: CF 2018/603Funding provided by: Swedish Research CouncilCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100004359Award Number: 2018-02874Funding provided by: Svenska Sällskapet för Medicinsk ForskningCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100003748Award Number: P16-0083Funding provided by: Stiftelsen Clas Groschinskys MinnesfondCrossref Funder Registry ID: http://dx.doi.org/10.13039/501100006350Award Number: M2002Funding provided by: Swedish Cancer FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100012538Award Number: 2018/603Funding provided by: Swedish Cancer FoundationCrossref Funder Registry ID: http://dx.doi.org/10.13039/100012538Award Number: 2018/829Funding provided by: National Institutes of HealthCrossref Funder Registry ID: http://dx.doi.org/10.13039/100000002Award Number: GM126901The deposited ZIP folders are described in following groups: @HTimages_1 high-throughput imaging datasets of cellular GFP-G3BP condensation assays presented in Figure 5 of the manuscript ZIP folders: img_plate2171.zip / img_plate2185.zip / img_plate21691.zip @HTimages_2 high-throughput imaging datasets of cellular GFP-Caprin condensation assays presented in Figure 7 of the manuscript. ZIP folders: img_plate1734.zip / img_plate1735.zip / img_plate1737.zip / img_plate1740.zip @HTimages_3 high-throughput imaging datasets of in-vitro reconstituted GFP-G3BP condensation assays (LLPS), presented in Figure 5 of the manuscript. ZIP folders: LLPS_GFP-G3BP1.zip @HTimages_4 high-throughput imaging datasets of in-vitro reconstituted GFP-Caprin condensation assays (LLPS), presented in Figure 7 of the manuscript. ZIP folders: LLPS_GFP-Caprin-1.zip @CPpipelines_1 Cellprofiler pipelines to analyse @HTimages_1 ZIP folders: pipelines_G3BP1.zip t @CPpipelines_2 Cellprofiler pipelines to analyse @HTimages_2 ZIP folders:pipelines_Caprin1.zip @Images_1 raw image files used for presentation of Blots or IF data in main and supplemental figures ZIP folders: Images_Blots.zip The collection of datasets is described in the methods section of the associated manuscript (ms, https://royalsocietypublishing.org/doi/10.1098/rsob.220369) @HTimages_1/@HTimages_2: ms section: 4.9.1. High-content microscopy: "Images were recorded with a Molecular Devices ImageXpress Micro microscope, equipped with a 20x or 40x objective, and illuminated with a mercury lamp and standard filters for DAPI, FITC, Cy3 and Cy5. Images were captured using a four-megapixel sCMOS digital camera with the manufacturer's software MetaXpress, and raw TIF files were analyzed using CellProfiler (CP), ImageJ and Rstudio." @HTimages_3/@HTimages_4 ms section: 4.9.4. In vitro reconstituted condensate assays "Images were taken with a Supercontinuum Confocal Leica TCS SP5 X, equipped with a pulsed white light laser and a Leica HCX PL Apo 63x/1.40 oil objective. " @Images_1 ms section 4.8. Immunoprecipitation and immunoblotting ms section: 4.9. Immunofluorescence analysi

Large scale discovery of coronavirus-host factor protein interaction motifs reveals SARS-CoV-2 specific mechanisms and vulnerabilities

Viral proteins make extensive use of short peptide interaction motifs to hijack cellular host factors. However, most current large-scale methods do not identify this important class of protein-protein interactions. Uncovering peptide mediated interactions provides both a molecular understanding of viral interactions with their host and the foundation for developing novel antiviral reagents. Here we describe a viral peptide discovery approach covering 23 coronavirus strains that provides high resolution information on direct virus-host interactions. We identify 269 peptide-based interactions for 18 coronaviruses including a specific interaction between the human G3BP1/2 proteins and an ΦxFG peptide motif in the SARS-CoV-2 nucleocapsid (N) protein. This interaction supports viral replication and through its ΦxFG motif N rewires the G3BP1/2 interactome to disrupt stress granules. A peptide-based inhibitor disrupting the G3BP1/2-N interaction dampened SARS-CoV-2 infection showing that our results can be directly translated into novel specific antiviral reagents