key: cord-0887522-dsog1b0v authors: Gransagne, Marion; Aymé, Gabriel; Brier, Sébastien; Chauveau-Le Friec, Gaëlle; Meriaux, Véronique; Nowakowski, Mireille; Dejardin, François; Levallois, Sylvain; Dias de Melo, Guilherme; Donati, Flora; Prot, Matthieu; Brûlé, Sébastien; Raynal, Bertrand; Bellalou, Jacques; Goncalves, Pedro; Montagutelli, Xavier; Di Santo, James P.; Lazarini, Françoise; England, Patrick; Petres, Stéphane; Escriou, Nicolas; Lafaye, Pierre title: Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein date: 2021-10-20 journal: J Biol Chem DOI: 10.1016/j.jbc.2021.101290 sha: 9b701068d530250d118a41ae04daf8446d22a743 doc_id: 887522 cord_uid: dsog1b0v The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus, and are required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, 7 VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs. . NTD and CTD are both able to bind RNA (7, 8) , while CTD also serves as a dimerization domain (9) . Despite many studies, the mechanism by which the RNA genome is encapsidated by N has not been fully unraveled. Indeed, the structure of full-length N is not known probably due to the flexibility of the LKR region. The structures of the NTD and the CTD have only been determined by X-ray crystallography for MERS-CoV (10, 11) and very recently for SARS-CoV-2 (12) . The interaction of the NTD with RNA has also been recently characterized by nuclear magnetic resonance (NMR) spectroscopy (13) . and for at least 21 days at 4°C ( Figure 1C ). Finally, two distinct protein species were detected by sedimentation velocity analysis: the main one (96%) at a sedimentation coefficient of 3.6S compatible with a dimeric organization and the minor one (5%) at 5.5S compatible with a tetrameric organization ( Figure 1D ). Altogether, these data reveal that N is stable at 4°C and mainly forms a dimer in solution. Selection and characterization of VHHs and NTD B6-1 the lowest (46.5 nM). An ELISA was then performed on infected and uninfected cell extracts ( Figure 2C ) The two VHHs selected against NTD, namely NTD B6-1 and NTD E4-3, interacted with distinct and non-overlapping epitopes. As shown in Figure (#4, #5, #7, #12, #14, #22, #30, #45, #47, #58, #60, #64, #67). We observed a good correlation between the 2 techniques ( Several variants have emerged recently B.1.1.7/alpha, B.1.351/beta and P1/gamma and have spread in multiple countries due to increased transmission (44, 45) . These variants of concern harbour mutations in the spike but also in the N protein, which could affect their detection in antibody-based tests. Therefore, we analyzed the binding of VHH G9-1 and VHH NTD E4-3 on these variants. We tested the ability of VHHs NTD E4-3 and G9-1 to detect the N protein on fixed tissues. Mice were infected with the B.1.351 and P1 variants as described in (46) The VHHs were monitored at 20°C just after their purification. Polyacrylamide Gel electrophoresis (PAGE) was performed using NuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) according to the manufacturer's instructions. PageRuler Prestained Protein ladder was used as molecular weight maker and Instant Blue (Expedeon UK) was used to stain the SDS-PAGE gel. Recombinant N was diluted to 0.2 µM in 0.15 % formic acid (pH 2.5). 50 µL (10 pmol The blood of the immunized animal (about 300 ml) was collected and the peripheral blood lymphocytes were isolated by centrifugation on a Ficoll (Cytiva, Velizy, France) discontinuous gradient and stored at -80 • C until further use. Total RNA and cDNA were obtained as previously described (52) . A nested PCR was performed with IgG-specific primers designed in our lab. In the first step, five sets of PCR primers were used to amplify the VH-CH1-CH2 and VHH-CH2 fragments. The bands corresponding to the VHH-CH2 regions were purified on an agarose gel. Next, VHH regions were specifically re-amplified with three sets of VHH-specific PCR primers The VHHs were assessed for quality using the protocol described above for N. The VHHs were biotinylated using the EZ-linkSulfo-NHS-biotin kit (Thermo) according to manufacturer's instructions. A modified version of a standard ELISA was used to test for the presence of VHH. Maxisorp Nunc-Immuno plates (Thermo Scientific) were coated with 1 μg/ml of recombinant The Rapid SARS-CoV-2 Antigen Test Card (MP biomedicals) was used for the detection of N according to manufacturer's instructions. Epitope mapping by hydrogen deuterium exchange mass spectrometry. A summary of the main HDX-MS experimental conditions is provided in Table S1 (54) . The quality and purity of N were assessed by intact mass analysis (Figure 1 ). All labeling were performed at room temperature in deuterated PBS 1X buffer, pD 7.4 (labeling buffer), unless specified. Table S1 ). The MEMHDX software (56) was used to visualize and statistically validate HDX-MS datasets (Wald test, false discovery rate of 1%, biological threshold sets to 3%, Table S1 ). Experiments were performed using a Biacore T200 instrument (GE Healthcare) equilibrated at 25°C in SPR buffer (PBS-300mM NaCl containing 0.1% Tween-20, 0.2mg/ml BSA and 100µM EDTA). Approximately 500 RU (1RU≈1 pg.mm -2 ) of N were captured non-covalently on an NiCl2loaded NTA sensor chip (GE Healthcare). VHHs were then injected at 30µl/min for 300s (E10-3, D12-3, NTD B6-1 and NTD E4-3) or 700s (E7-2, G9-1 and H3-3) to monitor the association of the VHH-N complexes, after which SPR buffer was injected for another 300s or 1200s to monitor the dissociation of the complexes. Finally, the surface of the sensor chip was regenerated by injecting sequentially EDTA 0.5M and SDS 0.1% for 60s. Association and dissociation profiles were analyzed with the BiacoreT200 evaluation software, assuming a 1:1 interaction, which allowed to determine the association (kon) and dissociation (koff) rates of the interactions, as well as their equilibrium constants (Kd). Immunofluorescence assays Mouse experiments on variants of concern were supported by ANR grants HUMOCID (ANR-20-COVI-0028-01) and IBEID (ANR-10-LABX-62-IBEID) and by the EU Horizon 2020 RECOVER The authors declare that they have no conflicts of interest with the contents of this article. Figure 1a shows the SDS Page gel with lanes 5 to 12 representing the eluted fractions containing the purified SARS-CoV-2 N and lane 13 to 18 are separated contaminants; Figure 1b represents the intact mass measurement. The measured molecular weight (48 752 .80 +/-1.96 Da) is consistent with the expected average mass calculated from the full-length SARS-CoV-2 N primary sequence (48 752.13 Da, m = +0.67 Da (+13.7 ppm)) thereby confirming the structural integrity of the protein; Figure 1c shows one main homogeneous population by DLS with a hydrodynamic radius of 6 nm. No aggregates are detectable at 37°C; Figure 1d represents the AUC measurement where 96% of the sample is under a dimeric form. J o u r n a l P r e -p r o o f A) Binding of the different VHHs to SARS-CoV-2 recombinant Nucleoprotein determined by ELISA. N was coated at 1 µg/ml and VHHs at different concentrations were then added. B) Real-time monitoring of the VHH/N interaction by SPR. The determined kinetic parameters of the VHHS are provided in Table 1 . C) Binding of the VHHs by ELISA on cell extracts. The VHH concentration leading to maximal difference obtained between infected and uninfected cell extracts are indicated by grey variations. a) Linear representation (Left) of full-length SARS-CoV-2 N with NTD in gray and CTD in light blue. The cartoon representations of the NTD domain (pdb # 7CDZ) and the CTD dimer (pdb # 7CE0) are shown on the right panel, with one CTD monomer colored light blue and the other colored black. The RNA-binding residues identified by NMR are also reported on the linear (Left, blue bars) and the cartoon representations of the NTD domain (Right, blue spheres). b,c) Comparison of the epitopes identified in the CTD (b) and the NTD (c) domains by HDX-MS. Red and orange patches correspond to regions were major and minor reductions in solvent accessibility were observed upon VHH binding. HDX-MS results are mapped onto the cartoon and surface representations of the NTD and CTD domains. Representative staining with biotinylated VHHs at 1ug/mL of subconfluent layer of FRhK4 infected cells. A rabbit antibody against the SARS-CoV-2 N was used as a control of the infection. 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The differences in uptake between the apo-and VHH-bound states were calculated for each peptide and time point and plotted as a function of peptide position. A positive uptake difference is indicative of a VHH-induced protective effect on the exchangeable amide hydrogens. Peptides displaying statistically significant uptake differences were removed from the statistical analysis due to either poor fitting quality to the Mixed Effect Model or poor MS signal Immunofluorescence labeling of SARS-CoV-2 virus in the lung of infected syrian Hamster Representative staining of lung slices with biotinylated VHHs at 1/500. Scales bar: 50 µm We want to thank Nicolas Wolff and Baptiste Colcombet-Cazenave for their technical help for the production of the different VHHs.The strain BetaCoV/France/IDF0372/2020 was supplied by the National Reference Centre for Respiratory Viruses hosted by Institut Pasteur (Paris, France) and headed by Pr. Sylvie van derWerf. The human sample from which strain BetaCoV/France/IDF0372/2020 was isolated was provided by Dr. X. Lescure and Pr. Y. Yazdanpanah from the Bichat Hospital, Paris, France.Moreover, the strain BetaCoV/France/IDF0372/2020 was supplied through the European Virus Archive goes Global (Evag) platform, a project that has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 653316.We are grateful to the National Reference Center for Respiratory Viruses for providing the variants of concern used in mouse experiments, to Sylvie van der Werf and Etienne Simon-Lorière for support and scientific advice, and to Grégory Jouvion for histological evaluation. We thank the Institut Pasteur Histology platform for histological slides preparation. Mouse experiments on variants of concern were supported by ANR grants HUMOCID (ANR-20-COVI-0028-01) and IBEID (ANR-10-LABX-62-IBEID) and by the EU Horizon 2020 RECOVER project (No. 101003589). P.G. and J.P.D. received support from the ANR grant 'COVARIMM'.The authors would like to thank the DIM 1HEALTH région Ile-de-France scheme for funding the Centrifection project that allowed to purchase the Optima ultracentrifuge. The CACSICE Equipex ANR-11-EQPX-0008 is acknowledged.