WO2022069232A1

WO2022069232A1 - Single domain antibodies against the nucleoprotein of sars-cov-2

Info

Publication number: WO2022069232A1
Application number: PCT/EP2021/075398
Authority: WO
Inventors: Pierre Lafaye; Nicolas Escriou; Marion GRANSAGNE; Gabriel AYME
Original assignee: Institut Pasteur
Priority date: 2020-10-02
Filing date: 2021-09-15
Publication date: 2022-04-07

Abstract

Single domain VHH antibodies that bind to the SARS-CoV-2 Nucleoprotein with nanomolar Kd, kits comprising the VHH antibodies, and methods of detecting a SARS-associated coronavirus.

Description

SINGLE DOMAIN ANTIBODIES AGAINST THE NUCLEOPROTEIN OF SARS-COV-2

BACKGROUND

Coronavirus is a well-defined virus family that causes diseases in birds and mammals. To date, 7 human coronaviruses have been identified. Common human coronaviruses, including types 229E and NL63 (both alpha coronaviruses), and OC43 and HKU1 (both beta coronaviruses) usually cause mild to moderate illnesses like the common cold. People around the world commonly get infected with these coronaviruses (1 ). Three epidemic events have been observed and are caused respectively by SARS-CoV-1 , MERS-CoV and SARS-CoV-2, three closely related coronaviruses. SARS-CoV-1 emerged in China in 2002-2003 and spread in this country and is known as SARS epidemic. MERS-CoV caused an epidemic that began in Saudi Arabia in 2012 and was limited in the Middle East and Korea (2). SARS-CoV-2, first isolated in December 2019 in Wuhan, China is a virus very similar to SARS and MERS. SARS-CoV-2 spread from China as a global pandemic, known as the Covid 19 pandemic (3), causing many casualties in the human population through Severe and Acute Respiratory Syndrome.

Coronaviruses are enveloped viruses with a positive-sense RNA genome and with a nucleocapsid of helical symmetry. Coronavirus nucleoproteins (N) localize to the cytoplasm and the nucleolus, a subnuclear structure, in both virus-infected primary cells and in cells transfected with plasmids that express N protein. Coronavirus N protein is required for coronavirus RNA synthesis, and has RNA chaperone activity that may be involved in template switch. Nucleocapsid protein is the most abundant protein of coronavirus. During virion assembly, Nucleoprotein binds to viral RNA and leads to formation of the helical nucleocapsid (for a review, (4)). The coronavirus Nucleoprotein is a homodimer formed by 2 monomers of 48 kDa. Each monomer is organized into two folded domains that are called the N-terminal domain (NTD) and the C-terminal domain (CTD). They are separated by a disordered region (called LKR) containing a serine/arginine stretch which could regulate the functions of N upon phosphorylation (5). NTD and CTD are both capable of RNA binding (6,7) and CTD in addition serves as a dimerization domain (8). 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 owing to the flexibility of the LKR, and the structures of the NTD and the CTD have been explored separately. The structures of the MERS-CoV NTD and CTD have been determined using X-ray crystallography by (9) and (10) respectively and recently the crystal structures of SARS-CoV-2 NTD and CTD have been obtained (11 ). Comparisons with the structures of the C-terminal domains of N from other coronaviruses reveals a high degree of structural conservation despite low sequence conservation, and differences in electrostatic potential at the surface of the protein (12). The interaction of NTD with RNA has been characterized using nuclear magnetic resonance (NMR) spectroscopy (13).

The current situation of the Covid-19 pandemic shows the importance of obtaining reliable solutions for the rapid and specific detection of SARS-CoV-2. Several solutions have been developed in record time. The reference method for diagnosis remains the RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) which allows to detect the RNA of the SARS-CoV-2, from nasopharyngeal, salivary or pulmonary samples. This is an expensive method, requiring the transport of the sample to a well-equipped laboratory and qualified personnel to carry out the analyzes. As the virus can be detected in the body for a short time (typically 7-21 days after infection) (14), diagnostic tests can only be efficient during a very short time window after the onset of symptoms: this means that one must be able to perform a test as soon as the first symptoms appear. It is therefore desirable for diagnostic tests to be widely available and accessible highlighting the importance to develop an antibody-based assay. Moreover, this assay would be useful if it is specific and does not cross-react with common human coronaviruses (229E, NL63, OC43, HKU1). Nucleocapsid protein being the most abundant protein of coronavirus (5) it is of utmost importance to develop antibodies to detect this protein in a diagnostic test. Nucleocapsid protein is a highly immunogenic phosphoprotein. SARS-CoV infection causes a highly restricted, IgG-dominated antibody response that is directed most frequently and predominantly at the N (15). In the diagnosis/screening hCoV-OC43, rabbit polyclonal antibodies demonstrated greater immunoreactivity to the central (LKR) region and CTD than the NTD of N protein in serum samples (16).

Camelids produce two kinds of antibodies: (i) conventional antibodies made of dimers of heavy and light chains and (ii) a class of IgG devoid of light chain and made of dimers of heavy chains only (HC-IgGs) (17). The HC-IgGs comprise two antigen-binding domains (referred to as VHH or nanobodies). VHHs are the smallest available intact antigen binding fragments with a MW of only 15 kDa, 2.5 nm in diameter and ~ 4 nm in height. They act as fully functional binding moieties and are easily produced in high amounts and in active form in E. coli. In addition, they exhibit unique characteristics, such as enlarged complementarity determining regions (CDRs) and the substitution of three to four hydrophobic framework residues (which interact with the VL in conventional antibodies) by more hydrophilic amino acids. To stabilize the enlarged CDRs, VHHs often possess an additional disulfide bond between CDR1 and CDR3 in dromedaries, and CDR2 and CDR3 in llamas (18,19). In particular the extended CDR3 loop can adopt a protruding conformation, which can interact with concave epitopes (20), whereas conventional antibodies recognize only convex or flat structures (19). These unique features allow nanobodies to recognize novel epitopes that are poorly immunogenic for conventional antibodies (21 ). Over the last decades, VHHs have received progressively greater interest due to their specific properties. Indeed, they combine the high affinity and selectivity of conventional antibodies with the advantages of small molecules: in particular, they diffuse more readily into tissues owing to their small size and bind intracellular antigens (22-26) and they are widely used for imaging (for a review, (27)).

VHHs have been raised to numerous viruses (reviewed in (28,29)) including: HIV (30,31 ); Influenza A (32-34); rabies virus (32); Poliovirus (35); Foot and Mouth Disease Virus (18); Rotavirus (36), HCV(37) and recently SARS-CoV1 , MERS-CoV and SARS-CoV-2 spike proteins (38-42). Although VHHs are monovalent they frequently exhibit comparable biological activities to conventional bivalent antibody molecules (35). For example VHHs can bind to the SARS-CoV-2 spike protein and prevent infection of cells (43,44).

There is a need in the art for reagents and methods for the detection of SARS-CoV-2 in a sample. This invention meets these and other needs.

SUMMARY OF THE INVENTION

The examples demonstrate the isolation and characterization of ten anti-SARS-CoV-2 alpaca nanobodies raised by immunization of an alpaca with SARS-CoV-2 nucleoprotein. These VHHs recognize either NTD or CTD with an affinity at the nanomolar level. The epitope mapping by Hydrogen Deuterium eXchange-Mass spectrometry (HDX-Ms) has been performed. Some of these VHHs are able to recognize SARS-CoV-2 virus in infected cells or in infected hamster tissues. An ELISA sandwich has been performed by using one anti-NTD VHH and one anti-CTD VHH to detect the nucleoprotein in solution and on a permeabilized virus. As low as 20 ng/ml and even as low as 4 ng/ml of nucleoprotein has been detected in solution. No detection of human common coronavirus nucleoproteins has been shown by using this sandwich immunoassay.

Accordingly, in a first aspect this invention provides an isolated single domain VHH antibody that binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 41. The isolated single domain VHH antibody comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively). The number and location of CDR region amino acid residues herein comply with the known CDR numbering criteria such as Kabat (Kabat, EA, etc. 1991 Sequences of Proteins of Immunological Interest, 5th Ed), IMGT (IMGT®: the international ImMunoGeneTics information system® http://www.imgt.org) or Chothia (Chothia C., Lesk A.M. Canonical structures for the hypervariable regions of immunoglobulins. Mol. Biol. 1987;196:901-917. doi: 10.1016/0022-2836(87)90412-8. ), preferably IMGT. In some embodiments the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOS: 11 -20 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 11 -20; the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOS: 21 -30 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 21 -30; and the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOS: 31 -40 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 31 -40.

In some embodiments of the isolated single domain VHH antibody, the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOS: 11 - 20; the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOS: 21 -30; and the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOS: 31 -40.

In some embodiments of the isolated single domain VHH antibody, the single domain VHH antibody comprises an amino acid sequence that is at least 90%, 91%, 92%, 93% or 94% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69. In some embodiments of the isolated single domain VHH antibody, the single domain VHH antibody comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69.

In some embodiments of the isolated single domain VHH antibody, the single domain VHH antibody comprises an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69. In some embodiments of the isolated single domain VHH antibody, the single domain VHH antibody consists of an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69.

In some embodiments the isolated single domain VHH antibody binds a protein comprising the amino acid sequence of the SARS-CoV-2 Nucleoprotein of NCBI

QHO62884.1. In some embodiments the isolated single domain VHH antibody binds to the C-terminal domain (CTD) of SARS-CoV-2 Nucleoprotein, as for example D12-3 having the amino acid sequence SEQ ID NO: 1 , H3-3 having the amino acid sequence SEQ ID NO: 2, E7-

2 having the amino acid sequence SEQ ID NO: 3, E7-2bis having the amino acid sequence SEQ ID NO: 69, G9-1 having the amino acid sequence SEQ ID NO: 4 or E10-

3 having the amino acid sequence SEQ ID NO: 5.

In some embodiments the isolated single domain VHH antibody binds to the N-terminal domain (NTD) of SARS-CoV-2 Nucleoprotein, as for example C7-1 having the amino acid sequence SEQ ID NO: 6, F11 -1 having the amino acid sequence SEQ ID NO: 7, E4-3 having the amino acid sequence SEQ ID NO: 8, H7-1 having the amino acid sequence SEQ ID NO: 9 or B6-1 having the amino acid sequence SEQ ID NO: 10.

In some embodiments the isolated single domain VHH antibody binds to the SARS-CoV- 2 Nucleoprotein with a nanomolar KD. For example, it may bind with a KD of as low as 50 nM, 40nM, 30 nM, 20 nM, 10, nM, 5 nM, 1 nM, 0.5 nM, or even as low as 0.1 nM.

In some embodiments, it is also provided a fusion protein comprising one of the single domain VHH antibodies, fused at its C-terminus to a Fc fragment, preferably a human Fc fragment. In some embodiments the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 60-61 .

In some embodiments, the isolated single domain VHH antibody is in the form of a monomer.

In some embodiments multimeric VHH antibody comprising at least two single domain VHH antibodies according to the invention and/or fusion protein according to the invention are provided. The multimeric VHH antibody may be homodimers or heterodimers. In some embodiments, it is also provided a dimeric VHH antibody comprising two fusion proteins according to the invention. In some embodiments the dimeric VHH antibody comprises two copies of the same fusion protein. In other embodiments the dimeric VHH antibody comprises two different fusion proteins.

In some embodiments, the single domain VHH antibody according to the invention, the fusion protein according to the invention and/or the multimeric VHH antibody according to the invention further comprises a label.

In some embodiments, the single domain VHH antibody according to the invention and/or the fusion protein according to the invention and/or the multimeric VHH antibody according to the invention is covalently attached to a substrate.

In another aspect an isolated nucleic acid sequence that encodes the single domain VHH antibody or fusion protein or multimeric VHH antibody is provided.

In another aspect a recombinant cell comprising the isolated nucleic acid sequence is provided.

In another aspect methods of producing the single domain VHH antibody or the fusion protein or the multimeric VHH antibody are provided. The methods comprise culturing the recombinant cell comprising the isolated nucleic acid sequence that encodes the single domain VHH antibody or the fusion protein or the multimeric VHH antibody under conditions sufficient for production of the single domain VHH antibody or the fusion protein or the multimeric VHH antibody.

In another aspect, methods for detection of a SARS-associated coronavirus in a biological sample are provided. The methods may comprise providing a single domain VHH antibody according to the invention and/or a fusion protein according to the invention and/or a multimeric VHH antibody according to the invention; providing a biological sample from a subject suspected to be infected with a SARS-associated coronavirus; contacting the single domain VHH antibody and/or the fusion protein and/or the multimeric VHH antibody with the biological sample; and visualizing the antigen-antibody complexes formed. In some embodiments the methods comprise an ELISA, lateral flow immunoassay, bead-based immunoassay, or multiplex bead-based immunoassay.

In some embodiments, the method for detection of a SARS-associated coronavirus in a biological sample comprise providing a first antibody directed against the SARS-CoV-2 Nucleoprotein, attached to a solid support; providing a biological sample from a subject suspected to be infected with a SARS-associated coronavirus; contacting the solid support with the biological sample under conditions sufficient to allow formation of first antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein in the biological sample and the antibody attached to the solid support to form first antigenantibody complexes; contacting the solid support with a second antibody directed against the SARS-CoV-2 Nucleoprotein under conditions sufficient to allow formation of second antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein and the second antibody; and visualizing the second antigen-antibody complexes wherein at least one of said first and second antibody is a single domain VHH antibody according to the invention and/or a fusion protein according to the invention and/or a multimeric VHH antibody according to the invention. In some embodiments the method for detection of a SARS-associated coronavirus in a biological sample comprise providing a first single domain VHH antibody according to the invention and/or a first fusion protein according to the invention and/or a first multimeric VHH antibody according to the invention, attached to a solid support; providing a biological sample from a subject suspected to be infected with a SARS-associated coronavirus; contacting the solid support with the biological sample under conditions sufficient to allow formation of first antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein in the biological sample and the VHH antibody and/or the fusion protein and/or the multimeric VHH antibody attached to the solid support to form first antigen-antibody complexes; contacting the solid support with a second single domain VHH antibody according to this invention and/or a second fusion protein according to the invention and/or a second multimeric VHH antibody according to the invention under conditions sufficient to allow formation of second antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein and the second single domain VHH antibody and/or the fusion protein and/or the multimeric VHH antibody; and visualizing the second antigen-antibody complexes. In some embodiments, the second single domain VHH antibody according to this invention and/or the second fusion protein according to the invention and/or the second multimeric VHH antibody according to the invention is labeled and visualizing the second antigen-antibody complexes comprises visualizing the label. In some embodiments, the first single domain VHH antibody is an anti-NTD VHH antibody, as for example C7-1 having the amino acid sequence SEQ ID NO: 6, F11 -1 having the amino acid sequence SEQ ID NO: 7, E4-3 having the amino acid sequence SEQ ID NO: 8, H7-1 having the amino acid sequence SEQ ID NO: 9 or B6-1 having the amino acid sequence SEQ ID NO: 10, and the second single domain VHH antibody is an anti-CTD VHH antibody, as for example D12-3 having the amino acid sequence SEQ ID NO: 1 , H3-3 having the amino acid sequence SEQ ID NO: 2, E7-2 having the amino acid sequence SEQ ID NO: 3, E7-2bis having the amino acid sequence SEQ ID NO: 69, G9-1 having the amino acid sequence SEQ ID NO: 4 or E10-3 having the amino acid sequence SEQ ID NO: 5. In some embodiments, the first single domain VHH antibody is an anti-CTD VHH antibody, as for example D12-3 having the amino acid sequence SEQ ID NO: 1 , H3-3 having the amino acid sequence SEQ ID NO: 2, E7-2 having the amino acid sequence SEQ ID NO: 3, E7-2bis having the amino acid sequence SEQ ID NO: 69, G9-1 having the amino acid sequence SEQ ID NO: 4 or E10-3 having the amino acid sequence SEQ ID NO: 5 and the second single domain VHH antibody is an anti-NTD VHH antibody, as for example C7-1 having the amino acid sequence SEQ ID NO: 6, F11 -1 having the amino acid sequence SEQ ID NO: 7, E4-3 having the amino acid sequence SEQ ID NO: 8, H7-1 having the amino acid sequence SEQ ID NO: 9 or B6-1 having the amino acid sequence SEQ ID NO: 10. In some embodiments the first single domain VHH antibody is VHH NTD E4-3 having the amino acid sequence SEQ ID NO: 8 or a variant thereof and the second single domain VHH antibody is VHH G9-1 having the amino acid sequence SEQ ID NO: 4 or a variant thereof. In some embodiments the first fusion protein according to the invention is VHH E4-3 fused to human Fc having the amino acid sequence SEQ ID NO: 61 or a variant thereof. In some embodiments the second fusion protein according to the invention is VHH G9-1 fused to human Fc having the amino acid sequence SEQ ID NO: 60 or a variant thereof. In some embodiments the first fusion protein according to the invention is VHH E4-3 fused to human Fc having the amino acid sequence SEQ ID NO: 61 or a variant thereof and the second fusion protein according to the invention is VHH G9-1 fused to human Fc having the amino acid sequence SEQ ID NO: 60 or a variant thereof. In a preferred embodiment of the methods for detection of a SARS-associated coronavirus the method detects SARS-CoV-2.

In a preferred embodiment of the methods for detection of a SARS-associated coronavirus the method detects SARS-CoV-2 Nucleoprotein in a sample.

In some embodiments, the detection sensitivity of the method allows detection of as low as 20 ng/ml and even as low as 4 ng/ml of the SARS-CoV-2 Nucleoprotein in a sample.

In another aspect a kit for detection of a SARS-associated coronavirus in a biological sample is provided. In some embodiments the kit comprises a single domain VHH antibody according to the invention and/or a fusion protein according to the invention and/or a multimeric VHH antibody according to the invention. In some embodiments of the kit, the single domain VHH antibody and/or fusion protein and/or multimeric VHH antibody further comprises a label. In some embodiments of the kit, the single domain VHH antibody and/or fusion protein and/or multimeric VHH antibody is covalently attached to a solid support. In some embodiments the kit comprises a first antibody directed against the SARS-CoV-2 Nucleoprotein, attached to a solid support; and a second antibody, attached to a label wherein at least one of said first or second antibody is a single domain VHH antibody according to the invention and/or a fusion protein according to the invention and/or a multimeric VHH antibody according to the invention. In some embodiments the kit comprises a first single domain VHH antibody according to the invention and/or a first fusion protein according to the invention and/or a first multimeric VHH antibody according to the invention, attached to a solid support; and a second single domain VHH antibody according to the invention and/or a second fusion protein according to the invention and/or a second multimeric VHH antibody according to the invention, attached to a label. In some embodiments, the first single domain VHH antibody is an anti-NTD VHH antibody, as for example C7-1 having the amino acid sequence SEQ ID NO: 6, F11 -1 having the amino acid sequence SEQ ID NO: 7, E4-3 having the amino acid sequence SEQ ID NO: 8, H7-1 having the amino acid sequence SEQ ID NO: 9 or B6-1 having the amino acid sequence SEQ ID NO: 10, and the second single domain VHH antibody is an anti-CTD VHH antibody, as for example D12-3 having the amino acid sequence SEQ ID NO: 1 , H3-3 having the amino acid sequence SEQ ID NO: 2, E7-2 having the amino acid sequence SEQ ID NO: 3, E7-2bis having the amino acid sequence SEQ ID NO: 69, G9-1 having the amino acid sequence SEQ ID NO: 4 or E10-3 having the amino acid sequence SEQ ID NO: 5. In some embodiments, the first single domain VHH antibody is an anti-CTD VHH antibody, as for example D12-3 having the amino acid sequence SEQ ID NO: 1 , H3-3 having the amino acid sequence SEQ ID NO: 2, E7-2 having the amino acid sequence SEQ ID NO: 3, E7-2bis having the amino acid sequence SEQ ID NO: 69, G9-1 having the amino acid sequence SEQ ID NO: 4 or E10-3 having the amino acid sequence SEQ ID NO: 5, and the second single domain VHH antibody is an anti-NTD VHH antibody, as for example C7-1 having the amino acid sequence SEQ ID NO: 6, F11 -1 having the amino acid sequence SEQ ID NO: 7, E4-3 having the amino acid sequence SEQ ID NO: 8, H7-1 having the amino acid sequence SEQ ID NO: 9 or B6-1 having the amino acid sequence SEQ ID NO: 10. In some embodiments the first single domain VHH antibody is VHH NTD E4-3 having the amino acid sequence SEQ ID NO: 8 or a variant thereof and the second single domain VHH antibody is VHH G9-1 having the amino acid sequence SEQ ID NO: 4 or a variant thereof. In some embodiments the first fusion protein according to the invention is VHH E4-3 fused to human Fc and has the amino acid sequence SEQ ID NO: 61 or is a variant thereof. In some embodiments the second fusion protein according to the invention is VHH G9-1 fused to human Fc and having the amino acid sequence SEQ ID NO: 60 or a variant thereof. In some embodiments the first fusion protein according to the invention is VHH E4-3 fused to human Fc and having the amino acid sequence SEQ ID NO: 61 or a variant thereof and the second fusion protein according to the invention is VHH G9-1 fused to human Fc and having the amino acid sequence SEQ ID NO: 60 or a variant thereof. In some embodiments the kit further comprises reagents for detecting the label. In some embodiments the kit further comprises a recombinant SARS-CoV-2 Nucleoprotein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : amino acid sequences of the VHHs. The complete sequence of each of the ten VHHs is presented in two rows. The sequences of CDR1 , CDR2, and CDR3 are bolded. The cysteines forming the extra disulfide bond are underlined.

Figure 2: Sequence Identification Numbers for VHHs presented in 1. The table lists sequence identification numbers for the complete VHH sequences (SEQ ID NOS: 1 - 10 and 69), the CDR1 sequences (SEQ ID NOS: 11 -20), the CDR2 sequences (SEQ ID NOS: 21 -30), and the CDR3 sequences (SEQ ID NOS: 31 -40).

Figure 3: Binding analysis of the different VHHs for Nucleoprotein. A) Nucleoprotein has been coated on ELISA plate at 1 pg/ml and VHHs at different concentrations were then added. The binding was revealed with anti-strep tag antibody. B) 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. Figure 4: Comparison of the binding of VHHs on SARS-CoV-1 and SARS-CoV- 2 Nucleoproteins. An ELISA was performed using the VHHs diluted at different concentrations on coated recombinant Nucleopreoteins from seasonal coronaviruses (OC43, HKU1 , 229E and NL63), SARS-CoV-1 or SARS-CoV-2 and the Spike protein of SARS-CoV-2 was used as control. The signal corresponding to 50% of the maximal OD measured was used as a reference and the concentration of VHH needed to reach this OD was determined. We represented here the inverse of those concentrations, thus a high value corresponds to a low concentration to achieve 50% of the maximal OD.

Figure 5: Hydrogen exchange behavior of full-length SARS-CoV-2 Nucleoprotein, a) Fractional deuterium uptake profile of full-length SARS-CoV-2 Nucleoprotein generated in 88.4% excess deuterium, pD 7.4, at room temperature. The deuterium content calculated for each peptide and at each time point is plotted as a function of peptide position. Each dot corresponds to the average uptake value of three independent technical replicates. The position of the different SARS-CoV-2 Nucleoprotein domains (N-arm, NTD, LKR, CTD and C-tail) is indicated, b) Deuterium uptake behavior of full-length SARS-CoV-2 Nucleoprotein after 10 sec and 120 min labeling plotted on the NTD (pdb # 6vyo) and CTD (pdb # 6wzo) crystal structures. The three disordered regions (N-arm, LKR and C-tail) are presented as long and flexible random lines. The position and orientation of the different domains within the homodimer were randomly selected.

Figure 6: Identification of the VHH binding sites by HDX-MS. a) Differential fractional uptake plots showing the relative variations in deuterium incorporation imposed by the binding of each VHH to full-length SARS-CoV-2 Nucleoprotein. 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 (wald test, p< 0.01 ) are highlighted in gray (See Figure 20). Peptides 340-353 (panel VHH E10) and 337-353 (panel VHH E7) were both found statistically significant by MEMHDX due to a poor fitting quality to the Mixed Effect Model and therefore removed from the final statistical analysis. The general organization of full-length SARS-CoV-2 Nucleoprotein is indicated above the plots, b) Surface and ribbon representations of the SARS-CoV-2 Nucleoprotein CTD domain (pdb # 6wzo) showing the position of the different HDX-MS defined VHH epitopes. The uptake difference values calculated for each CTD peptide and at each time point were summed and plotted on the CTD crystal structure. The color code was normalized using the highest summed value obtained for each condition (Max = 10 Da for VHHs G9, D12, E10; Max = 3.0 Da for VHH E7; Max = 2.8 Da for VHH H3).

Figure 7: Immunofluorescence labeling of SARS-CoV-2 virus in infected cells.

Figure 8: Immunofluorescence labeling of SARS-CoV-2 virus in the lung of infected Syrian Hamster.

Figure 9: Detection of Nucleoprotein by ELISA sandwich. VHH NTD E4-3 (2 pg/ml) was coated on the plate, Nucleoprotein was then added at different concentrations and was revealed by adding biotinylated anti-CTD VHH G9-1 followed by peroxidase labelled streptavidin. All measures were performed in triplicate.

Figures 10-14 illustrate the purification of the recombinant N protein of SARS-CoV-

2. Figure 10. SDS PAGE analysis of fractions collected from 1 st PURIFICATION STEP:

AFFINITY MAC (AKTAPure) according to example 1. Peak of the fractions from A5 to C12 are boxed (Estimated quantity on unicorn 162 mg). Figures 11 -14. 2ND PURIFICATION STEP: Hiload 16/60 Superdex 200 pg column filtration gel according to example 1 . Selection of peaks on histograms and integration of peaks for estimation of protein quantity (Figures 11 and 12). SDS-Page Fraction Removal and Deposition on SDS-Page Gel. GEL FILTRATION 3 on AKTA 1 (Figure 13). GEL FILTRATION 6 on AKTA 2 (Figure 14).

Figure 15: Intact mass measurement of full-length SARS-CoV-2 Nucleoprotein. Intact mass measurement was performed on a Waters SynaptG2-Si HDMS mass spectrometer equipped with a standard ESI source. Prior to mass measurement, the protein concentration was adjusted to 0.28 pM in 0.15 % formic acid, pH 2.5. A total of 14 pmoles (i.e. , 50 pL) was loaded onto a ACQUITY UPLC BEH C4 trap column (2.1 pm x 5 mm; Waters Corporation) pre-equilibrated in 0.15 % formic acid and desalted for 2 min at 100 pL/min and room temperature. The protein was directly eluted into the mass spectrometer using a quick linear gradient of acetonitrile (supplemented with 0.15% formic acid) from 5 to 90% in 2 min at 60 pL/min. The positive-ion mass spectrum of full-length SARS-CoV-2 Nucleoprotein shows a well resolved Gaussian distribution of the different charge states from +33 to +74. 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 Nucleoprotein primary sequence (48 752.13 Da, Am = +0.67 Da (+13.7 ppm)) thereby confirming the structural integrity of the protein.

Figure 16: Peptide Map of full-length SARS-CoV-2 Nucleoprotein (SEQ ID NO: 41 ). The peptide map was generated after 2 min digestion at 20°C with immobilized pig pepsin. Each blue bar corresponds to a unique peptide identified by MS/MS. A total of 51 peptides (blue bar) covering 94.4% of the protein sequence with a 2.43 redundancy value were used to extract HDX data.

Figure 17-19: Uptake plots for all individual SARS-CoV-2 Nucleoprotein peptides generated in the absence (Apo) and presence of VHHs. Only one charge state was selected per peptide to extract the relative uptakes values. The last time point in the apo state corresponds to the fully deuterated control obtained after 21 h incubation at room temperature in deuterated PBS 1X buffer, pD 7.4, and 7.1 M final urea-d4.

Figure 20: Detection of Nucleoprotein by sandwich ELISA.

VHH NTD-E4 or NTD-B6 were coated on the plate, Nucleoproteins from SARS- CoV-2 (A) or permeabilized SARS-CoV-2 virus (B) were then added at different concentrations and were revealed by adding a biotinylated anti-CTD followed by peroxydase labelled streptavidin. Controls without Nucleoprotein or virus were performed and their values were substracted to the data.

Figure 21 : Detection of Nucleoprotein by sandwich ELISA. VHH NTD-E4 or NTD-B6 were coated on the plate, and nasal swabs diluted 1/3 were added (A). In parallel a reference curve was performed with recombinant Nucleoprotein (B).

Figure 22: Alignment of amino acid sequences of 229E, NL63, OC43, HKU1 , SARS-CoV-1 and SARS-CoV-2 nucleoproteins. The different proteins are identified by their UniProt identifier. The alignment was performed by using the Clustal Omega software https://www.ebi.ac.uk/Tools/msa/clustalo/. The epitope recognized by VHH E4- 3 is underlined, the B6-1 epitope is double underlined. The epitopic regions recognized by anti-CTD VHHs are in bold. The differences between the SARS N epitopes are highlighted in grey. Figure 23: Immunofluorescence labeling of N protein in lung sections of mice infected with the B.1-351 and P1 SARS-Cov-2 variants. Figure 24 shows representative staining of lung slices with biotinylated VHHs at 1/500. The scales bar is 50 pm. The left panel shows uninfected control and the right panels show infected mice.

Figure 24: Detection of Nucleoprotein from variants by sandwich ELISA. VHH NTD-E4-3 was coated on the plate. Permeabilized SARS-CoV-2 virus variants were then added at different concentrations and were revealed by adding a biotinylated VHH G9-1 followed by peroxydase labelled streptavidin. Control without virus were performed and their values were substracted from the data.

Figure 25: Detection of Nucleoprotein by sandwich ELISA. VHH NTD E4-3 Fc protein was coated on the plate, SARS-CoV-2 N was then added at different concentrations and was revealed by adding a biotinylated VHH G9-1 Fc protein followed by peroxydase labelled streptavidin.

DETAILED DESCRIPTION

Coronaviruses are enveloped viruses with a positive-sense RNA genome and with a nucleocapsid of helical symmetry. Coronavirus nucleoproteins (N) localize to the cytoplasm and the nucleolus. Coronavirus Nucleoprotein is required for coronavirus RNA synthesis. During virion assembly, Nucleoprotein binds to viral RNA and leads to formation of the helical nucleocapsid. Nucleoprotein is the most abundant protein of coronavirus and is a highly immunogenic phosphoprotein. Because of the conservation of Nucleoprotein sequence and its strong immunogenicity, the Nucleoprotein of coronavirus has been chosen by the inventors as a diagnostic tool.

As reported in the examples, an alpaca was immunized with the recombinant

Nucleoprotein of SARS-CoV-2. Phage display and additional reported experiments demonstrate that 5 VHHs recognized the C terminal domain (CTD) (VHHs E7-2, G9-1 , H3-3, D12-3, E10-3) and 5 VHHs the N terminal domain (NTD B6-1 , NTD C7-1 , NTD F11 - 1 , NTD H7-1 , and NTD E4-3). The VHHs had an affinity in the nanomolar range. Some of these VHHs are able to recognize SARS-CoV-2 virus in infected cells or on infected hamster tissues.

An ELISA sandwich assay has been performed by using the anti NTD E4-3 VHH and the anti-CTD G9-1 to detect the nucleoprotein in solution. As low as 20 ng/ml and even as low as 4 ng/ml of nucleoprotein has been detected.

Based in part on these experimental results, several aspects of the invention are provided herein.

A. Single Domain VHH Antibodies

In a first aspect this invention provides a single domain VHH antibody. In some embodiments the single domain VHH antibody is raised against a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 , which is a recombinantly produced SARS- CoV-2 Nucleoprotein protein. In certain embodiments, the single domain VHH antibody also binds to naturally occurring SARS-CoV-2 Nucleoprotein. In certain embodiments, the single domain VHH antibody also binds to other species of SARS-CoV-1 Nucleoprotein. Therefore, the antibodies are useful, among other things, to detect the presence of SARS- CoV-2 Nucleoprotein in a sample. In certain embodiments the antibodies are useful, to detect a SARS-CoV-2 infection in a subject. Therefore, in certain embodiments the single domain VHH antibody may be used to identify a subject having COVID19.

The examples describe isolation and characterization of the following single domain VHH antibodies: D12-3 (D12-3 is alternatively referred to as D12-1 ), H3-3, E7-2 (E7-2 is alternatively referred to as E7), G9-1 , E10-3, NTD-C7-1 , NTD-F11-1 , NTD-E4-3, NTD- H7-1 , NTD-B6-1 . The amino acid sequences of the antibodies are provided below. In the sequences of the VHHs, the CDR1 , CDR2, and CDR3 domains are bolded and disulfide bonded C residues underlined.

The single domain VHH antibody D12-3 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCTVSEFSLRWNAIGWFRQAPGKEREGVSCISSNGAYTYIADSVK GRFAISTDSVKKMVYLQMNMLKPEDTAVYYCATGSPGCYSAVDEFPYWGRGTQVTVSS ( SEQ ID NO : 1 ) .

D12-3 comprises the following CDR domains:

CDR1 : SEFSLRWNAIG ( SEQ ID NO : 11 ) ;

CDR2: SCISSNGAYTYIADSVKG ( SEQ ID NO : 21 ) ;

CDR3: ATGSPGCYSAVDEFPY ( SEQ ID NO : 31 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 ). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 )-

In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 11 , a CDR2 domain having the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 21 , and a CDR3 domain having the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 31. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 . In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to

SEQ ID NO: 1.

The single domain VHH antibody H3-3 has the following amino acid sequence:

EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAIGWMVGSIYYADSVK DRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAELGGSYLSWRDYGMDYWGKGTLVTVSS ( SEQ ID NO : 2 ) .

H3-3 comprises the following CDR domains

CDR1 : SGRTFSSYAMG ( SEQ ID NO : 12 ) ;

CDR2: AAIGWMVGSI YYADSVKD ( SEQ ID NO : 22 ) ;

CDR3: AAELGGSYLSWRDYGMDY ( SEQ ID NO : 32 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2).

In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 12, a CDR2 domain having the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 22, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 32. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2. In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2.

The single domain VHH antibody E7-2 has the following amino acid sequence: EVQLVESGGGLVQAGDSLRLSCAASGRTFSNYAMGWFRQAPGKEEREFVAAISRDGGFKFYAESV KGRFTISRD IARDTVYLQMNSLKPEDTAVYYCAAKSNTYFSDGIITSRTQYDYWGQGTQVTVS S ( SEQ ID NO : 3 ) .

The single domain VHH antibody E7-2bis has the following amino acid sequence: E VQL VE S GGGLVQAGD S LRL S CAASGRTFSNYAMGWFRQAP GKE REF VAAI SRDGGFKF YAESVK GRFTISRDIARDTVYLQMNSLKPEDTAVYYCAAKSNTYFSDGIITSRTQYDYWGQGTQVTVS S ( SEQ ID NO : 69 ) .

E7-2 and E7-2bis comprise the following CDR domains

CDR1 : SGRTFSNYAMG ( SEQ ID NO : 13 ) ;

CDR2: AAISRDGGFKFYAESVKG ( SEQ ID NO : 23 ) ;

CDR3: AAKSNTYFSDGI ITSRTQYDY ( SEQ ID NO : 33 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3 or 69. In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3

Or 69. In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 13, a CDR2 domain having the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 23, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 33. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3). In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 3 or 69.

The single domain VHH antibody G9-1 has the following amino acid sequence: EVQLVESGGGLVePGGSLRLSCAASGFTWDYYDIGWFRQAPGKEREGVACISSSGSSTNYGDSVK GRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAADIVDYGLESASCMWIDRGYWGQGTQVTVSS ( SEQ ID NO : 4 ) .

G9-1 comprises the following CDR domains

CDR1 : SGFTWDYYDIG ( SEQ ID NO : 14 ) ; CDR2: ACISSSGSSTNYGDSVKG ( SEQ ID NO : 24 ) ;

CDR3: AADIVDYGLESASCMWIDRGY ( SEQ ID NO : 34 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 14, a CDR2 domain having the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 24, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 34. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4). In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4.

The single domain VHH antibody E10-3 has the following amino acid sequence: EVQLVESGGGLVQPGGSLRLSCAASGFGLDYYAIGWFRQAPGKEREGVSCISNSGRSTNPADSVK GRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAATAWRHACTHISNEYDYWGQGTQVTVSS ( SEQ ID NO : 5 ) .

E10-3 comprises the following CDR domains

CDR1 : SGFGLDYYAIG ( SEQ ID NO : 15 ) ;

CDR2: SCISNSGRSTNPADSVKG ( SEQ ID NO : 25 ) ;

CDR3: AATAWRHACTHI SNEYDY ( SEQ ID NO : 35 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

15).

In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 15, a CDR2 domain having the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 25, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 35 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 35. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5. In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5.

The single domain VHH antibody NTD-C7-1 has the following amino acid sequence:

EVQLQASGGGLVQPGGSLRLSCAASGFTLGYYRIGWFRQAPGKEREGVSCLSSSGRSTNYADSV KGRF T I S TDNAKNT VYLQMD S LKP ED TAVY YCAADFTPGPRLCS ILSLNEYSAWGQGTQVTVS S ( SEQ ID NO : 6 ) .

NTD-C7-1 comprises the following CDR domains

CDR1 : SGFTLGYYRIG ( SEQ ID NO : 16 ) ;

CDR2: SCLSSSGRSTNYADSVKG ( SEQ ID NO : 26 ) ; CDR3: AADFTPGPRLCSILSLNEYSA ( SEQ ID NO : 36) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6).

In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 16, a CDR2 domain having the amino acid sequence of SEQ ID NO: 26 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 26, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 36 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 36. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6). In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 6).

The single domain VHH antibody NTD-F11 -1 has the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTSDYYVIGWFRQAPGKEREGVSCISSGGGSTNYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAALNRIHYYSCSVLMGDYGSWGQGTQVTVSS ( SEQ ID NO : 7 ) .

NTD-F11 -1 comprises the following CDR domains

CDR1 : SGFTSDYYVIG ( SEQ ID NO : 17 ) ;

CDR2: SCISSGGGSTNYADSVKG ( SEQ ID NO : 27 ) ;

CDR3: AALNRIHYYSCSVLMGDYGS ( SEQ ID NO : 37 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

7). In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 17, a CDR2 domain having the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 27, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 37 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 37. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7). In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7).

The single domain VHH antibody NTD-E4-3 has the following amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIYWFRQAPGKEREGVSCISSSGGSTNYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGPSECGYSDYLDYWGQGTQVTVSS ( SEQ

ID NO : 8 ) .

NTD-E4-3 comprises the following CDR domains CDR1 : SGFTLDYYAIY ( SEQ ID NO : 18 ) ;

CDR2: SCISSSGGSTNYADSVKG ( SEQ ID NO : 28 ) ;

CDR3: AAGPSECGYSDYLDY ( SEQ ID NO : 38 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8).

In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 18, a CDR2 domain having the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 28, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 38 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 38. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8). In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8).

The single domain VHH antibody NTD-H7-1 has the following amino acid sequence: EVQLQASGGGLVQAGGSLRLSCAASGRTFSSYAMGWFReAPGKEREFVAAISWSGAGTYYADSV KGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAPSAWAGTYVADYDYWGQGTQVTVSS ( SEQ ID NO : 9 ) .

NTD-H7-1 comprises the following CDR domains

CDR1 : SGRTFSSYAMG ( SEQ ID NO : 19 ) ;

CDR2: AAISWSGAGTYYADSVKG ( SEQ ID NO : 29 ) ;

CDR3: AAPSAWAGTYVADYDY ( SEQ ID NO : 39 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:

9)- In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 19, a CDR2 domain having the amino acid sequence of SEQ ID NO: 29 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 29, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 39 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 39. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9). In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 9).

The single domain VHH antibody NTD-B6-1 has the following amino acid sequence:

QVQL VE S GGGLVQAGGS LRL S CAASGRSFSNYNTAWF RQAP GKE REF VALI SWTVGNTP YADSV KGRFTISRDNAKNTVYLQMNSLNAEDTAVYYCAAGRPSIYYRTYDRYDYWGQGTQVTVSS ( SEQ ID NO : 10 ) .

NTD-B6-1 comprises the following CDR domains CDR1 : SGRSFSNYNTA ( SEQ ID NO : 20 ) ;

CDR2: ALISWTVGNTPYADSVKG ( SEQ ID NO : 30 ) ;

CDR3: AAGRP SIYYRTYDRYDY ( SEQ ID NO : 40 ) .

Therefore, in some embodiments this invention provides a single domain VHH antibody that comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10). In some embodiments this invention provides a single domain VHH antibody that consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10).

In some embodiments this invention provides a single domain VHH that comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively), wherein the single domain VHH comprises a CDR1 domain having the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 20, a CDR2 domain having the amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 30, and a CDR3 domain having the amino acid sequence of SEQ ID NO: 40 or an amino acid sequence having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NO: 40. In some embodiments the single domain VHH antibody also comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10). In some embodiments the single domain VHH antibody also consists of an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 10).

In some embodiments of the isolated single domain VHH antibody the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOS: 11 - 20 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 11 -20; the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOS: 21-30 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 21 -30; and the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOS: 31 -40 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 31 -40.

In some embodiments of the isolated single domain VHH antibody, the single domain VHH antibody comprises an amino acid sequence that is at least 90%, 91%, 92%, 93% or 94% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69. In some embodiments of the isolated single domain VHH antibody, the single domain VHH antibody comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69.

In some embodiments the isolated single domain VHH antibody binds to the C-terminal domain (CTD) of SARS-CoV-2 Nucleoprotein.

In some embodiments the isolated single domain VHH antibody binds to the N-terminal domain (NTD) of SARS-CoV-2 Nucleoprotein.

In some embodiments, the isolated single domain VHH antibody further comprises a label.

In some embodiments, the isolated single domain VHH antibody is covalently attached to a substrate. Unless otherwise specified the expression “single domain VHH antibody” encompasses monomers and multimers (homomers or heteromers) of the single domain VHH antibody as well as fusion protein comprising the single domain VHH antibody

B. Fusion Proteins

In another aspect a fusion protein comprising a single domain VHH antibody of the invention and a second polypeptide or protein is provided. The VHH antibody may be any antibody described in Section A above.

As used herein a "fusion protein" refers to a polypeptide having two portions covalently linked together, where each of the portions is a polypeptide having a different property. The property may be a biological property, such as activity in vitro or in vivo. The property may also be a simple chemical or physical property, such as binding to a target antigen, catalysis of a reaction, etc. The two portions may be linked directly by a single peptide bond or through a peptide linker containing one or more amino acid residues. Generally, the two portions and the linker will be in reading frame with each other. Preferably, the two portions of the polypeptide are obtained from heterologous or different polypeptides. In the context of this invention, one of the portions is a single domain VHH antibody of the invention.

In fusion protein of the present invention, the single domain VHH antibody may be directly fused or linked via a linker moiety to the other elements of the fusion protein. The linker may be a peptide, peptide nucleic acid, or polyamide linkage. Suitable peptide linkers may include a plurality of amino acid residues, for example, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 amino acids, such as (Gly)4, (Gly)5, (Gly)4Ser, (Gly)4(Ser)(Gly)4, or combinations thereof or a multimer thereof (for example a dimer, a trimer, or a tetramer, or greater). For example, a suitable linker may be (GGGGS)3. Alternative linkers include (Ala)3(His)6 or multimers thereof. Also included is a sequence which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto.

In some embodiments of the fusion protein, the second polypeptide or protein is selected from a Fab, Fc, F(ab’)2 (including chemically linked F(ab’)2 chains), Fab’, scFv (including multimer forms thereof, i.e. di-scFv, or tri-scFv), sdAb, or BiTE (bi-specific T-cell engager). In some embodiments the second polypeptide is a Fc fragment of a mammalian immunoglobulin. In some embodiments the mammal is a human. In some embodiments the Fc fragment has the amino acid sequence of SEQ ID NO: 68.

RSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTI SKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ( SEQ ID NO : 68 ) .

In some embodiments, the first polypeptide is the single domain VHH antibody comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1 -10 or 69 or variants thereof having at least 95%, 96%, 97%, 98%, 99% of identity with SEQ ID NO: 1 -10 or 69 and the second polypeptide is a Fc fragment of a mammalian immunoglobulin, preferably a human Fc fragment, more preferably the Fc fragment having the amino acid sequence of SEQ ID NO: 68.

In some embodiment, the first protein of the fusion protein is VHH G9-1 and the second protein of the fusion protein is a human Fc. In this embodiment, the fusion protein may have the amino acid sequence SEQ ID NO: 60.

MAEVQLVESGGGLVQPGGSLRLSCAASGFTWDYYDIGWFRQAPGKEREGVACI

SSSGSSTNYGDSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCAADIVDYGLE

SASCMWIDRGYWGQGTQVTVSSAAARSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 60)

In some embodiment, the first protein of the fusion protein is VHH E4-3 and the second protein of the fusion protein is a human Fc. In this embodiment, the fusion protein may have the amino acid sequence SEQ ID NO: 61 .

MAEVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIYWFRQAPGKEREGVSCIS SSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAGPSECGYS DYLDYWGQGTQVTVSSAAARSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 61 )

C. Multimeric VHH Antibodies

Multimeric VHH antibodies are also provided. The multimeric VHH antibody comprises at least two VHHs of the invention. Each VHH present in a multimeric VHH antibody may be in the form of a fusion protein or may not be in the form of a fusion protein. For example, the multimeric VHH antibody may comprise VHH1 and VHH2, or may comprise VHH1 -Fc and VHH2-FC.

In some embodiments, the isolated single domain VHH antibody is in the form of a homomultimer, such as a homodimer or a homotrimer. In some embodiments, the isolated single domain VHH antibody is in the form of an heteromultimer, such as a heterodimer or a heterotrimer. In a particular embodiment the isolated single domain VHH antibody is in the form of an heteromultimer VHHE4-VHH B6 and more particularly VHHE4-(G4S)3-VHH B6.

In some embodiments dimers of single domain VHH antibodies according to the invention are provided. The dimers may be homodimers of a single domain VHH antibody or heterodimers, comprising two different single domain VHH antibodies.

In some embodiments, it is also provided a dimeric VHH antibody comprising two fusion proteins according to the invention. In some embodiments the dimeric VHH antibody comprises two copies of the same fusion protein. In other embodiments the dimeric VHH antibody comprises two different fusion proteins.

Each VHH present in the multimeric VHH antibody may be the same as at least one other VHH in the multimeric VHH antibody. Alternatively, each VHH present in the multimeric VHH antibody may be different than all other VHH antibodies present in the multimeric VHH antibody.

In certain embodiments the multimeric VHH antibodies are Fc fusion proteins. In such embodiment the Fc portion may be responsible for linking the VHHs together into the multimeric form, for example via fusion or linkage of the Fc.

In certain embodiments the VHH antibodies may be linked together via one or more type of linker, such as a Gly-Ser linker. Therefore, in some embodiments the dimeric VHH antibody is a homodimer. In other embodiments, the dimeric VHH antibody is a heterodimer. Mixtures comprising VHH antibody homodimers, mixtures comprising VHH antibody heterodimers, and mixtures comprising VHH antibody homodimers and heterodimers are also provided.

In some embodiments the multimeric VHH antibody is a homodimer of a VHH comprising or consisting of an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69. The VHHs in the homodimer may be in the form of fusion proteins.

In some embodiments the multimeric VHH antibody is a heterodimer of two different VHH comprising or consisting of an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69. The VHHs in the heterodimer may be in the form of fusion proteins.

In some embodiments the multimeric VHH antibody is a homotrimer of a VHH comprising or consisting of an amino acid sequence selected from SEQ ID NOS: 1-10 or 69.

In some embodiments the multimeric VHH antibody is a heterotrimer of three VHH comprising or consisting of an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69.

In an embodiment, the multimeric VHH antibody is a heterodimer of VHH G9-1 having the amino acid sequence SEQ ID NO: 4 and of VHH E4-3 having the amino acid sequence SEQ ID NO: 8.

In an embodiment, the multimeric VHH antibody is a heterodimer of fusion protein VHH G9-1 -Fc having the amino acid sequence SEQ ID NO: 60 and of fusion protein VHH E4- 3-Fc having the amino acid sequence SEQ ID NO: 61 .

D. Nucleic Acids and Cells In another aspect an isolated nucleic acid sequence that encodes the single domain VHH antibody or a fusion protein comprising a single domain VHH antibody is provided. The VHH antibody may be any antibody described in Section A above, any fusion protein described in Section B above, or any multimeric VHH described in Section C above.

Also provided are recombinant vectors comprising the isolated nucleic acid sequence. The recombinant vector can be a vector for eukaryotic or prokaryotic expression, such as a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector and especially a retroviral vector, or any expression vector. An expression vector as defined herein is chosen to enable the production of single domain VHH antibody, either in vitro or in vivo.

In one embodiment, the expression vector comprises a single domain VHH antibody cDNA cloned into the Expression Vector pHEN6 or pASK.

In one embodiment, the expression vector encodes a protease cleavage site, such as TEV cleave site, inserted between the single domain VHH antibody protein coding sequence and a protein purification Tag, such as polyHis tag. In a preferred embodiment, the expression vector encodes a His tag. In one embodiment, a TEV cleavage site is positioned to remove the His tag, for example, after purification.

The expression vector can comprise transcription regulation regions (including promoter, enhancer, ribosome binding site (RBS), polyA signal), a termination signal, a prokaryotic or eukaryotic origin of replication and/or a selection gene. The features of the promoter can be easily determined by the man skilled in the art in view of the expression needed, i.e., constitutive, transitory or inducible (e.g. IPTG), strong or weak. The vector can also comprise sequence enabling conditional expression, such as sequences of the Cre/Lox system or analogue systems.

In various embodiments, the expression vector is a plasmid, a phage for bacterium introduction, a YAC able to transform yeast, a viral vector, or any expression vector. An expression vector as defined herein is chosen to enable the production of a VHH of the invention, either in vitro or in vivo.

The nucleic acid molecules according to the invention can be obtained by conventional methods, known per se, following standard protocols such as those described in Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc., Library of Congress, USA). For example, they may be obtained by amplification of a nucleic sequence by PCR or RT-PCR or alternatively by total or partial chemical synthesis.

The vectors are constructed and introduced into host cells by conventional recombinant DNA and genetic engineering methods which are known per se. Numerous vectors into which a nucleic acid molecule of interest may be inserted in order to introduce it and to maintain it in a host cell are known per se; the choice of an appropriate vector depends on the use envisaged for this vector (for example replication of the sequence of interest, expression of this sequence, maintenance of the sequence in extrachromosomal form or alternatively integration into the chromosomal material of the host), and on the nature of the host cell.

In another aspect methods of producing the single domain VHH antibody are provided. The methods comprise culturing the recombinant cell comprising the isolated nucleic acid sequence that encodes the single domain VHH antibody under conditions sufficient for production of the single domain VHH antibody.

E. coli comprising a pASK vector encoding a single domain VHH antibody of the invention were deposited under the terms of the Budapest Treaty at the Collection Nationale de Culture de Microorganismes (CNCM), at Institut Pasteur, 25, Rue de Docteur Roux F- 75724 Paris Cedex 15 FRANCE on October 7, 2020. The following deposits were made and assigned the following reference numbers.

E. coli expressing VHH N-CTD C7-1 (CNCM 1-5601).

E. coli expressing VHH N-CTD E7-2 (CNCM I-5602).

E. coli expressing VHH N-CTD G9-1 (CNCM I-5603).

E. coli expressing VHH N-CTD H3-3 (CNCM I-5604).

E. coli expressing VHH N-NTD B6-1 (CNCM I-5605).

E. coli expressing VHH N-NTD E4-3 (CNCM I-5606).

E. coli comprising a pASK vector encoding a fusion protein of the invention were deposited under the terms of the Budapest Treaty at the Collection Nationale de Culture de Microorganismes (CNCM), at Institut Pasteur, 25, Rue de Docteur Roux F-75724 Paris Cedex 15 FRANCE on September 7, 2021. The following deposits were made and assigned the following reference numbers.

E. coli expressing VHH N-SARS2 E04-2 Fc hu corresponding to the VHH E04-3 fused to human Fc (CNCM I-5745).

E. coli expressing VHH N-SARS2 G09-1 Fc hu corresponding to the VHH G09-1 fused to human Fc (CNCM I-5744). E. Methods of Use, Detection methods, Diagnostic methods

In another aspect, methods for detection of a SARS-associated coronavirus in a biological sample are provided. The methods may comprise providing a single domain VHH antibody according to this disclosure or a fusion protein according to this disclosure ; providing a biological sample from a subject suspected to be infected with a SARS- associated coronavirus; contacting the single domain VHH antibody with the biological sample; and visualizing the antigen-antibody complexes formed. In some embodiments the methods comprise an ELISA, lateral flow immunoassay, bead-based immunoassay, or multiplex bead-based immunoassay. Typically, the method of detection is an in vitro method.

In some embodiments the method for detection of a SARS-associated coronavirus in a biological sample comprise providing a first single domain VHH antibody according to this disclosure or a first fusion protein according this disclosure, attached to a solid support; providing a biological sample from a subject suspected to be infected with a SARS- associated coronavirus; contacting the solid support with the biological sample under conditions sufficient to allow formation of first antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein in the biological sample and the VHH antibody or the fusion protein attached to the solid support to form first antigen-antibody complexes; contacting the solid support with a second single domain VHH antibody according to any this disclosure or a second fusion protein according this disclosure under conditions sufficient to allow formation of second antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein and the second single domain VHH antibody or the second fusion protein; and visualizing the second antigen-antibody complexes. In some embodiments, the second single domain VHH antibody according to this disclosure or the second fusion protein according this disclosure is labeled and visualizing the second antigen-antibody complexes comprises visualizing the label. In some embodiments, the first single domain VHH antibody is an anti-NTD VHH antibody or the first fusion protein is a fusion protein comprising an anti-NTD VHH antibody and the second single domain VHH antibody is an anti-CTD VHH antibody or the second fusion protein is a fusion protein comprising an anti-CTD VHH antibody. In some embodiments, the first single domain VHH antibody is an anti-CTD VHH antibody or the first fusion protein is a fusion protein comprising an anti-CTD VHH antibody and the second single domain VHH antibody is an anti-NTD VHH antibody or the second fusion protein is a fusion protein comprising an anti-NTD VHH antibody. In some embodiments the first single domain VHH antibody is VHH NTD E4-3 which has the amino acid sequence SEQ ID NO: 8 or a variant thereof and the second single domain VHH antibody is VHH G9-1 which has the amino acid sequence SEQ ID NO: 4 or a variant thereof.

In some embodiments the first fusion protein according to the invention is VHH E04-3 fused to human Fc and has the amino acid sequence SEQ ID NO: 61 or is a variant thereof. In some embodiments the second fusion protein according to the invention is VHH G9-1 fused to human Fc and has the amino acid sequence SEQ ID NO: 60 or is a variant thereof. In some embodiments the first fusion protein according to the invention is VHH E04-3 fused to human Fc and has the amino acid sequence SEQ ID NO: 61 or is a variant thereof and the second fusion protein according to the invention is VHH G9-1 fused to human Fc and has the amino acid sequence SEQ ID NO: 60 or is a variant thereof.

In a preferred embodiment the method detects SARS-CoV-2. In a preferred embodiment the method detects SARS-CoV-2 Nucleoprotein in a sample. In some embodiments, the method allows detection of as low as 20 ng/ml and even as low as 4 ng/ml of the SARS-CoV-2 Nucleoprotein in a sample.

In some embodiments, the method does not detect human common coronavirus Nucleoproteins.

In a preferred embodiment, the methods utilize a single domain VHH antibody that binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 . The single domain VHH antibody comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively). In some embodiments the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOS: 11 -20 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 11 -20; the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOS: 21 -30 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 21 -30; and the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOS: 31 -40 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 31 -40.

In some embodiments the method utilizes a single domain VHH antibody comprising an amino acid sequence comprising a CDR1 selected from the amino acid sequences of SEQ ID NOS: 11 -20; an amino acid sequence comprising a CDR2 selected from the amino acid sequences of SEQ ID NOS: 21 -30; and an amino acid sequence comprising a CDR3 selected from the amino acid sequences of SEQ ID NOS: 31 -40. In some embodiments the method utilizes a single domain VHH antibody that comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93% or 94% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69. In some embodiments the single domain VHH antibody comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69.

In some embodiments the method utilizes a single domain VHH antibody comprising an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69. In some embodiments the single domain VHH antibody consists of an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69.

In some embodiments the method utilizes a single domain VHH antibody that binds to the C-terminal domain (CTD) of SARS-CoV-2 Nucleoprotein.

In some embodiments the method utilizes a single domain VHH antibody that binds to the N-terminal domain (NTD) of SARS-CoV-2 Nucleoprotein.

In some embodiments the method utilizes a single domain VHH antibody that binds to the SARS-CoV-2 Nucleoprotein with a nanomolar KD. For example, it may bind with a KD of as low as 50 nM, 40nM, 30 nM, 20 nM, 10, nM, 5 nM, 1 nM, 0.5 nM, or even as low as 0.1 nM.

In some embodiments the method utilizes a single domain VHH antibody that further comprises a label.

In some embodiments the method utilizes a single domain VHH antibody that is covalently attached to a substrate. A skilled artisan will appreciate that the single domain VHH antibodies can be used for the diagnosis of a SARS-associated coronavirus infection using any other suitable antigenic assay format known in the art that is designed to utilize antibodies. In particular by an immunoassay, such as an immunoenzymatic method (e.g., ELISA).

The invention encompasses methods comprising identifying a patient infected with a SARS-associated coronavirus infection, providing a sample from the patient, contacting the sample with a single domain VHH antibody of this disclosure or a fusion protein according to this disclosure, and visualizing the antigen-antibody complexes. The sample may be a nasopharyngeal sample (for example, mucus from the back of throat or nose collected using a swab), saliva (including gargling), etc. In a preferred embodiment, the SARS-associated coronavirus infection is identified as SARS-CoV-2. In some embodiments, the patient has been identified as being infected with a SARS-associated coronavirus infection, but lacks detection of the virus by PGR or another nucleic acid amplification technique.

The invention encompasses a composition comprising a single domain VHH antibody for the use of a single domain VHH antibody for detection and/or diagnosis of a SARS-CoV- 2 coronavirus in a biological sample.

The invention encompasses a composition comprising a single domain VHH antibody for the use of a single domain VHH antibody for detection and/or diagnosis of a SARS-CoV- 1 coronavirus in a biological sample.

The ability of the single domain VHH antibodies to bind with high affinity to Nucleoprotein of SARS-CoV-2 allows its use in such methods, particularly for diagnostics of a SARS-

CoV-2 infection. The single domain VHH antibody according to the invention is useful for the direct diagnosis of a SARS-associated coronavirus infection; the detection of the protein(s) of a SARS coronavirus is carried out by an appropriate technique, in particular EIA, ELISA, RIA, immunofluorescence, in a biological sample collected from an individual capable of being infected.

In some embodiments, the patient has been shown to be infected by SARS-CoV-1 or SARS-CoV-2 by a nucleic acid detection test, such as a PGR or other nucleic acid amplification test.

In some embodiments, the patient has not been shown to be infected by SARS-CoV-1 or SARS-CoV-2 by a nucleic acid detection test, such as a PGR or other nucleic acid amplification test.

In one embodiment, the invention comprises a method for the detection of a SARS- associated coronavirus, from a biological sample, which method is characterized in that it comprises bringing a biological sample from a patient infected with or suspected to be infected with a SARS-CoV-1 coronavirus with a single domain VHH antibody according to the invention, and visualizing the antigen-antibody complexes formed. Preferably, the antigen-antibody complexes are visualized by EIA, ELISA, RIA, or by immunofluorescence.

In one embodiment, the single domain VHH antibody is attached to an appropriate support, in particular a microplate or a bead.

In one embodiment, the method comprises bringing a biological sample from a subject, preferably a human, infected with or suspected to be infected with a SARS-CoV-1 or SARS-CoV-2 coronavirus into contact with the single domain VHH antibody, which is attached to an appropriate support, in particular a microplate or bead, to allow binding to occur; washing the support to remove unbound proteins; adding a detection reagent that binds to Nucleoprotein of SARS-CoV-2 and/or Nucleoprotein of SARS-CoV-1 protein; and detecting the Nucleoprotein of SARS-CoV-2 and/or Nucleoprotein of SARS-CoV-1 protein-antibody complexes formed.

In one embodiment, the method for the detection of a SARS-associated coronavirus in a biological sample comprises providing a single domain VHH antibody of this disclosure or a fusion protein according to this disclosure; providing a biological sample from a patient infected with or suspected to be infected with a SARS-CoV-2 coronavirus; contacting said single domain VHH antibody or said fusion protein with said biological sample; and visualizing the antigen-antibody complexes formed. Preferably, the method comprises an ELISA.

Preferably, the protein-antibody complexes are detected with a second single domain VHH antibody that binds to Nucleoprotein of SARS-CoV-2.

Preferably, the second single domain VHH antibody comprises a label selected from a chemiluminescent label, an enzyme label, a fluorescence label, and a radioactive (e.g., iodine) label.

Preferred labels include a fluorescent label, such as FITC, a chromophore label, an affinity-ligand label, an enzyme label, such as alkaline phosphatase, horseradish peroxidase, or galactosidase, an enzyme cofactor label, a hapten conjugate label, such as digoxigenin or dinitrophenyl, a Raman signal generating label, a magnetic label, a spin label, an epitope label, such as the FLAG or HA epitope, a luminescent label, a heavy atom label, a nanoparticle label, an electrochemical label, a light scattering label, a spherical shell label, semiconductor nanocrystal label, wherein the label can allow visualization with or without a secondary detection molecule.

Preferred labels include suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase or acetylcholinesterase; members of a binding pair that are capable of forming complexes such as streptavidin/biotin, avidin/biotin or an antigen/antibody complex including, for example, rabbit IgG and antirabbit IgG; fluorophores such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, tetramethyl rhodamine, eosin, green fluorescent protein, erythrosin, coumarin, methyl coumarin, pyrene, malachite green, stilbene, lucifer yellow, Cascade Blue, Texas Red, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, fluorescent lanthanide complexes such as those including Europium and Terbium, cyanine dye family members, such as Cy3 and Cy5, molecular beacons and fluorescent derivatives thereof, as well as others known in the art; a luminescent material such as luminol; light scattering or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive material include 14C, 1231, 1241, 1251, 32P, 33P, 35S, or 3H.

Preferably, the method comprises comparing the results obtained with a patient serum to positive and negative controls.

In another aspect, the use of a single domain VHH antibody or a fusion protein according to this disclosure for detection of a SARS-associated coronavirus in a biological sample is provided. The use may comprise providing a single domain VHH antibody or a fusion protein according to this disclosure; providing a biological sample from a subject suspected to be infected with a SARS-associated coronavirus; contacting the single domain VHH antibody with the biological sample; and visualizing the antigen-antibody complexes formed. In another aspect, the use of a single domain VHH antibody according to this disclosure or a fusion protein according to this disclosure in the preparation of a diagnosis reagent for detecting the presence of a Nucleoprotein, in particular a Nucleoprotein of SARS-associated coronavirus in a sample obtained from a subject, preferably a human, infected with or suspected to be infected with SARS-CoV-1 or SARS- CoV-2.

F. Kits

In another aspect a kit for detection of a SARS-associated coronavirus in a biological sample is provided. In some embodiments the kit comprises a single domain VHH antibody according to this disclosure or a fusion protein according to this disclosure. In some embodiments of the kit, the single domain VHH antibody further comprises a label. In some embodiments of the kit, the single domain VHH antibody is covalently attached to a solid support. In some embodiments the kit comprises a first antibody directed against the SARS-CoV-2 Nucleoprotein, attached to a solid support; and a second antibody, attached to a label wherein at least one of said first or second antibody is a single domain VHH antibody or fusion protein according to this disclosure. In some embodiments the kit comprises a first single domain VHH antibody according to this disclosure or a first fusion protein according to this disclosure, attached to a solid support; and a second single domain VHH antibody according to this disclosure or a second fusion protein according to this disclosure, attached to a label. In some embodiments, the first single domain VHH antibody or this first fusion protein is an anti-NTD VHH antibody and the second single domain VHH antibody or the second fusion protein is an anti-CTD VHH antibody. In some embodiments, the first single domain VHH antibody or the first fusion protein is an anti- CTD VHH antibody and the second single domain VHH antibody or the second fusion protein is an anti-NTD VHH antibody. In some embodiments the first single domain VHH antibody is VHH NTD E4-3 which has the amino acid sequence SEQ ID NO: 8 or a variant thereof. In some embodiment the second single domain VHH antibody is VHH G9-1 which has the amino acid sequence SEQ ID NO: 4 or a variant thereof. In some embodiments the first single domain VHH antibody is VHH NTD E4-3 which has the amino acid sequence SEQ ID NO: 8 or a variant thereof and the second single domain VHH antibody is VHH G9-1 which has the amino acid sequence SEQ ID NO: 4 or a variant thereof. In some embodiments the kit further comprises reagents for detecting the label. In some embodiments the kit further comprises a recombinant SARS-CoV-2 Nucleoprotein. In some embodiments the first fusion protein according to the invention is VHH E04-3 fused to human Fc and has the amino acid sequence SEQ ID NO: 61 or a variant thereof. In some embodiments the second fusion protein according to the invention is VHH G9-1 fused to human Fc and has the amino acid sequence SEQ ID NO: 60 or a variant thereof. In some embodiments the first fusion protein according to the invention is VHH E04-3 fused to human Fc and having the amino acid sequence SEQ ID NO: 61 or a variant thereof and the second fusion protein according to the invention is VHH G9-1 fused to human Fc and having the amino acid sequence SEQ ID NO: 60 or a variant thereof.

In a preferred embodiment, the kits comprise a single domain VHH antibody that binds a polypeptide comprising the amino acid sequence of SEQ ID NO: 41. The single domain VHH antibody comprises four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively). In some embodiments the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOS: 11 -20 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 11 -20; the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOS: 21 -30 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 21 -30; and the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOS: 31 -40 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 31 -40.

In some embodiments the kit comprises a single domain VHH antibody comprising an amino acid sequence comprising a CDR1 selected from the amino acid sequences of SEQ ID NOS: 11 -20; an amino acid sequence comprising a CDR2 selected from the amino acid sequences of SEQ ID NOS: 21 -30; and an amino acid sequence comprising a CDR3 selected from the amino acid sequences of SEQ ID NOS: 31 -40.

In some embodiments the kit comprises a single domain VHH antibody that comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93% or 94% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69. In some embodiments the single domain VHH antibody comprises an amino acid sequence that is at least 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69.

In some embodiments the kit comprises a single domain VHH antibody comprising an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69. In some embodiments the single domain VHH antibody consists of an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69.

In some embodiments the kit comprises a single domain VHH antibody that binds to the C-terminal domain (CTD) of SARS-CoV-2 Nucleoprotein.

In some embodiments the kit comprises a single domain VHH antibody that binds to the N-terminal domain (NTD) of SARS-CoV-2 Nucleoprotein.

In some embodiments the kit comprises a single domain VHH antibody that binds to the SARS-CoV-2 Nucleoprotein with a nanomolar KD. For example, it may bind with a KD of as low as 50 nM, 40nM, 30 nM, 20 nM, 10, nM, 5 nM, 1 nM, 0.5 nM, or even as low as 0.1 nM.

In some embodiments the kit comprises a single domain VHH antibody that further comprises a label.

In some embodiments the kit comprises a single domain VHH antibody that is covalently attached to a substrate.

EXAMPLES

Example 1 : Materials and Methods

Production of recombinant Nucleoprotein from SARS-CoV-2

Optimized synthetic gene (GenBank MN908947) was cloned in the pETM11 expression vector allowing the production of N fused to an N-terminal (His)e tag. Production and purification of N has been described by Grzelak et al (45).

Quality assessment of SARS-CoV-2 nucleoprotein

Prior to use in experiments, both VHHs and the protein N undergo a battery of tests by checking their integrity, solubility and stability. The method is adapted from a previously published approach (46) and ARBRE-MOBIEU P4EU recommendations (https://arbre-mobieu.eu/guidelines-on-protein-quality-control/).

UV spectroscopy quantification

Protein quantification at 280 nm was carried out by recording a full spectrum between 240 and 340 nm. Detection of nucleotides at 260 nm and scattering at 340 nm were also checked. Measurements were done at room temperature in a 1 cm quartz cell, reference 105.202-QS.10 (Hellma, France), using a JASCO V-650 spectrophotometer (JASCO Corporation, Japan). A baseline subtraction at 340 nm was performed with the Spekwin32 software (F. Menges "Spekwin32 - optical spectroscopy software", Version 1.72.2, 2016, http://www.effemm2.de/spekwin/) to accurately calculate the protein concentration.

Dynamic light scattering (PLS) experiments

DLS was performed on a DynaPro Plate Reader III (Wyatt, Santa Barbara, CA, USA) to confirm that the samples did not contain aggregates. Experiments were performed in triplicate in a 384-well microplate (Corning ref 3540, New-York, USA), with 20 acquisitions of 10 s each, monitored with the DYNAMICS version V7.9.1.3 software (Wyatt, Santa Barbara, CA, USA). The Nucleoprotein stored at 4°C was monitored for 3 weeks. In parallel, an overnight experiment at 37°C was perform on it. The VHHs were monitored at 20°C just after their purification.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE)

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 SDS-PAGE gel. Intact mass measurement by LC-ESI-MS

Recombinant N was diluted to 0.2 pM in 0.15 % formic acid (pH 2.5). 50 pL (10 pmol) was loaded onto an ACQUITY UPLC BEH C4 Trap column (2.1 pm x 5 mm, Waters Corporation, Milford, MA), and desalted for 2 min at 100 pL/min with 0.15 % formic acid, pH 2.5. The protein was eluted into the mass spectrometer with a quick linear gradient of acetonitrile from 5 to 90 % in 2 min, at 60 pL/min. Mass spectra were acquired in resolution and positive ion-mode (m/z 400-2000) on a Synapt G2-Si HDMS mass spectrometer (Waters Corporation, Milford, MA). To ensure mass accuracy, a Glu-1 - Fibrinopeptide B solution (100 fmol/pL in 50% acetonitrile, 0.1 % formic acid) was continuously infused through the reference probe of the electrospray source.

Sedimentation velocity analysis.

Sedimentation velocity experiments were carried out at 42,000 rpm and 20°C in a Optima analytical ultracentrifuge, using 12-mm aluminum-Epon double-sector centerpieces in an An55Ti rotor. Protein concentrations were recorded in continuous mode using absorbance at 230, 271 nm. N proteins were studied at 0.15mg/ml. The partial specific volume, solvent density, and viscosity, was calculated with SEDNTERP (75), were 0.724 ml/g, 1.012 g/cm³, and 0.01045 poise, respectively. The data recorded from moving boundaries were analyzed in terms of continuous size distribution function of sedimentation coefficient C(S) using the program SEDFIT (47).

Production of the N terminal Domain (NTD) of Nucleoprotein

The gene encoding residues 1 to 200 of N (GenBank: YP_009724397.2) was retrieved by polymerase chain reaction (PGR). The amplicon was sub-cloned into a pET23-derived plasmid encoding an His6 tag at 3’end. Sequencing verified that no mutations were introduced during the process. The recombinant protein was expressed in E.coli SHuffle C3029H cells (New England Biolabs) and purified from a soluble cytoplasmic extract, as described above for the whole nucleoprotein. About 15 mg of purified protein was systematically obtained from 1 liter of culture medium.

Production of native Nucleoprotein: cell extracts and virus inactivation

Vero-NK (African Green Monkey Kidney) cells were infected with the SARS-CoV-2 virus (BetaCoV/France/IDF0372/2020) at a MOI of 10^-2. An uninfected control was also produced in the same conditions. After 24 hours of incubation the cells were washed with 150mM NaCI and 50mM Tris HCI pH7.5 (TBS), and the cell monolayer was scratched. The cells were centrifuged and the pellet was resuspended in TBS-2% Triton X100 and incubated at 37°C for 15min before being sonicated. Cells were centrifuged and |3- propiolactone (1/50) was added to the supernatant before being incubated for 24 hours at 4°C then 24 hours at 20°C. The virus inactivation was controlled before the use of cell extracts for ELISA.

The SARS-CoV-2 virus was also inactivated with p-propiolactone (1/50) in TBS for 24 hours at 4°C then 24 hours at 20°C. The virus inactivation was then controlled. To permeabilize the viral membrane, the virus was incubated for 15min at 37°C in PBS-2% Triton X100.

Alpaca immunization

Animal procedures were performed according to the French legislation and in compliance with the European Communities Council Directives (2010/63/UE, French Law 2013-118, February 6, 2013). The Animal Experimentation Ethics Committee of Pasteur Institute (CETEA 89) approved this study (2020-27412). One young adult male alpaca (Lama pacos) was immunized at days 0, 17 and 24 with 150 ng of the nucleoprotein. The immunogen was mixed with Freund complete adjuvant for the first immunization and with Freund incomplete adjuvant for the following immunizations. The immune response was monitored by titration of serum samples by ELISA on coated nucleoprotein. The bound alpaca antibodies were detected with polyclonal rabbit anti-alpaca IgGs (48)

Library construction and phacie display

The blood of the immunized animal was collected and the peripheral blood lymphocytes were isolated by centrifugation on a Ficoll (GE Healthcare) discontinuous gradient and stored at -80 ° C until further use. Total RNA and cDNA were obtained as previously described (48). A nested PGR was performed with IgG specific primers. In the first step, five sets of PGR 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 reamplified with three sets of PGR primers specific for VHH complementary to the 5’ and 3’ ends of the amplified product and incorporating Sfil and Notl restriction sites at the ends of the VHH genes (Table 3). The PGR products were digested and ligated into phage vector pHEN 6.

Table 3: List of the primers used for the construction of the VHH libraries

Phage Display technology allows the selection of antigen specific phage-VHHs. A large number of phage-VHHs (10¹³) were used to perform a round of panning. A different blocking agent was used at each of the three rounds of panning: 2% skimmed milk, Licor blocking buffer (Biosciences) diluted with PBS in the ratio 1 :4, and 4% BSA were respectively used. After blocking step, phage-VHHs were incubated with antigen precoated immunotubes for 2h on wheel at room temperature. To remove non-specific binders, a 6 x PBS Tween 0.1% and 4 x PBS washing procedure was performed, specific phage-VHHs were then eluted in 100 mM TEA (triethylamine) during 5 min on wheel and the excess TEA was neutralized immediately in 0.1 M Tris-HCI, pH 7.6. E. coli TG1 at exponential growth phase was then infected with eluted phage-VHHs and then incubated for 30 min at 37°C without stirring then 30 min at 37°C under stirring. The remaining bacteria were centrifuged at 4000 rpm for 15 min and the pellet was resuspended in 1 ml of 2YT and spread on 2YT+A Bio-assay dish (24 cm x24 cm) for an overnight incubation at 30°C and bacteria were recovered the next day with 4ml of 2YT containing 8% of DMSO (Dimethyl sulfoxide Sigma-Aldrich) and were stored at -80°C in aliquots of 1 ml.

Selection of specific phage-VHHs by ELISA

Individual colonies from the second and the third round of panning were picked from Petri dishes and were cultured in a 96 well plate (Plate I) (Cell star, Greiner Bio-one) containing 200 pl of 2YT +A /well overnight at 37°C with shaking. This plate (Plate I) was used to seed a secondary plate (Plate II) in order to express phage-VHH. Three microliters of each colony were cultured in 200 pl of 2YT A+G in a 96 deepwell plate (MasterBlock, Greiner Bio-one) (Plate II). After an incubation of 1 h30 min at 37°C with shaking, the bacteria present in each well were infected with 1x10⁹ VCS M13 helper phages. The plate II was then incubated for 30 min at 37°C without shaking followed by 30 min at 37°C with shaking and then centrifuged at 2500 rpm for 10 min. The pelleted cells in each well were resuspended in 500 pl of 2YT+A+K+IPTG. The cultures were then incubated overnight at 30°C with shaking to allow expression of phage-VHHs by bacteria. Each well contained a single selected phage-VHH. In parallel, plate III (Nunc Thermo Scientific) was coated with antigen overnight at 4°C.

Between each ELISA step, plates were washed 6x with PBS-Tween 0.1%. The following day, plates III were first saturated with PGT (PBS-Gelatin 0.5%-Tween 0.1%) for 30 min at 37°C (100 pl/well). Plates II were centrifuged at 2500 rpm for 10 min to precipitate bacteria and to retrieve the supernatant containing phage-VHHs. Phage-VHHs were then diluted with PGT in a ratio of 1/5 and transferred into the plates III and incubated at 37°C for 1 h. Anti-phage M13 IgG conjugated to HRP (GE Healthcare) diluted in PGT (1 OOpI) at 1/5000 were added for 1 h at 37°C. Subsequently, the reactions were developed by adding 100 pl of OPD (o-Phenylenediamine, Dako) and stopped by adding 50 pl of 3M HOI. The optical density was measured spectrophotometrically at 490 nm using Magellan microplate reader (Sunrise Tecan). A clone was considered as positive when SNR (signal-to-noise ratio) was greater than or equal to 10.

Expression of VHHs pHEN6 vector contained a His tag and a c-myc tag that allow the expression of VHHs in the periplasm without the phage context and their purification. Transformed E. coli TG1 cells expressed VHH in the periplasm after overnight induction with 0.25mM IPTG at

16°C. Purified VHHs were isolated by immobilized-metal affinity chromatography (IMAC) from periplasmic extracts treated by 10 U/ml Benzonase Nuclease (Merck, NJ) and Complete protease inhibitor (Roche) using a chelating agarose column charged with ²⁺ Protino Ni-NTA Agarose (Macherey-Nagel), according to the manufacturer’s instructions.

The coding sequences of the selected VHHs in the vector pHEN6 were sub-cloned into a bacterial expression vector pASK (IBA) containing a C terminal strep tag using Ncol and Notl restriction sites. Transformed E. coli L cells expressed VHH in the periplasm after overnight induction with anhydrotetracycline (200 pg/L) at 30°C. Purified VHHs were isolated on StrepTactin affinity columns from periplasmic extracts treated by 10 U/ml Benzonase Nuclease (Merck, NJ) and Complete protease inhibitor (Roche), according to the manufacturer’s instructions, followed by size exclusion chromatography with a Superdex 75 column (GE Healthcare).

Biotinylation of VHHs

The VHHs have been biotinylated by using the EZ-linkSulfo-NHS-biotin kit (Thermo) according to manufacturer’s instructions.

Enzyme-linked Immunosorbent Assay (ELISA)

A modified version of a standard ELISA was used to test for the presence of VHH. Maxisorp Nunc-lmmuno plates (Thermo Scientific) were coated with 1 pg/ml of Nucleoprotein or cell extracts (1/1000) overnight at 4°C. Plates were washed with buffer 0.1% Tween 20 in PBS. His & c-myc tagged VHHs were diluted in buffer 0.5% gelatin 0.1% Tween 20 in PBS. After 2 hours incubation at 37 °C, plates were washed again before adding a peroxidase labeled mouse anti-c-myc tag antibody (clone 9E10, Abeam). OPD (o-Phenylenediamine Dako) was used as substrate. Alternatively, plates were washed with buffer 0.1% Tween 20 in PBS. Strep-tagged VHHs were diluted in PBS containing 1% BSA and 0.1 % Tween 20. After 1 hour incubation at 37 °C, plates were rewashed before adding an anti-strep-tag mouse antibody followed by a peroxidase- labeled goat anti-mouse immunoglobulins (SouthernBiotech). TMB (3,3’-5’5- tetramethylbenzidine, SeraCare) was used as substrate. The optical density was measured spectrophotometrically at 490 or 450 nm, respectively, using a Magellan microplate reader (Sunrise Tecan). An anti strep-tag mouse antibody followed by a peroxidase labeled goat anti-mouse immunoglobulins (Vector labs) were used for the detection of the strep-tagged VHHs.

The recombinant proteins used for the coating were : SARS-CoV-1 and SARS-CoV- 2 N produced in E.coliand described above, seasonal human coronaviruses N from Sino Biological, and SARS-CoV-2 Spike protein was previously described (45).

Nasopharynx swab

Patients were sampled for nasopharyngeal swabs after a median duration of 9 days (interquartile range, 2 to 39) after disease onset. Nasopharynx specimens were obtained with sterile dry swabs (COPAN LQ Stuart Transport Swab, COPAN Italia SpA, Brescia, Italy), which were rotated 5 times around the inside of each nostril while applying constant pressure. Nasopharynx swabs were collected in the office under strict aseptic conditions. Prior to ELISA analysis nasopharyngeal swabs (1 ml) were treated in a P3 laboratory for viral decontamination. Briefly, samples were treated with Triton X100 (TX100) 1% (v/v) for 2hrs at RT. Nasopharyngeal viral loads were determined using RdRp-IP4 quantitative RT-PCR designed at the Institut Pasteur (National Reference Center for Respiratory Viruses) to target a section of the RdRp gene based on the first sequences of SARS- CoV-2 made available on the Global Initiative on Sharing All Influenza Data database on Jan 1 1 , 2020 (49). Primer and probe sequences: nCoV_IP4-14059Fw GGTAACTGGTATGATTTCG (SEQ ID NO: 57); nCoV_IP4-14146Rv CTGGTCAAGGTTAATATAGG (SEQ ID NO: 58); nCoV_IP4-14084Probe(+) TCATACAAACCACGCCAGG [5']Fam [3']BHQ-1 (SEQ ID NO: 59). The work described was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.

ELISA sandwich

VHH NTD-E4 has been coated on Maxisorp Nunc-lmmuno plates at 2 pg/ml. After washing with buffer 0.1% Tween 20 in PBS, nucleoprotein diluted at different concentrations was added for 1 hour at 37°C. Then biotinylated VHHs (0,5 pg/ml) have been added for 1 hour at 37°C followed by the addition of a peroxidase labeled streptavidin (Jackson ImmunoResearch).

Detection of Nucleoprotein in solution by an immunochromatographic assay

Rapid SARS-CoV-2 Antigen Test Card (MP biomedicals) have been used for the detection of N according to manufacturer’s instructions.

Sodium dodecyl sulfate-polvacrylamide gel electrophoresis (SDS- PAGE)

Polyacrylamide Gel electrophoresis (PAGE) was performed using NuPAGE Novex 4- 12% Bis- Tris gel (Invitrogen) according to manufacturer’s instructions. PageRuler Prestained Protein ladder was used as molecular weight maker and Instant Blue (Expedeon UK) was used to stain SDS-PAGE gel.

Epitope mapping by hydrogen deuterium exchange mass spectrometry.

A summary of the main HDX-MS experimental conditions is provided in Table 4 (50).

The quality and purity of the SARS-CoV-2 Nucleoprotein construct was assessed by intact mass analysis (Figure 15). All labeling were performed at room temperature in deuterated PBS 1X buffer, pD 7.4 (labeling buffer), unless specified.

Table 4: HDX summary data

^& considering a 1 to 1 binding stoichiometry between the nucleocapsid monomer and each VHH

# one unique charge state was selected per peptide

* MEMHDX (www.c3bi.pasteur.fr) Sample preparation.

The SARS-CoV-2 Nucleoprotein was labeled in the presence and absence of each VHH. Each complex was formed by mixing 6.6 pL of SARS-CoV-2 Nucleoprotein (12.7 pM in PBS 1X, monomer concentration) with 1.4 pL of VHH (stock solution at ~ 70 pM in PBS 1X). A control sample (Apo-state) was prepared in parallel by replacing the VHH solution with PBS 1X. After 30 min incubation at room temperature, the labeling was initiated by adding 61.9 pL of labeled buffer (final D2O/H2O ratio = 88.4/1 1.6%). The final concentration of each protein (SARS-CoV-2 Nucleoprotein = 1.2 pM; VHH = -1.4 pM) was carefully selected to avoid the dissociation of the homodimer (52) and to ensure than > 95% of SARS-CoV-2 Nucleoprotein remains in complex during the exchange reaction. Continuous labeling was performed for t = 0.16, 1 , 5, 10, 30, 60 and 120 min. Aliquots of 10 pL (i.e., 12 pmol of SARS-CoV-2 Nucleoprotein with or without 14 pmol of VHH) were removed and quenched upon mixing with 50 pL of an ice cold solution of 2% formic acid, 3M urea to decrease the pH to 2.50 (final D2O/H2O ratio = 14.7/85.3%). Quenched samples were immediately snap frozen in liquid nitrogen and stored at -80°C.

Undeuterated samples were obtained following the same experimental procedure. A fully deuterated control was prepared by mixing 6.6 pL of SARS-CoV-2 Nucleoprotein (12.7 pM, monomer concentration) with 1.4 pL of PBS 1X and 61.9 pL of labeled buffer supplemented with 8 M urea-d4 (final D2O/H2O ratio = 88.4/1 1.6%). After 21 h incubation at room temperature, samples were quenched as described above using a cold solution of 2% formic acid, 1 .6 M urea. All samples were prepared in triplicate for each time point and condition (with the exception of the 120 min time point for both VHH E10 and H3 where two replicates were acquired).

LC-MS Data acquisition.

Quenched samples were thawed and immediately injected onto an HDX manager connected to two nanoACQUITY UPLC M-Class pumps (Waters Corporation, Milford, MA). The temperature of the HDX manager was maintained at 0°C to minimize back exchange. 50 pL of each labeled sample (i.e., 10 pmol of SARS-CoV-2 Nucleoprotein with or without 11.7 pmol of VHH) were digested using an in-house packed column (2.0 x 20 mm, 63 pL bed volume) of immobilized pig pepsin agarose beads (Thermo Scientific, Rockford, IL) for 2 min at 20°C. Peptides were directly trapped and desalted onto a C18 Trap column (VanGuard BEH 1.7 pm, 2.1 x 5 mm, Waters Corporation, Milford, MA) at a flow rate of 100 pL/min (0.15% formic acid) and separated by a 8 min linear gradient of 5-30% acetonitrile followed by a short 2 min increase from 30% to 40% of acetonitrile at 40 pL/min using an ACQUITY UPLC BEH C18 analytical column (1.7 pm, 1 x 100 mm,

Waters Corporation, Milford, MA). After each run, the pepsin column was manually cleaned with two consecutive injections of 1% formic acid, 5% acetonitrile, 1.5 M guanidinium chloride, pH 1.7. Blank injections were performed between each run to confirm the absence of carry-over.

Mass spectra were acquired in resolution and positive ion-mode (m/z 50-2000) on a Synapt G2-Si HDMS mass spectrometer (Waters Corporation, Milford, MA) equipped with a standard ESI source and lock-mass correction. Peptic peptides were identified in undeuterated samples by a combination of data independent acquisition (MS^E) and exact mass measurement (below 10 ppm mass error) using the same chromatographic conditions than for the deuterated samples. Four distinct MS^E trap collision energy ramps were employed to optimize the efficiency of the fragmentation: 10-30V (low), 15-35V (medium), 20-45V (high), and 10-45V (mixed mode).

Data processing.

The initial SARS-CoV-2 Nucleoprotein peptide map was generated by database searching in ProteinLynX Global server 3.0 (Waters corporation, Milford, MA) using the following processing and workflow parameters: low and elevated intensity thresholds set to 100.0 and 50.0 counts; intensity threshold sets to 750.0 counts; automatic peptide and fragment tolerance; non-specific primary digest reagent; false discovery rate sets to 4%. Each fragmentation spectrum was manually inspected for assignment confirmation. The N-arm (residues 1 -45), LOK (residues 180-246), the charge-rich CTD N-terminal region (residues 247-267) and the C-tail (residues 363-419) domains of SARS-CoV-2 Nucleoprotein contain a high proportion of residues not tolerated by pig pepsin (i.e., Proline, Lysine, Histidine or Arginine) resulting in a lack of sequence coverage or resolution (Figure 16). The use of Type XIII protease from Aspergillus saitoi (Sigma Aldrich) either immobilized on POROS 20-AL beads (Applied Biosystems, Bedford, MA) or in solution did not improve the final sequence coverage and resolution. Pig pepsin was therefore selected to perform local HDX analysis. The peptide map was further refined in DynamX 3.0 (Waters corporation, Milford, MA) using the following Import PLGS results filter: minimum intensity = 3000; minimum products per amino acid = 0.15; minimum score = 6.5; maximum MH+ error (ppm) = 10; file threshold = 2.

DynamX 3.0 was used to extract the centroid masses of all peptides selected for HDX-MS. One unique charge state was used per peptide and no back-exchange correction was performed. HDX-MS results are reported as relative deuterium exchange level expressed in either mass unit or fractional exchange. Fractional exchange data were calculated by dividing the experimental uptake value by the theoretically maximum number of exchangeable backbone amide hydrogens that could be replaced into each peptide in 88.4% excess deuterium. Overlapping peptides covering the same region were only used to increase the spatial resolution if their experimental back-exchange values were similar (difference <10%, Table 4). The MEMHDX software (52) was used to visualize and statistically validate HDX-MS datasets (Wald test, false discovery rate of 1 %, biological threshold sets to 3%, Table 4).

Kinetic characterization by Surface Plasmon Resonance (SPR)

Experiments were performed using a Biacore T200 instrument (GE Healthcare) equilibrated at 25°C in SPR buffer (PBS-300mM NaCI containing 0.1% Tween-20 , 0.2mg/ml BSA and 100pM EDTA).

Approximately 500 RU ((1 RU«1 pg. mm ²) of Nucleoprotein were captured non covalently on an NiCh-loaded NTA sensor chip (GE Healthcare). VHHs were then injected at 30| l/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- Nucleoprotein 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 (K_on) and dissociation (k_Off) rates of the interactions, as well as their equilibrium constants (Kd).

Biolaver interferometry (BLI)-analysis

Association and dissociation of recombinant N protein to VHHs was analyzed via BLI- analysis using Octet HTX-equipment (Fortebio, Reading, UK). VHHs (50 pg/ml) diluted into 10 mM Acetate buffer (pH 5.0) were immobilized onto amine reactive biosensors as recommended by the manufacturer (Fortebio), unoccupied amine-reactive sites were quenched by incubating with ethanolamine (1 M). VHHs-coated biosensors were incubated with various concentrations of N protein (0-205 nM) in PBS/0.1 Tween-20 for 15 min to allow association. Subsequently, biosensors were put in PBS/0.1 % Tween 20 for 15 min to initiate dissociation. All incubations were performed at 30°C under continuous shaking (1 ,000 rpm). Data were analyzed using Octet Software version HT10.0 using a 1 :1 fitting model.

Immunofluorescence

RhK4 (Fetal Rhesus monkey Kidney) cells were grown in 96 wells plate coated with poly-D-lysine. Infection was performed at 37°C on exponentially growing cells at a multiplicity of infection of 10² in order to have approximately one out of two cell infected with SARS-CoV-2 virus after 24 hours. Cells were fixed 20 min at 4°C with 2% PFA in PBS (v/v) and permeabilized 10 min at 4°C with 0.2% Triton X100 in PBS (v/v). Cells were incubated at room temperature with PBS-BSA 3% 1 hour without VHH then 1 hour with biotinylated VHH at 1 ug/mL or rabbit polyclonal antibodies against Nucleoprotein - SARS-CoV-2 as a control of cell infection. To label the VHH a streptavidin Alexa Fluor 488 (Thermofisher) in PBS-BSA 3% was incubated for 1 hour at room temperature according to manufacturer’s instructions. Immunofluorescence was observed at 40X on a fluorescent microscope (Zeiss).

SARS-CoV-2 intranasal inoculation and tissue imaging

Animal procedures were performed according to the French legislation and in compliance with the European Communities Council Directives (2010/63/UE, French Law 2013-118, February 6, 2013). The Animal Experimentation local Ethics Committee (CETEA 89) approved this study (2020-0023). The animals were housed and manipulated in isolators in a Biosafety level-3 facility. Male Mesocricetus auratus Syrian hamsters of 5-6 weeks of age (Janvier, Le Genest Saint Isle, France) were intranasally inoculated under anesthesia (intraperitoneal injection of ketamine (200mg/kg) and xylazine (10mg/kg)) with 100 pl of SARS-CoV-2 (50 pl/nostril) (isolate IDF0372/2020, EVAg collection, Ref-SKU: 014V-03890) at 6x10⁴ PFU (plaque-forming units))- or physiological solution as previously described (53).

Lungs were collected at 4 days post-infection, formalin-fixed after transcardial perfusion of hamsters with a physiological solution containing heparin (5 X_ 10³ U/ml, Sanofi) followed by 4% paraformaldehyde in phosphate buffer. Tissues were postfixed by incubation in the same fixative during one week, cryoprotected by incubation in 30% sucrose in PBS overnight, and then embedded in Tissue-tek (Sakura). Lung 20-pm-thck transverse sections were obtained using a cryostat (CM3050S, Leica) and were thawmounted onto coated glass slides (Superfrost Plus). Antigen retrieval was performed by incubating sections for 20 minutes in citrate buffer 0.1 M pH 6.0 at 96°C and then blocked in 0.4% Triton, 4% fetal bovine serum (Sigma) and 10 % goat serum (ThermoScientific). They were incubated overnight at 4°C with biotinylated VHHs diluted 1/500, rinsed in PBS and followed by a 2 hour-incubation step with Alexa 568-conjugated streptavidin (Jackson ImmunoResearch Laboratories) at room temperature. Fluorescent sections were stained with the nuclear dye HOESCHT and then mounted in Fluoromount solution (Invitrogen).

Example 2: Nucleoprotein Production and characterization cDNAs encoding the native nucleoprotein antigen (NPJSARS2) from 2019-nCoV (SARS-CoV-2) was designed base on the Genbank MN908947 sequence publicly available from NBCBI on 20th January 2020. This sequence was then processed to generate an optimized nucleotide sequences for high expression in E coli. Optimization process includes codon adaptation, mRNA de novo synthesis and stability, transcription and translation efficiency. Bsal and Xhol/EcoRI/Notl restriction sites were then added at the 5’ and 3’ ends, respectively, of the nucleotide sequences. The resulting optimized cDNA named "N-Ecoli optimized gene" was synthesized. The Bsal-Xhol fragment of the "N-Ecoli optimized gene" has been inserted into Ncol/Xhol-digested pETM-11 vector and the resulting pETM1 1-Necoli_2019-nCoV (= pETM11/N-nCov E. coli) has been used to produce a fusion polypeptide between the SARS-CoV-2 protein and a N-terminally located poly-histidine tag (6 histidine), separated by a TEV cleavage site.

The resulting His6-N_2019-nCoV (NJSARS2) polypeptide has the sequence: 1 MKHHHHHHPM SDYDIPTTEN LYFQGAMSDN GPQNQRNAPR ITFGGPSDST GSNQNGERSG

61 ARSKQRRPQG LPNNTASWFT ALTQHGKEDL KFPRGQGVP I NTNSSPDDQI GYYRRATRRI

121 RGGDGKMKDL SPRWYFYYLG TGPEAGLPYG ANKDGI IWVA TEGALNTPKD HIGTRNPANN 181 AAIVLQLPQG TTLPKGFYAE GSRGGSQASS RSSSRSRNSS RNSTPGSSRG TSPARMAGNG

241 GDAALALLLL DRLNQLESKM SGKGQQQQGQ TVTKKSAAEA SKKPRQKRTA TKAYNVTQAF 301 GRRGPEQTQG NFGDQELIRQ GTDYKHWPQI AQFAPSASAF FGMSRIGMEV TPSGTWLTYT

361 GAIKLDDKDP NFKDQVILLN KHIDAYKTFP PTEPKKDKKK KADETQALPQ RQKKQQTVTL

421 LPAADLDDFS KQLQQSMSSA DSTQA** (SEQ ID NO : 41 ) .

Nucleoprotein coding sequences (WT-CoV-2 SARS DNA and E. coli optimized CoV- 2 SARS DNA,) are cloned into pETM11 vector (EMBL; Dummler et al (2005), Microb Cell Fact 13;4:34) or plVEX2-3 (Roche vector) vectors. The N-recombinant Nucleoprotein of CoV-2-SARS is produced in E. coli BL21 (DE3) pDIA17 as a fusion protein comprising an N- or C-terminal (His)e polyhistidine label. Concerning the production of N-recombinant Nucleoprotein with a (His)e N-terminal label, the following recombinant vectors are used for the transformation of E. coli strain BL21 (DE3) pDIA17: pETM11/N-nCov WT4-(His)₆-Nter pETM11/N-nCov WT6-(His)₆-Nter pETM11/N-nCov E. coli 3 -(His)e-Nter pETM11/N-nCov E. coli 4 -(His)₆ -Nter pIVEX/nCov WT-(His)₆-Nter Clone 1 pIVEX/nCov WT-(His)₆-Nter Clone 2 E. coli strain BL21 (DE3) pDIA17 transformed with recombinant plasmid pETM11/N- nCov E. coli 3 -(His)e-Nter or pETM11/N-nCov E. coli 4 -(His)e-Nter (Bacterium_N- Cov_Ecoli_PETM11_coli3 and Bacterium_N-Cov_Ecoli_PETM11_coli4) were deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) at the Institut Pasteur, 25, Rue du Docteur Roux, 75724 Paris, FR, on May 11 , 2020 , under the deposit numbers CNCM 1-5510 and CNCM 1-5511 , respectively.

Cultures in Thomson flasks shaken in LB medium (IPTG induction) and NZytech medium (self-inducible) of E. coli BL21 (DE3) pDIA17 strains transformed by the pETM11 vector or by the pIVEX 2.3 vector.

The Thomson flasks are 2.5 L notched flasks allowing cultures of 1 litre of medium to be aerated under good aeration conditions in stirrers.

Production in LB environment (IPTG induction)

The 4 strains of E. coli BL21 (DE3) pDIA17 transformed by the pETM11 vector (DMSO no. 1535, 1536, 1537, 1538) are spread on an agar LB Petri dish containing 50 pg/ml kanamycin and 30 pg/ml chloramphenicol. The 2 strains of E. coli BL21 (DE3) pDIA17 transformed by the vector plVEX2.3 (DMSO n° 1539, 1540) are spread on an agar LB Petri dish containing 100 pg/ml ampicillin and 30 pg/ml chloramphenicol. All plates of LB Agar Petri LB are incubated overnight at 37°C in an oven.

From each of the 6 Petri LB agar plates are inoculated with a platinum handle, 6 precultures of 500 ml of LB medium in 2.5 L Thomson flasks (LB medium plus antibiotics appropriate to each recombinant vector pETM11 and plVEX2.3). These pre-cultures are shaken at 180 rpm in a Multitron Infors shaker for 15 h at 30°C. From the 4 LB pre-cultures of BL21 (DE3) pDIA17 strains transformed by the pETM11 vector (DMSO No. 1535, 1536, 1537, 1538) are seeded at an initial cell density equivalent to DOA600 = 0.2, cultures of 1 L of LB medium containing 50 pg/ml kanamycin and 30 pg/ml chloramphenicol.

From the 2 LB pre-cultures of BL21 (DE3) pDIA17 strains transformed by the pIVEX 2.3 vector (DMSO No. 1539, 1540) are seeded at an initial cell density equivalent to DOA600 = 0.2, cultures of 1 L of LB medium containing 100 pg/ml ampicillin and 30 pg/ml chloramphenicol.

All these cultures in LB medium are placed under agitation at 180 rpm and 30°C. When the cell density, equivalent to DOA600 = 0.8 is reached the cultures are induced by addition of 1 mM IPTG and the temperature is maintained at 30°C.

After 2 hours at 30°C in the presence of the inducer the cultures are stopped. A 10 ml sample of each culture is centrifuged and will be used for analysis on SDS-Page of the total soluble and insoluble protein fractions. The remainder of each culture is centrifuged (15 min at 6000 rpm) and the pellets stored at -80°C.

Production in NZvtech medium (self-inducible)

From the 4 LB pre-cultures of BL21 (DE3) pDIA17 strains transformed by the pETM11 vector (DMSO No. 1535, 1536, 1537, 1538) are seeded at an initial cell density equivalent to DOA600 = 0.2, 1 L cultures in NZytech (self-inducible) medium containing 50 pg/ml kanamycin and 30 pg/ml chloramphenicol.

From the 2 LB pre-cultures of BL21 (DE3) pDIA17 strains transformed by the pIVEX

2.3 vector (DMSO No. 1539, 1540) are seeded at an initial cell density equivalent to DOA600 = 0.2, 1 L cultures in NZytech (self-inducible) medium containing 100 pg/ml ampicillin and 30 pg/ml chloramphenicol.

Cultures in NZytech medium (self-inducible) are carried out at 37°C with stirring at 180 rpm.

After 4 hours at 37°C, the cultures are placed at 18°C.

After 15 hours of culture at this temperature of 18°C, the bacterial cultures are stopped. A 10 ml sample of each culture is centrifuged and will be used for analysis on SDS-Page of the total soluble and insoluble protein fractions. The remainder of each culture is centrifuged (15 min at 6000 rpm) and the pellets stored at -80°C.

Cultures in BioPod F200 microfermenters in high cell density HDM medium ( I PTG induction) of E. coli BL21 (DE3) pDIA17 strains transformed by the pETM1 1 vector or by the pIVEX 2.3 vector:

The HDM medium is a complex culture medium developed by our Platform specifically designed for the large production of E.coli biomass in a bioreactor during batch culture. This buffered medium does not require a regulation of the pH value in culture.

Microfermenters are miniaturized bioreactors allowing to realize 100 ml cultures in high density medium (HDM medium). These micro-fermenters are equipped with mass flow meters and sinter allowing a very efficient micro-bubbling by air progressively enriched with oxygen according to the bacterial growth. These bioreactors are also equipped with Peltier system and PT1000 probe which allow a very reliable regulation of the growth temperature and fast passages from 37°C to 16°C during the induction phase. This system of miniaturized bioreactors is a tool for optimizing the culture conditions allowing with a high rate of reliability a scale-up of 100 ml cultures to larger volume reactors (4L and 16 L in our Platform).

The 2 strains of E. coli BL21 (DE3) pDIA17 transformed by the pETM11 vector (DMSO n° 1535 and 1537) are spread on an agar LB Petri dish containing 50pg/ml kanamycin and 30pg/ml chloramphenicol.

The 2 strains of E. coli BL21 (DE3) pDIA17 transformed by the vector plVEX2.3 (DMSO no. 1539, 1540) are spread on an agar LB Petri dish containing 100pg/ml ampicillin and 30pg/ml chloramphenicol. All LB agar plates are incubated overnight at 37°C in an oven.

About 1 .5 ml of antibiotic-free LB medium is deposited on each of the agar plates. The bacterial mat of each LB plate is scraped off with a sterile rake. Each bacterial suspension collected is used to inoculate a micro-fermentor containing 100 ml of HDM medium plus antibiotics appropriate for E. coli BL21 (DE3) pDIA17 strains transformed by the recombinant pETM11 or plVEX2.3 vectors. The initial cell density of the bioreactors is equivalent to DOA600= 0.8 to 1 .

The cultures are grown at a temperature of 37°C, and aeration is set at 0.5 VVM. When the cell density equivalent to DOA600 = 18 to 20 is reached, the temperature is lowered to 16°C and IPTG (1 mM) is added to the cultures.

After 15 hours of culture at 16°C in the presence of the inducer, the bacterial cultures are stopped. A 1 ml sample of each culture is centrifuged and will be used for analysis on SDS-Page of the total soluble and insoluble protein fractions. The remainder of each culture is centrifuged (15 min at 6000 rpm) and the pellets stored at -80°C.

Purification from pETM11/N-nCov E. coli-(His)6-Nter/NZytech cultures Data prot param:

- MW = 48.7 KDa

- pl = 9.9

- Ext. coefficient

- Abs 0.1 % (=1 g/l) : 0.961

Bacterial pellet breakage

Take the 9g pellet with 50 ml buffer A: 50mM phosphate, 300mM NaCI, 20mM imidazole pH8 with 1 Roche EDTA free protease tablet and 5 pl benzonase in the blender/wait incubation at room temperature for approx. 20 min.

1. Cold breaking with the Cell D 1.3 kbar Cell Disrupter.

2. Addition of eNASR A (250 pl to 10 mg/ml or 2.5mg). Incubation at room temperature for about 20 min.

3. Centrifugation 19000rpm rotor SS34 1 hour 4°C

4. Recovery of the soluble fraction = supernatant for affinity purification on nickel resin.

Treatment of the soluble fraction

- 1 st STEP OF PURIFICATION: AFFINITY IMAC (AKTAPure): 1 column Nickel 5ml

■ 1 New 5 ml Protino Ni-NTA column (Macherey Nagel) mounted on AKTA Pure (room temperature) o Washing of the column in H2o: 10CV o Column equilibration buffer: Phosphate 50mM, NaCI 300mM, imidazole 20mM pH8: 10 CV

■ Loading the 60 ml crude extract onto the 5 ml IMAC column at a rate of 1 ml/min with the AKTA pump o Flow rate: 1 ml/min o Washing with Phosphate buffer 50mM, NaCI 300mM, imidazole 20mM, pH8: 10CV ■ Elution : o Elution Buffer: Phosphate 50mM, NaCI 300mM, imidazole 250mM pH8 o Gradient from 20 to 250 mM imidazole = 100% buffer B at 2 ml/min on 10 HP. o Fractions of 1 .5 ml were recovered ■ Histogram peak integration for protein quantity estimation

Peak of the fractions from A5 to C12, i.e. 48 ml at 3,3 mg/ml. Estimated quantity on unicorn162 mg

■ Fraction analysis at this stage on SDS-Page (Figure 10)

DQ280nm measurement at 1/10 of the A5 to C9 pool

DO280 = 0.364. For 1 g/l, the OD is 0.96 3.64 / 0.96 = 3.7 mg/ml. Either for 42 ml: 42 ml x 3.7 mg/ml = 159 mg total

The elution volume will be injected in 8 x 5 ml on 2 gel filtration columns with 5 ml loops. The columns are installed on the 2 pure AKTAs.

4 runs of gel filtration will be performed per column.

2ND PURIFICATION STEP: Hiload 16/60 Superdex 200 pg (120ml) column filtration Equilibration of the columns with 50mM Phosphate buffer, 500mM NaCI pH8,

Flow rate 0.5 ml/min

Protein injection with 5 ml and 10 ml loops.

Elution volume for each run 1 .4 column volume. Fractions of 1 .8 ml. ■ Selection of peaks on histograms and integration of peaks for estimation of protein quantity (Figure 11 and 12)

SDS-Paqe Fraction Removal and Deposition on SDS-Paqe Gel

[0002] GEL FILTRATION 6 on AKTA 2 (Figure 14)

[0003] Formation of the gel fraction pool filtration

GF 1 : 1 B1 TO 1 B8

GF 2: 2A6 TO 2A12 GF 3 : 2H6 TO 2H12

GF 4 : 3G6 TO 3G12

GF 5: 1A11 TO1 B6

GF 6: 1 H8 TO 2A2

GF 7: 2G5 TO 2G11 GF 8: 3F2 TO 3F8

■ 280nm OP measurement of the pool

DO280 = 0.597. For 1 g/l, the OD is 0.96. 0.597/ 0.96 = 0.62 mg/ml. Either for 100 ml: 00 ml x 0.6 mg/ml = 60 mg total

PURIFICATION AND STORAGE BALANCE NP_SARS2 purified protein (concentration 0.62 mg/ml in phosphate 50mM NaCI 00mM pH8). Pool gel filtration 80 ml split into 4 parts: 20 ml filtered without antiproteases 7 aliquots of 0.5 ml stored at +4°C (box in cold room; 20 ml filtered with antiproteases 6 aliquots of 0.5 ml stored at +4°C (box in cold room); 20 ml unfiltered 40 aliquots of 0,5 ml stored at -80°C (box in freezer room); 20 ml unfiltered + glycerol 50%final 42 aliquots of 1 ml stored at -20°C (box in freezer room).

E. coli optimized SARS-CoV-2 DNA cloned into pETM-1 1 expression vector gave highest protein production yields in E.coli. Unexpectedly, the clones pETM11/N-nCov E. coli 3 -(His)6-Nter and pETM11/N-nCov E. coli 4 -(His)6-Nter were able to achieve high level production without protein aggregation.

Example 3: Selection and characterization of VHHs recognizing Nucleoprotein

One alpaca was immunized with the recombinant SARS-CoV-2 nucleoprotein and a VHH-specific library was constructed from cDNA encoding VHH domains isolated from lymphocytes. The total size of the library contained 5.85x10⁷ different phage-VHHs. VHHs were selected by phage display through 3 panning cycles with nucleoprotein at different buffer and washing conditions. Four hundred individual clones were tested by ELISA using Nucleoprotein. Five different VHHs were obtained, called D12-3, E7-2, E10-3, G9- 1 , H3-3 respectively (Figure 1 ).

The first VHHs were recognizing the CTD domain of nucleoprotein (see below). So another panning was performed with NTD by using the same library. The same panning procedure was performed and 5 different VHHs were isolated, called NTD E4-3, NTD H7- 1 , NTD C7-1 , NTD F11 -1 and NTD B6-1 .

C terminal Strep-tagged VHHs were obtained after subcloning of their genes in pASK vector. VHHs had production yields about 0,1 mg/L and 1 mg/L of culture after streptactin affinity chromatography from periplasmic extracts. NTD H7-1 and NTD F11 -1 were produced at a very low yield and were not studied anymore. Quality control was performed and VHHs are not aggregated, have the expected molecular mass and are pure by MS and SDS-Page. By ELISA, the different VHHs showed strong binding to Nucleoprotein (Fig. 3). A comparison of binding of the five VHHs for nucleoprotein from SARS-CoV-1 and SARS-CoV-2 was performed by ELISA. The amount of VHHs giving 50% of binding was determined for both nucleoproteins. Even if the different VHHs can recognized both proteins, D12-3 and E10-3 recognized preferentially SARS-CoV-2 nucleoprotein (Table 1 )-

Table 1 : Comparison of the binding of VHHs on SARS and SARS-CoV 2 Nucleoproteins. An ELISA was performed by using the VHHs diluted at different concentrations on coated Nucleopreoteins. The amount of VHHs (ng/ml) giving 50% of the binding was calculated.

Determination of kinetic constants was performed by Biolayer Interferometry and Biacore. The different VHHs showed high affinities to the Nucleoprotein with apparent KD constant (KD app) in the nanomolar range (Table 2 and Table 5). The measured kinetic constants correlate with the binding observed by ELISA with VHH E7-2 giving the highest signal and an affinity of 0.206 nM and NTD B6-1 having the lowest signal and an affinity of 46.5 nM.

Table 2: Kinetic parameters of the interaction between the SARS-CoV-2 Nucleoprotein and the different VHHs (D12-3, E7-2, E10-3, G9-1 , H3-3, NTD B6-1 , NTD E4- 3).

Table 5: Kinetic analysis by Biolayer Interferometry of SARS-Cov-2 Nucleoprotein binding to different VHHs (D12-3, E7-2, E10-3, G9-1, H3-3, NTD B6, NTD C7, NTD E4).

Then an ELISA was performed on infected and uninfected cell extracts, the maximal difference of OD obtained between infected and uninfected is represented in Figure 3B. All the VHHs recognized the Nucleoprotein present in infected cell extracts. The different bar colors represent the concentration of VHH for which the maximal difference was obtained. VHHs can be classified in different groups: E7-2 shows the better signal for infected cells at a concentration as low as 4 ng/ml; VHH H3-3 presenting the optimal signal at 0.25 pg/ml, VHHs G9-1 and NTD-E4-3 at 1 pg/ml and finally E10-3, D12-3 and NTD-B6-1 at 4 pg/ml. These variations can be explained by the signal observed at high concentrations on uninfected cells.

We explored the VHHs’ specificity to SARS-CoV-2 N in ELISA by comparing their binding to the seasonal human coronaviruses (OC43, HKLI1 , 229E and NL63), the SARS- CoV-1 and SARS-CoV-2 using SARS-CoV-2 spike protein as a control (Figure 4). No binding was observed with the Nucleoprotein of seasonal coronaviruses, suggesting a high specificity of VHHs to SARS-Cov-2 Nucleoprotein. VHHs NTD-E4-3, D12-3 and E10- 3 present a better recognition of SARS-CoV-2 Nucleoprotein than SARS-CoV-1 Nucleoprotein. Nevertheless VHH E7-2 interacts with the Nucleoprotein of seasonal coronavirus as well as with the spike protein but to a lesser extent than for SARS-CoV-1 and SARS-CoV-2. This non specific binding is difficult to explain because E7-2 presents a high affinity for SARS-CoV-2 N.

Example 4: Identification of the SARS-CoV-2 Nucleoprotein antigenic regions recognized by each VHH

HDX-MS was used to locate the binding sites of each VHH on full-length SARS-CoV- 2 Nucleoprotein. The quench and pepsin conditions were first optimized to generate a peptide map with high sequence coverage and peptide redundancy. A total of 51 unique peptides covering 94.4 % of the SARS-CoV-2 Nucleoprotein sequence with a 2.43 redundancy value were selected and used for HDX-MS (Figure 16).

The deuterium uptake profile of SARS-CoV-2 Nucleoprotein in the apo-sate reveals the presence of two main structural contents. Dynamic HDX-MS behavior, resulting from stable secondary structural elements, was observed throughout the NTD and CTD domains, confirming that both regions are well-folded (Figure 5a). A few CTD peptides, namely 315-322 (oc5-(31 ), 316-322 (oc5-(31 ) and 330-336 (|32), give no dynamic HDX-MS pattern and exhibit very low incorporation (< 20%) after 120 min incubation time. These peptides locate at the dimerization interface and contain hydrophobic residues identified as important in the stabilization of the SARS-CoV N CTD homodimer (7). Moreover, the absence of bimodal isotopic pattern at the dimerization interface confirms that the homodimer does not dissociate and remain stable during labeling. The backbone amide hydrogens covering the N-arm, the LKR and the C-tail domains on the other hand are fully exchanged from the first time point to the last (Figure 5a). This result provides direct evidence that, under our experimental conditions, the N-arm, the LKR and the C-tail regions are fully disordered and do not contain stable secondary structural elements. The two structured CTD and NTD domains of SARS-CoV-2 Nucleoprotein appear therefore flanked by three long intrinsically disordered segments (Figure 5b), as previously observed with the SARS-CoV Nucleoprotein (54).

Epitope mapping was performed by comparing the SARS-CoV-2 Nucleoprotein deuterium exchange profiles between the apo- and the VHH-bound states. The relative fractional uptake difference plots obtained with each VHH are presented in Figure 6a. A positive uptake difference value indicates a VHH-induced protective effect on the exchangeable amide hydrogens (i.e., uptake reduction within the complex). As reported in Figure 6a, the binding of all VHHs reduces the solvent accessibility of elements located in the CTD domain only, with no effect on the other regions of the protein. This result reveals that the CTD domain contains the antigenic regions recognized by the five VHHs. The formation of the G9-1 D12-3 and E10-3/SARS-CoV-2 Nucleoprotein complexes affects the solvent accessibility of the same peptides. The main differences are observed in peptides 268-269 (oc2), 274-291 (Ioop-oc3), 315-330 (oc5-(31 -loop-(32), 323-330 and 323- 331 ((31 -loop-(32). Inspection of the different overlapping peptides reveals that the main reduction of solvent accessibility is restricted segment 274-291 and 323-330. These two regions are mainly composed of solvent accessible loops that locate on the same side of the CTD domain and in close vicinity (Figure 6b). Based on this observation, the epitope recognized by G9-1 , D12-3 and E10-3 appears to be conformational and formed by elements of the same monomer. The binding of H3-3 shows no effect on the solvent accessibility of region 323-330 but slightly reduces the uptake of peptides 268-269 (oc2), 274-291 (Ioop-oc3) , 315-322 (oc5-

P1 ), 315-330 (oc5-(31 -loop-|32) and 316-322 (oc5-(31 ) (Figure 6a). The different overlapping peptides covering these two regions allow confining the effect of H3 to regions 274-291 and 315-322. The 315-322 segment lies just beneath the 274-291 region and does not appear to be solvent accessible on the CTD crystal structure (Figure 6b). Based on this observation, the slight decrease in solvent accessibility observed in 315-322 might be attributed to a change in dynamics rather than a masking effect. The epitope recognized by H3-3 is therefore linear and formed by the long loop connecting oc2 to oc3.

Finally, the epitope recognized by E7-2 shares elements with G9-1 , D12-3, E10-3 and H3-3 (Figure 6a). The main variation in deuterium uptake takes place in regions 274-291 , 315-322 and 323-330 (Figure 6b). As observed with G9-1 , D12-3, E10-3, the epitope recognized by E7-2 is probably conformational.

Example 5: Recognition of infected cells by Immuno Fluorescence

FRhK4 cells were infected with the SARS-CoV-2 virus. After 24 hours, the subconfluent layer of cells was fixed and permeabilized. A control with rabbit polyclonal antibodies against SARS-CoV-2 N labelled with an anti-rabbit Alexa Fluor 488 allowed us to evaluate the cell infection around 50%. Biotinylated VHHs were used at a concentration of 1 pg/mL and labelled with streptavidin Alexa-Fluor 488. All the fluorescent VHHs labelled the infected cells, as shown in Figure 7, whereas no labelling was observed on uninfected cells (data not shown), suggesting that they all recognized the SARS-CoV-2 virus in situ. The exposition for imaging needed to be adjusted for each VHH. Those variabilities in sensitivity are consistent with the different affinities observed between the VHHs.

Example 6: Recognition of SARS-CoV-2 virus on infected hamster tissues

Syrian hamster has been infected with SARS-CoV-2 virus. The lungs were recovered and VHH has been used 1/100 (5pg/ml) and 1/500 (1 pg/ml). A control was performed with non-infected hamster lung. Nice labeling of virus present in broncho-alveolar epithelium was observed with biotinylated VHH G9-1 (Figure 8).

However, significant variations of labelling intensity were observed between the different VHHs used. Even if we cannot exclude that these could be due to the differences in affinity of the VHHs or to the accessibility of their epitopes, the most likely explanation is that the number of infected cells varied somewhat between different sections of lung tissue. Each VHH was indeed incubated within a different section of the lung, and although all sections were adjacent in the tissue, the size of the foci of infection varied from one section to another.

Example 7: Detection of Nucleoprotein in solution by ELISA sandwich

An ELISA sandwich was set-up to detect in solution the full-length SARS-CoV-2 Nucleoprotein. The three best VHHs against CTD (E7-2, G9-1 and H3-3) were used in combination with the two anti-NTD VHHs (NTD-B6-1 and NTD-E4-3) to determine which was the optimal couple for the detection of N. One couple give the best results: VHH NTD E4-3 coated on the plate and biotinylated VHH G9-1 directed against CTD for the detection (Figure 20). VHH G9-1 and NTD-E4-3 gave respectively the better signal for the anti-CTD VHHs and the anti-NTD VHHs (Figure 21 A). As little as 4 ng/ml of SARS- CoV-2 N could be detected. The sandwich ELISA E4-3/G9-1 is specific of SARS-CoV-2 N because no detection of seasonal human coronaviruses Nucleoprotein was observed (data not shown).

Those VHHs were also able to detect inactivated and permeabilized SARS-CoV-2 virus. The same ranking was observed: NTD-E4-3 in combination with the CTD G9-1 was the best couple to detect the SARS-CoV-2 Nucleoprotein in native conditions (Figure 20B).

SARS-CoV-2 replicate mainly in human upper respiratory tract (56,57). Therefore, human nasopharyngeal swabs from healthy controls and COVID-19 patients have been tested for the detection of N by the sandwich ELISA using VHHs NTD E4-3 and G9-1 (Figure 21 A). The results from the sandwich ELISA were compared to an available commercial immunochromatographic assay. The immunochromatographic assay can detect as little as 1 ng/ml of N (Data not shown). Quantification of Nucleoprotein by ELISA was made possible by using a reference curve (Figure 21 B). 5 samples were considered as negative by PCR (#1 , #2, #3, #6, #79) and 12 positive (#4, #5, #7, #12, #14, #22, #30, #45, #47, #58, #60, #64, #67). We observed a good correlation between the 2 techniques with the different samples (Table 6).

Table 6: Comparison of the presence of Nucleoprotein detected in human nasal swabs by an immunochromatographic assay and a sandwich ELISA

No detection of Nucleoprotein was observed in samples #1 , #2 and #3 either by the immunochromatographic assay or by ELISA. However detection of Nucleoprotein was observed in samples #6 (healthy control, PGR neg) by immunochromatographic assay but not by VHH ELISA. On the other hand, the ELISA is able to detect Nucleoprotein in sample #79 even if were considered as negative by PGR (39 days post-infection). For positive samples, no detection of Nucleoprotein was observed for samples #14, #22 and #30 by using both techniques. We observed a discrepancy between sample #60 with the presence of a band in the immunochromatographic assay and no detection by ELISA.

Example 8: Detection of SARS-CoV-2 variants of concern

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 (62, 63). 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 (64). Sections of formalin-fixed lungs were incubated with 2 pg/ml of biotinylated VHHs NTD E4-3 and G9-1. Uninfected mouse lung was used as control. Strong labelling was observed with both VHHs in mice infected with either variants (Figure 22).

We also performed a sandwich immunoassay on uninfected and infected cell extracts using Wuhan, B.1 .1 .7 and B.1 .351 variants (Figure 23) and found that both VHHs NTD E4-3 and G9-1 recognized the N protein in all infected cell extracts.

Example 9: Expression of VHH-Fc fusion proteins and detection of SARS CoV- 2 Nucleoprotein

Genes encoding for VHH NTD E4-3 and VHH G9-1 have been cloned previously in pHEN6 plasmids. These plasmids were used as templates for the cloning of the nanobodies into the pFuse-hlgG1 -Fc2 expression vector (66) for the expression of the dimeric VHH-Fc fusion proteins. The proteins have been expressed in eukaryotic Expi293TM cells by using the ExpiFectamineTM 293 Tranfection kit (thermofisher) according to the manufacturer’s instructions. The VHH-Fc fusion proteins have been purified on Protein G column followed by a gel filtration. The VHH-Fc proteins have been biotinylated by using the EZ-Link Sulfo-NHS-Biotin kit (Thermofisher) according to manufacturer’s instructions.

A sandwich ELISA was set-up to detect in solution the full-length SARS-CoV-2 N. VHH NTD E4-3 Fc hu was coated on an ELISA plate and biotinylated VHH G9-1 Fc hu was added to detect the Nucleoprotein (Figure 24). As little as 1 ng/ml of SARS-CoV-2 N could be detected.

Discussion

We have obtained 10 different VHHs that recognize the SARS-CoV-2 Nucleoprotein. To this date, these are the first described VHHs directed against this protein. N was expressed in E. coli as a dimer. By HDX-MS we have confirmed that the NTD and CTD regions are structured unlike the N-arm, the LKR region and the C tail. N was then used for immunization of an alpaca. The first VHHs isolated after panning with the whole protein were directed against CTD. Another panning with NTD was required to isolate VHHs specific of this domain. This bias might be due either to the fact that N coated on polystyrene tubes might expose CTD preferentially or the selection process being based on competition, the VHHs against NTD presenting a lower affinity are counter selected. VHHs directed against CTD (E7-2, H3-3, G9-1 , E10-3 and D12-3) recognized overlapping epitopes and Biacore experiments showed a steric hindrance between these VHHs suggesting that the existence of an immunodominant epitopic region in the CTD. Two anti-NTD VHHs B6-1 and E4-3 recognized two different epitopes. None of the VHHs recognized the non-structured regions.

Different VHHs have been tested on different human coronavirus Nucleoprotein. They recognized SARS-CoV-1 and SARS-CoV-2 Nucleoprotein but not the other nucleoproteins. The comparison of the protein sequences of the different Nucleoprotein showed that a high homology between SARS-CoV-1 and SARS-CoV-2 nucleoproteins with 90% identity and a large difference with the other nucleoproteins with 28-33% identity (Figure 23). The epitopes recognized by the tested VHHs are different between the common human and SARS coronaviruses explaining why the VHHs recognized specifically SARS Nucleoprotein. As SARS-CoV-1 virus is not circulating anymore, VHHs are compelling to set up a specific detection test. Interestingly, the VHH E4-3 recognized preferentially SARS-CoV-2 Nucleoprotein. The epitope as defined by HDX-MS is located between aa 11 1 and 133. The sequence YYLGTGP being common to all nucleoproteins, the epitope could be restricted to aa 118-133. Three aa differences are observed in this region: in position 120 a glycine is present for SARS-CoV-2 while it is a serine for SARS- CoV-1 , an aspartic acid instead of a glutamic acid in position 128 and an isoleucine is in place of a valine in position 131 suggesting that these positions are important for the binding of VHH E4-3. The HDX-defined NTD B6-1 epitope contained the positively charged R149 residue recently identified as important for RNA binding (data not shown)(13). Among the regions recognized by the anti CTD VHHs, only one mutation at position 290 with an aspartic acid instead of a glutamic acid is observed between the 2 SARS nucleoproteins. This position is probably not involved in the binding as some anti CTD VHHs present the same binding for both nucleoproteins. Recently, some variants emerged in United Kingdom, South Africa and Brazil (57,58). These variants are more efficiently transmitted. They present several mutations mostly in the Spike protein but also in the Nucleoprotein. The Nucleoprotein mutations are D3L and S235F for the variant B.1.1.7 from United Kingdom, T205I for the variant B.1.351 found in South Africa and P80R for the variant P.1 found in Brazil (59). Interestingly most of these mutations occur either in the N terminal arm (position 3) or in the LKR (positions 205 and 235) two intrinsic disordered regions. A mutation is also observed at position 80 at the N terminal end of NTD close to the N arm. These data suggest that the VHHs targeted conserved regions not prone to mutations that is important for the robustness of a diagnostic test.

The different VHHs recognized Nucleoprotein in infected cells and in infected hamster tissues showing their ability to recognize the native nucleoprotein. The development of an ELISA sandwich also allowed the detection of native Nucleoprotein. These data suggest that the presence of RNA on N does not seem to impede the VHH binding.

We determined the best combination of VHHs to detect the nucleoprotein in samples first on the recombinant protein, then on a permeabilized virus. We found that coating the anti-NTD E4-3 for the capture and the anti-CTD G9-1 to reveal the nucleoprotein is the best option. Moreover no cross reaction was observed with other human seasonal coronaviruses Nucleoprotein due to the exquisite specificity of both VHHs.

This assay has been used to test the presence of Nucleoprotein in human nasal swabs. In parallel an immunochromatographic assay (“Rapid SARS-CoV-2 Antigen test Card”) was used. Both tests can detect low amount of Nucleoprotein (4 ng/ml for ELISA, 1 ng/ml for dipstick test). 18 samples diluted 1/3 were tested and a correlation was observed for 16 out of 18 samples by using both techniques. These results validated the sandwich ELISA. Some PCR negative samples #6 and #79 were found positive while PCR positive samples #14, #22 and #30 were found negative with both techniques. These discrepancies will need to be further analyzed. But for example, the negative samples are patients in early phase of infection (3, 8 and 2 days post-infection, respectively) suggesting low concentration of Nucleoprotein. On the other hand, the ELISA is able to detect Nucleoprotein in the sample #7939 days post-infection, suggesting that this ELISA have a high sensitivity even after the recovery from infection. The development of a reliable test based on nanobodies will be performed in the future by using a large number of human samples. This test can be adapted to an ultra-sensitivity Simoa assay that can promote a nearly 3,000-fold increase of sensitivity compared with that of the commercially available N protein ELISA kit assay (60). The ultra-sensitivity of Simoa assays will provide a quantitative resolution of N concentrations and enables to measure even the earliest stages of infection. An alternative could be the use of a highly sensitive Luciferase-Linked Immunosorbent Assay (LuLISA) using anti N VHH expressed in tandem with the catalytic domain of the enzyme luciferase (nanoKAZ)(62).

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2009 9:14.

Claims

CLAIMS What is claimed is:

1 . An isolated single domain VHH antibody that binds to a polypeptide comprising the amino acid sequence of SEQ ID NO: 41 .

2. The isolated single domain VHH antibody according to claim 1 , comprising four framework regions (FR1 to FR4, respectively) and three complementary determining regions (CDR1 to CDR3, respectively); wherein the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOS: 1 1-20 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 11 -20; wherein the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOS: 21-30 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 21 -30; and wherein the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOS: 31-40 and variants thereof having up to two amino acid additions, deletions, and/or substitutions compared to SEQ ID NOS: 31 -40.

3. The isolated single domain VHH antibody according to claim 2; wherein the amino acid sequence of the CDR1 is selected from the amino acid sequences of SEQ ID NOS: 1 1-20; wherein the amino acid sequence of the CDR2 is selected from the amino acid sequences of SEQ ID NOS: 21-30; and wherein the amino acid sequence of the CDR3 is selected from the amino acid sequences of SEQ ID NOS: 31-40.

4. The isolated single domain VHH antibody according to any one of claims 1 to 3, wherein the single domain VHH antibody comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69.

5. The isolated single domain VHH antibody according to any one of claims 1 to 4, wherein the single domain VHH antibody comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 -10 or 69.

6. The isolated single domain VHH antibody according to any one of claims 1 to 5, wherein the single domain VHH antibody comprises an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69.

7. The isolated single domain VHH antibody according to any one of claims 1 to 6, wherein the single domain VHH antibody consists of an amino acid sequence selected from SEQ ID NOS: 1 -10 or 69.

8. The isolated single domain VHH antibody according to any one of claims 1 to 7 , wherein the single domain VHH antibody binds to the C-terminal domain (CTD) of

SARS-CoV-2 Nucleoprotein.

9. The isolated single domain VHH antibody according to any one of claims 1 to 7 , wherein the single domain VHH antibody binds to the N-terminal domain (NTD) of SARS-CoV-2 Nucleoprotein.

10. The isolated single domain VHH antibody according to any one of claims 1 to 9, wherein the single domain VHH antibody binds to the SARS-CoV-2 Nucleoprotein with a nanomolar KD.

11. A fusion protein comprising a single domain VHH antibody according to any one of claims 1 to 10 fused at its C-terminus to a Fc fragment.

12. A multimeric VHH antibody comprising at least two single domain VHH antibody according to any one of claims 1 to 10 and/or fusion protein according to claim 11.

13. The isolated single domain VHH antibody according to any one of claims 1 to 10 or a fusion protein according to claim 11 or a multimeric VHH antibody according to claim 12, wherein the single domain VHH antibody or the fusion protein or the multimeric VHH antibody according to claim 12 comprises a label.

14. The isolated single domain VHH antibody according to any one of claims 1 to 10 and 13 or the fusion protein according to claim 11 or 13 or the multimeric VHH antibody according to claim 12 or 13, wherein the single domain VHH antibody, or the fusion protein or the multimeric VHH antibody is covalently attached to a substrate.

15. An isolated nucleic acid sequence that encodes the single domain VHH antibody according to any one of claims 1 to 10, or the fusion protein according to claim 11 or the multimeric VHH antibody according to claim 12.

16. A recombinant cell comprising the isolated nucleic acid sequence according to claim 15.

17. A method of producing the single domain VHH antibody according to any one of claims 1 to 10, or a fusion protein according to claim 11 or a multimeric VHH antibody according to claim 12, comprising culturing the recombinant cell according to claim 14 under conditions sufficient for production of the single domain VHH antibody.

18. A method for detection of a SARS-associated coronavirus in a biological sample, comprising: providing a single domain VHH antibody according to any one of claims 1 to 10 and 13-14, ora fusion protein according to any one of claims 11 and 13-14 or a multimeric VHH antibody according to claim 12-14; providing a biological sample from a subject suspected to be infected with a SARS-associated coronavirus; contacting the single domain VHH antibody, or the fusion protein or the multimeric VHH antibody with the biological sample; and visualizing the antigen-antibody complexes formed.

19. The method of claim 18, comprising an ELISA, lateral flow immunoassay, bead-based immunoassay, or multiplex bead-based immunoassay.

20. A method for detection of a SARS-associated coronavirus in a biological sample, comprising: providing a first single domain VHH antibody according to any one of claims 1 to 10 or a fusion protein according to claim 11 or a multimeric VHH antibody according to claim 12, attached to a solid support; providing a biological sample from a subject suspected to be infected with a SARS-associated coronavirus; contacting the solid support with the biological sample under conditions sufficient to allow formation of first antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein in the biological sample and the VHH antibody or, the fusion protein or the multimeric VHH antibody attached to the solid support; contacting the solid support with a second single domain VHH antibody according to any one of claims 1 to 10, or a second fusion protein according to claim 11 or a multimeric VHH antibody according to claim 12, under conditions sufficient to allow formation of second antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein and the second single domain VHH antibody; and visualizing the second antigen-antibody complexes.

21 . The method of claim 20, wherein the second single domain VHH antibody, or the second fusion protein or the second multimeric VHH is labeled and wherein visualizing the second antigen-antibody complexes comprises visualizing the label.

22. The method of claim 20 or 21 , wherein the first single domain VHH antibody is VHH NTD E4-3 and wherein the second single domain VHH antibody is VHH G9-1.

23. The method of any one of claims 18 to 22, wherein the method detects SARS-CoV-2 Nucleoprotein.

24. The method of any one of claims 18 to 23, wherein the detection sensitivity of the method allows detection of as low as 4 ng/ml of the SARS-CoV-2 Nucleoprotein in a sample.

25. A kit for detection of a SARS-associated coronavirus in a biological sample, comprising a single domain VHH antibody according to any one of claims 1 to 10 and/or a fusion protein according to claim 11 and/or a multimeric VHH antibody according to claim 12.

26. The kit according to claim 25, wherein the single domain VHH antibody, and/or the fusion protein and/or the multimeric VHH antibody further comprises a label.

27. The kit according to claim 25 or 26, wherein the single domain VHH antibody, the fusion protein or the multimeric VHH antibody is covalently attached to a solid support.

28. A kit for detection of a SARS-associated coronavirus in a biological sample, comprising: a first single domain VHH antibody according to any one of claims 1 to 10, or a fusion protein according to claim 11 or a multimeric VHH antibody according to claim 12, attached to a solid support; and a second single domain VHH antibody according to any one of claims 1 to 10, or a fusion protein according to claim 11 or a multimeric VHH antibody according to claim 12, attached to a label.

29. The kit of claim 28, wherein the first single domain VHH antibody is VHH NTD E4-3 and wherein the second single domain VHH antibody is VHH G9-1 .

30. The kit of claim 28 or 29, further comprising reagents for detecting the label.

31 . The kit of any one of claims 25 to 30, further comprising a recombinant SARS-CoV-2 Nucleoprotein.

32. The kit of any one of claims 25 to 31 , wherein the kit allows detection of SARS-CoV-2 Nucleoprotein.

33. A method for detection of a SARS-associated coronavirus in a biological sample, comprising: providing a first antibody directed against SARS-associated coronavirus Nucleoprotein attached to a solid support; providing a biological sample from a subject suspected to be infected with a SARS-associated coronavirus; contacting the solid support with the biological sample under conditions sufficient to allow formation of first antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein in the biological sample and the first antibody; contacting the solid support with a second antibody directed against SARS- associated coronavirus Nucleoprotein, under conditions sufficient to allow formation of second antigen-antibody complexes between SARS-associated coronavirus Nucleoprotein and the second antibody; and visualizing the second antigen-antibody complexes, wherein the first antibody and/or the second antibody is a single domain VHH antibody according to any one of claims 1 to 10, and/or a fusion protein according to claim 11 and/or a multimeric VHH antibody according to claim 12.

34. A kit for detection of a SARS-associated coronavirus in a biological sample, comprising: a first antibody directed against SARS-associated coronavirus Nucleoprotein, attached to a solid support; and a second antibody directed against SARS-associated coronavirus Nucleoprotein attached to a label, wherein the first antibody and/or the second antibody is a single domain VHH antibody according to any one of claims 1 to 10, and/or a fusion protein according to claim 11 and/or a multimeric VHH antibody according to claim 12.