WO2024089627A1

WO2024089627A1 - Single domain antibody - sdab/vhh against the nucleoprotein of sars-cov-2 virus

Info

Publication number: WO2024089627A1
Application number: PCT/IB2023/060777
Authority: WO
Inventors: Alejandro ROJAS-FERNANDEZ; Ronald JARA; Guillermo VALENZUELA-NIETO; Anne BERKING
Original assignee: Berking Biotechnology SpA; Universidad Austral de Chile
Current assignee: Berking Biotechnology SpA; Universidad Austral de Chile
Priority date: 2022-10-26
Filing date: 2023-10-26
Publication date: 2024-05-02
Anticipated expiration: 2025-04-26
Also published as: EP4608860A1; AR130878A1; CL2025001244A1

Abstract

The invention provides a set of new single domain antibodies - sdAb/Vhh against the nucleoprotein (N) of SARS-CoV-2 and their use for diagnostic, therapeutic and prophylactic applications. Wherein the Vhh is selected from Vhh1 and/ or Vhh2 wherein the Vhh1 comprises 3 CDR having at least a 90% identity with aminoacidic sequence according to SEQ ID No 5, 6 and 7, and Vhh2 comprises 3 CDR having at least a 90% identity with aminoacidic sequence according to SEQ ID No 8, 9 and 10.

Description

Single Domain Antibody - sdAb/Vhh against the nucleoprotein of SARS-CoV-2 virus

Field of the invention

The invention relates to a set of Single Domain Antibody - sdAb/Vhh against the nucleoprotein of SARS-CoV-2 and their use for detection, diagnostic, prophylaxis and therapies against the wild type of SARS-CoV-2 virus and its variants.

State of the art

Coronavirus disease 2019 (COVID-19) is the name given to the illness caused by the SARS- CoV-2 infection, a novel coronavirus identified in China and related to pneumonia, that constitutes an unprecedented threat for health and economic systems. Many months have passed since it was first reported and we are still continuously learning about the virus and the associated clinical manifestations, its clinical implications, and possible treatments. By the end of June 2022, over 554 million people have been infected and more than 6 million have died worldwide, reinforcing the urgent need for better diagnostics and treatment procedures.

Genetic studies determined that the pathogen responsible for this outbreak belongs to the genus beta-coronavirus, sub-genus sarbecovirus, and the family coronaviridae. It has high sequence homology with the bat coronavirus RaTG13, providing evidence that the new virus may have originated in bats. There are eight coronaviruses associated with human disease: HKU1 , hCoV- OC43, hCoV-NL-63, hCoV-229E, and the pandemics SARS-CoV-1 , MERS and SARS-CoV-2.

The SARS-CoV-2 sequence contains 29903bp, which includes 14 open reading frames (ORFs) coding for the replicase ORFI ab, spike (S), envelope (E), membrane (M) and nucleocapsid (N) structural proteins, and several non-structural (nsps) and accessory proteins. The glycoprotein spike (S) on the surface of the virus is responsible for the attachment and invasion of host cells.

The angiotensin-converting-enzyme 2 protein (ACE2) appears to be the entryway to the host, as the SARS-CoV-1 spike binds to this receptor, the virus requires priming proteolysis by the TMPRRS2 serine protease, which attacks a furin-like cleavage site on the S protein. Several variants of the Spike protein have emerged during the pandemic reducing the binding to diagnostic and neutralising antibodies. In contrast, the nucleoprotein has been shown to remain less variable, thus, most antigen tests for rapid diagnostic are currently based on the detection of the nucleoprotein using conventional monoclonal antibodies.

Single domain Vhh antibodies

Some naturally occurring antibodies lack light chains, known as single-domain antibodies (HCAb, Heavy Chain only Antibodies). They are derivates of IgG and occur in the entire Camelidae family. The camelid family comprises of camels, dromedaries, llamas, vicunas, guanaco, and alpacas. The antigen-binding fragment of an HCAb contains a single variable Vhh domain consisting of 3 hypervariable regions (CDR). When isolated, the Vhh domain is also known as a Nanobody (©SANOFI). The target-specific Vhh derived from camelid HCAbs are obtained after immunization with the target protein, plus adjuvant. Our platform has developed an improved procedure for Vhh production using alpacas as a donor species. To isolate the genetic sequences of the target-specific Vhh produced after immunization, we must first isolate the peripheral B- lymphocytes to obtain total RNA, followed by cDNA preparation to finally amplify the Vhh region. The cDNA fragment encoding the Vhh is as short as 360nt, and up to ~ 3x10⁶ single clones can be obtained in a bacterial display library from 120mL of blood. We use a bacterial display system to clone the full single Vhh. Here, we have obtained and selected a set of Vhh against the nucleoprotein of SARS-CoV-2.

The target-specific Vhh derived from HCAbs of camelids are generally rapidly obtained after immunization with the target protein plus adjuvant. Analysis of the Vhh structure reveals how the hypervariable regions are projected in loops outside of the core structure. To isolate the genomic sequences of the target-specific Vhh, first peripheral B-lymphocytes must be obtained to isolate total RNA, followed by cDNA preparation to finally amplify the Vhh region from gene V of the repertoire. The fragment encoding the Vhh in gene V is as short as 360nt long. Vhh sequences are cloned in a bacterial display vector for transformation of competent bacteria. The bacterial display technology allows Vhh to be expressed on the surfaces of bacterium and, therefore, expressing the Vhh of interest allowing the affinity purification. The final isolated Vhh are recombinantly expressed in bacteria and their binding abilities can be characterized by ELISA. Vhh are then produced in a renewable and economical manner. Further advantages of Vhh are their small size, they can be humanized, their stable structure and behaviour in aqueous solutions, their specific and high-affinity binding to a single target protein and their natural production by camelids. Therefore, Vhh are the best tools available today for affinity-based diagnostics and therapies.

Advantages of single domain Vhh antibodies

Here, we summarise the advantages and uses of Vhh in detail.

Purification

Vhh purification is simple compared to any other antibody source. They are often expressed linked to an affinity tag, such as 6x histidine tags, to allow affinity purification. Enrichment is often set up in the bacterial periplasm where the oxidizing environment allows the formation of proper disulphide bonds. Several milligrams can be isolated from one liter of culture and the recombinant isolated Vhh can be further isolated by standard biochemical techniques.

Stability

Vhh are small and compact polypeptides and they are often expressed in the periplasm of bacteria. They are very stable at high temperatures, starting at 6°C compared to human VH, and they are also resistant to denaturing chemical agents. Furthermore, molecular engineering of the Vhh structure has shown that stability increases when introducing a cysteine at position 54 and 78 to form an extra disulphide bond. Interestingly, the resulting super stable Vhh is also more resistant to proteases such as pepsin or chymotrypsin. Immune invisibility

Vhh can be used as therapeutic bullets against tumors, pathogens, and chronic diseases, however, as foreign proteins they could trigger an immune response themselves. Fortunately, the small size, rapid clearance from the blood, and high homology to the human variable region of the heavy chain VH make them little immunogenic. Only a few amino acids differ between Vhh and the human VH, the substitution camelid amino acids for human amino acids has been used to humanize camelid Vhh and make them even safer for therapies.

Accessibility

Vhh are strict monomers, their affinity for substrates depends on the projection of the three hypervariable loops. In consequence, Vhh tent to interact with cavities of the spatial structure of polypeptides, but not efficiently with peptides. For instance, several identified Vhh directly block active enzyme sites. The some Vhh can even cross the blood-brain barrier by transcytosis and form partly bispecific antibodies used for therapeutic approaches. Finally, the molecular accessibility impacts access to macromolecular complexes.

Use of single domain Vhh antibodies

Diagnostics single domain Vhh antibodies are a superior tool for diagnostics. Their unlimited capacity of in vitro production makes Vhh more reliable than conventional antibodies and independent of batch preparation or animal serum limitations. Vhh can be produced as a protein fused with reporter peptides or proteins for direct staining or visualization, including affinity tags (Flag, HA, V5 and cMyc), fluorescent proteins (GFP, RFP, etc), and enzymes for colorimetric measurements such as horseradish peroxidase (HRP). For instance, ELISA assays can be improved by using Vhh for either specific immobilization or detection using a specific Vhh coupled to horseradish peroxidase (HRP).

Today, the best chances for cancer survival is earlier detection and opportune surgery. Thus, in vivo detection based on Vhh is one of the most promising future technologies to fight cancer. Therapies

Several Vhh have been developed in the context of different experimental therapeutic applications against different viruses: HIV, Hepatitis B virus, influenza virus, Respiratory Syncytial virus, Rabies virus, FMDV, Poliovirus, Rotavirus, and PERVs. Remarkably, Vhh can neutralize HIV infection; cell to cell spread has been inhibited using HIV isolated from patients. Due to the low immunoreactivity of Vhh in humans, they can be injected into patients with very little or no secondary effects. To make them more efficient and specific, Vhh can be linked to produce bivalent, multivalent, and/or multi-specific Vhh, or combined with other Vhh or circulating proteins such as albumin to increase their turn over and therapeutic effectiveness. Rabies virus causes lethal brain infection in people. Soon after exposure anti-rabies prophylaxis is provided with plasma-derived immunoglobulins and vaccines. Often, this occurs directly after the attack of an animal that could be infected. Anti-rabies Vhh can significantly prolong survival or even completely heal the disease in animal models. Respiratory Syncytial Virus, RSV, is one of the major causes of hospitalization in children, every year more than 1.9 million children under one year of age are infected, and there are over 0.3 million children under five years of age that are hospitalized. No current therapy is available against RSV. However, trivalent Vhh-based therapy is in phase II clinical trials. The absolute novelty of the RSV therapy developed by Ablynx (SANOFI), ALX-0171 , is the direct neutralization of the virus in the lung of infected experimentation animals. The Vhh is administered by nebulization, and it reduces the virus titter by 10.000 times. Vhh are also used for immunotherapies against cancer.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Immunization of alpacas and evaluation of immune conversion. Dot-blot analysis, immobilizing the Nucleoprotein of SARS-COV-2 to a nitrocellulose membrane and using alpaca serum as a source of primary antibodies. Figure 1 A, before immunization, no reaction was observed. Figure 1 B, after immunization, alpaca serum recognizes the N protein in the membrane.

Figure 2. Characterization of Vhh-1 (A147). A) Hela cells were transfected with GFP tagged Nucleoprotein of SARS-CoV-2 (Green) were immuno-stained with a myc-tagged Vhh-1 (A147)(Red), nuclei were stained with DAPI (blue) and scale bars represent 20 pm. B) Histogram of colocalization between GFP-Nucleoprotein and myc-Vhh-1 (A147) the curve indicates strong colocalization only in transfected cells. C) ELISA assays, Nucleoprotein was immobilised in ELISA plates and further incubated with dilutions of myc-tagged Vhh-1 (A147), finally anti-mouse HRP conjugated antibodies were used for the Vhh-1 (A147) detection.

Figure 3. Characterization of Vhh-2 (A21). A) Hela cells were transfected with GFP tagged Nucleoprotein of SARS-CoV-2 (Green) were immuno-stained with a myc-tagged Vhh-2 (A21 )(Red), nuclei were stained with DAPI (blue) and scale bars represent 20 pm. B) Histogram of colocalization between GFP-Nucleoprotein and myc- Vhh-2 (A21 ) the curve indicates strong colocalization only in transfected cells. C) ELISA assays, Nucleoprotein was immobilised in ELISA plates and further incubated with dilutions of myc-tagged Vhh-2 (A21 ), finally anti-mouse HRP conjugated antibodies were used for the Vhh-2 (A21 ) detection.

DETAILED DESCRIPTION OF THE INVENTION

The inventors obtained single domain Vhh antibodies that specifically recognize nucleoprotein of SARS-CoV-2, both nucleotide and amino acid sequences of the Vhh, are shown in the SEQ ID No. 1 to 4. The CDR sequences are shown in SEQ ID No. 5 to 10.

The invention was developed using the nucleoprotein of SARS-CoV-2 protein as the purified antigen. The alpaca was immunized and further single domain Vhh antibodies were cloned from peripheric lymphocytes and further characterised.

In an embodiment of the invention, the Vhh has an aminoacidic sequence having at least a 90% identity with SEQ ID No 2, or 4. In a prefer embodiment of the invention, the Vhh has an aminoacidic sequence having a 100% identity with SEQ ID No 2, or 4.

In an embodiment of the invention, the Vhh is encoded by a nucleotide sequence having at least a 90% identity to SEQ ID No 1 or 3. In a prefer embodiment of the invention, the Vhh has an aminoacidic sequence having a 100% identity with SEQ ID No 1 or 3.

The Vhh-1 against the nucleoprotein of SARS-CoV-2 according to the invention comprises 3 CDR having at least a 90% identity with the aminoacidic sequence according to SEQ ID No 5, 6 and 7.

In a further embodiment of the invention, the Vhh comprises 3 CDR having an aminoacidic sequence according to SEQ ID No 5, 6 and 7.

The Vhh-2 against the nucleoprotein of SARS-CoV-2 according to the invention comprises 3 CDR having at least a 90% identity with the aminoacidic sequence according to SEQ ID No 8, 9 and 10. In a further embodiment of the invention, the Vhh-2 comprises 3 CDR having an aminoacidic sequence according to SEQ ID No 8, 9 and 10.

A method for the detection of SARS-CoV-2, wherein a Vhh-1 and/or Vhh2 are used for detecting the presence of the virus in a sample.

A set of Vhh for detection of the nucleoprotein o SARS-CoV-2, wherein a Vhh-1 and/or Vhh2 are used for detecting the presence of the virus in a sample.

A set of Vhh, Vhh-1 and/or Vhh2, for the intracellular detection and proteasomal-mediated degradation of the nucleoprotein o SARS-CoV-2 and concomitant viral reduction leading to either prophylactic and therapeutic approaches.

For the experts working in this area, it will be evident that a Vhh that recognizes the nucleoprotein of SARS-CoV-2 can be useful in diagnosis, prophylactic measures and therapies of COVID-19. In a further embodiment the invention comprises a method of immunoassays using Vhh1 and/or Vhh2. Also the invention aim into a method for therapy of SARS-CoV-2, wherein a Vhh-1 and/or Vhh2 is used as therapeutic measures against the virus.

The Vhh of invention, Vhh-1 and/or Vhh2, can be used as to bind the Nucleoprotein in the intracellular compartments; or to capture the Nucleoprotein to a diagnostic device o sensor; or to detect the Nucleoprotein in a diagnostic device o sensor.

The single domain antibody of any one of the preceding claims, wherein the single domain antibody has been at least partly humanized.

The antigen binding molecule, Vhh-1 and/or Vhh2, wherein the antigen binding molecule is or comprises a monovalent single domain antibody, a multivalent single domain antibody, or a multispecific single domain antibody comprising one or more of the single domain antibodies Vhh- 1 and/or Vhh2. The invention also comprises the antigen binding molecule Vhh-1 and/or Vhh2, wherein the antigen binding molecule is or comprises an immunoconjugate.

A method of diagnosing or monitoring a SARS-CoV-2 virus infection and/or a disease, disorder or condition associated therewith in a subject, said method including the step of contacting the subject and/or a biological sample from the subject with the single domain antibody Vhh-1 and/or Vhh2.

In diagnostic, a Vhh that recognizes the nucleoprotein of of SARS-CoV-2 can be used for the detection of infected cells by flow cytometry, immunofluorescence, agglutination assays, image based in vivo technologies such as positron emission tomography, ELISA, dot-blot, western-blot, immunoblotting, immunohistochemistry, immunoprecipitation, lateral flow test, agglutination in latex, cytometry bases studies, Cytometric Bead Array (CBA), electronic sensors and all others systems comprehending antibodies for detection. Especially in portable diagnostic medical devices based on lateral-flow diagnostics and several diagnostic tools for SARS-CoV-2 the use of conventional monoclonal antibodies against either the Spike or the Nucleoprotein of SARS- CoV-2 has been demonstrated (Dankova Z, Novakova E, Skerenova M, Holubekova V, Lucansky V, Dvorska D, Brany D, Kolkova Z, Strnadel J, Mersakova S, Janikova K, Samec M, Pokusa M, Petras M, Sarlinova M, Kasubova I, Loderer D, Sadlonova V, Kompanikova J, Kotlebova N, Kompanikova A, Hrnciarova M, Stanclova A, Antosova M, Dzian A, Nosal V, Kocan I, Murgas D, Krkoska D, Calkovska A, Halasova E. Comparison of SARS-CoV-2 Detection by Rapid Antigen and by Three Commercial RT-qPCR Tests: A Study from Martin University Hospital in Slovakia. Int J Environ Res Public Health. 2021 Jul 1 ;18(13):7037. doi: 10.3390/ijerph 18137037. PMID: 34280974; PMCID: PMC8295881 ).

In prophylaxis or therapy, a Vhh that recognizes the nucleoprotein of SARS-CoV-2 can be used to target the nucleoprotein for proteasomal mediated degradation in the cytoplasm (Ibrahim AFM, Shen L, Tatham MH, Dickerson D, Prescott AR, Abidi N, Xirodimas DP, Hay RT. Antibody RING- Mediated Destruction of Endogenous Proteins. Mol Cell. 2020 Jul 2;79(1 ):155-166. e9. doi: 10.1016/j.molcel.2020.04.032. Epub 2020 May 25. PMID: 32454028; PMCID: PMC7332993; Fulcher LJ, Macartney T, Bozatzi P, Hornberger A, Rojas-Fernandez A, Sapkota GP. An affinity- directed protein missile system for targeted proteolysis. Open Biol. 2016 Oct;6(10): 160255. doi: 10.1098/rsob.160255. PMID: 27784791 ; PMCID: PMC5090066) and to control the disease in individuals. The infection of animals can eventually be avoided by the generation of transgenically modified animals that are fully resistant to SARS-CoV-2. Single domain Vhh antibodies can be produced as fusion with the Fc domain of human antibodies to also target viral proteins for recognition and/or neutralisation to trigger the immune reaction of the host and enhance recognition of the virus by the human or animal immune system.

In another embodiment of the invention, the Vhh, Vhh-1 and/or Vhh2, is bound to a carrier molecule, such as antibodies fusion, Polyethylene glycol, sialic acid polymers, beta carboxyterminal peptides, albumin or albumin binding peptides, other Vhh and others. Optionally, the one or more Vhh according are bound to an human Fc fragment to increase circulatory time and link the immune response of the host.

The specificity of an antibody, and of a Vhh, is given by the structural complementarity between the antibody combining site and the antigenic determinant. The antibody combining sites are hypervariable regions also known as complementarity-determining regions (CDRs). Vhh have three CDRs, so the specificity of each Vhh -produced by this invention- is given by its 3 CDRs. Typically, the CDRs can be identified by analysing the DNA or protein sequence of the antibody or Vhh in an appropriate computational system, there are several state-of-the-art systems available. Therefore, to identify the 3 CDRs of the Vhh sequences SEQ ID No. 1 to 4 would be a routine procedure for an expert in the field, after obtain the Vhh full sequence.

It will also be evident that since the specificity of Vhh is given by their CDRs, the Vhh from this invention could have changes in their "framework region", or "FR" (the name of the amino acid sequences inserted between the CDRs). Therefore, a Vhh produced by the invention is defined as a Vhh with the same CDRs of the Vhh, Vhh-1 and/or Vhh2, i.e., Vhh of SEQ ID NOS: 1 -4 Equally, these Vhh could be defined as a Vhh with at least a 90% identity with the 3 CDR sequences of a Vhh selected from the group contained in SEQ ID NOS: 1 -4.

Additionally, the invention comprises structures formed by linking the single-chain variable fragment (scFv) and/or the Vhh of the invention to the Fc fragment of the desired species, keeping their specificity, binding properties, and activity.

The Vhhs of invention could be used together or separately, in any method in which detect protein N of Sars-CoV-2 is useful, for example could be used as a tracer for in vivo imaging such as positron emission tomography, or into a method to target the nucleoprotein for proteasomal- mediated degradation. In addition, Vhhs of invention could be constitutively expressed in a transgenic animal to target the nucleoprotein for proteasomal-mediated degradation and resist the infection. Also, could be used in vivo neutralization of the virus allows to control the disease in an individual, and could been used in humanized form.

In another use, Vhhs of invention could be optimised for expression in any other animal species, could be used bound to a carrier molecule, for example bound to an human Fc fragment.

Examples

Detection of nucleoprotein of SARS-CoV-2 in human cells

An alpaca was immunized 4 times with 10Opg of the nucleoprotein of SARS-CoV-2. The immune response of the alpaca's serum before and after immunisation was compare. Then, after the third immunization, it was observed a significant increase of IgG antibodies in the alpaca's serum in a rapid qualitative manner by Dot-blot analysis, immobilizing the epitope to a nitrocellulose membrane and using alpaca serum as a source of primary antibodies (Figure 1 A and 1 B). Thus, the inventors rapidly constructed a bacterial display library consisting of 1 x10⁶ single Vhh clones and started the selection using Ficol gradient Vhh isolation. Immunization and Vhh library construction

The alpaca immunization process followed the protocol "Animal use in research" generated by the Bioethics Committee of the Austral University of Chile. A day before immunization, 5ml of blood was collected for pre-immune serum tests. For immunization (day 1 ), 100 pg of full-length Nucleoprotein was used. The nucleoprotein protein was dissolved in 2 ml adjuvant (Veterinary Vaccine Adjuvant, GERBU FAMA) diluted 1 :1 in sterile water and injected subcutaneously in a male alpaca (Vicugna pacos). A total volume of 4 ml was injected in four different locations in the alpaca. A 5ml blood sample was collected seven days after the first immunization. On day 14, the alpaca was immunized again with 100 pg nucleoprotein of SARS-CoV-2, and on day 15, a sample of 120 ml of blood was collected from the jugular vein in tubes containing 3.8% sodium citrate as an anti-coagulant. The uncoagulated blood sample was mixed with the same volume of HBSS medium without calcium (Gibco), divided into aliquots of 10 ml, and 5 ml of Ficoll-Paque Premium (GE Healthcare) was added on top of each aliquot in 15 ml sterile Falcon tubes. After centrifugation (1.200 x rpm, 80 min, RT), the PBMC fraction was recovered from the interphase, washed twice in HBSS by centrifugation (3.500 x rpm, 10 min), resuspended in 4 ml of sterile PBS 1 x (phosphate buffered saline Gibco). RNA extraction and cDNA production were performed using the commercial RNeasy Mini Kit (Qiagen) and QuantiTect Reverse Transcription Kit (Qiagen) respectively. Approximately, 2 pl of each synthesized cDNA was used as a template in a total PCR reaction volume of 50 pl with oligonucleotides CALL001 (5'-GTC CTG GCT CTC TTC TAC AAG G-3') and CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC- 3') (Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J, Wyns L, Muyldermans S. Betalactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob Agents Chemother. 2001 Qct;45(10):2807-12. doi: 10.1128/AAC.45.10.2807- 2812.2001 ). The amplified fragments of ~0.6 kb, corresponding to Vhh-CH2 domains, and -0.9 kb, corresponding to conventional VH-CH1 -CH2 domains, were separated in 1.2% (w/v) low melting agarose gel and the -0.6 kb band was purified (QIAEX II Gel Extraction kit, Qiagen). This fragment was used as a template in a second PCR reaction with oligonucleotides Vhh-Sfi2 (5'- GTC CTC GCA ACT GCG GCC CAG CCGGCC ATG GCT CAG GTG CAG CTG GTG GA-3’) and Vhh-Not2 (5'- GGA CTA GTG CGG CCG CTG AGG AGA CGG TGA CCT GGG T-3') to finally obtain the amplified fragments of ~0.4 kb, corresponding to Vhh domains. The amplified Vhh fragments were digested with Sfil and Notl (Thermo Scientific) restriction enzymes and ligated into the same sites of purified vector pNeae2 (Salema V, Lopez-Guajardo A, Gutierrez C, Mencia M, Fernandez LA. Characterization of nanobodies binding human fibrinogen selected by E. coli display. J BiotechnoL 2016 Sep 20;234:58-65. doi: 10.1016/j.jbiotec.2016.07.025.). Ligations were electroporated in E. coli DH10B-T1 R cells achieving a library size of -3 x 10⁶ individual clones, as determined by plating on LB-Chloramphenicol agar plates with 2% w/v glucose incubated at 30°C. Less than 0.7% re-ligated vectors were estimated from a control ligation performed in parallel without the DNA insert. Transformed bacteria were scraped from plates and storeed at -80 degrees in LB broth with 30% glycerol.

Once the library was obtained, the inventors applied the selection of Vhh based on a simple density gradient using Ficoll.

The bacterial display system expresses Vhh on the surface of bacteria fused to an intein protein and a myc tag. Buffer conditions were optimized to extract the Vhh-intein fusion from the bacterial membrane and used the bacterial extract directly for binding confirmation to the Nucleoprotein of SARS-CoV-2 by High-content microscopy. After Vhh selection using our simple density gradient protocol based on Ficoll, we obtained ~ 200 colonies on LB-agar plates from the sepharose- antigen coated fraction. The 200 colonies were used to inoculate liquid LB media and further induced for the expression of intein-Vhh. Cells were lysed under optimized conditions and the extract was used as a source of Vhh as primary antibodies for immunofluorescence using high content microscopy.

A single 10cm-plate of HeLa cells was transfected with the nucleoprotein of SARS-CoV-2 fused to the Green Fluorescent Protein (GFP) for 24h, and further cells were seeded onto 3 x 96 wellplates ~ 8000 cells per well. After 24 hours, the cells were fixed, permeabilized, and individual extracts of the 200 selected bacterial display clones were incubated as a source of Vhh acting as primary antibodies for immunofluorescent assays using a CellDiscoverer-7 microscope. The Vhh are fused top a myc tag, therefore after incubation with the individual Vhh the samples were incubated with mouse anti-myc antibody (Cell signalling) an anti-mouse Alexa647 secondary antibody was used for the immunofluorescence assays. Further colocalization of the GFP-tagged nucleoprotein of SARS-CoV2 and myc Vhhs was observed my immunofluorescence. The selected clones were sequenced, the alignment of the amino acid sequences showed two well defined families represented by the best of each family in intensity the Vhh-1 and Vhh-2, both Vhh are different and most likely recognize different epitopes on the nucleoprotein of SARS-CoV- 2. In conclusion, of the first 200 selected Vhh clones, two families of Vhh showed a strong binding capability for full-length Spike high content microscopy screening Vhh-1 (A147) (Figure-3A & 3B) and Vhh-2 (A21 ) (Figure-4A & 4B) (Vhh-1 SEQ ID No. 1 and SEQ ID No. 2 and Vhh-2 SEQ ID No. 3 and SEQ ID No. 4)

Claims

1 . A Vhh against the nucleoprotein of SARS-CoV-2, wherein the Vhh is selected from Vhh1 and/ or Vhh2 wherein the Vhh1 comprises 3 CDR having at least a 90% identity with aminoacidic sequence according to SEQ ID No 5, 6 and 7, and Vhh2 comprises 3 CDR having at least a 90% identity with aminoacidic sequence according to SEQ ID No 8, 9 and 10.

2. The Vhh according to claim 1 , wherein the CDR are contained in the Vhh1 with an aminoacidic sequence having at least a 90% identity with SEQ ID No 2.

3. The Vhh according to claim 1 , wherein the Vhh1 is codificated by a nucleotidic sequence having at least a 90% identity to SEQ ID No 1 .

4. The Vhh according to claim 1 , wherein the CDR are contained in the Vhh2 with an aminoacidic sequence having at least a 90% identity with SEQ ID No 4.

5. The Vhh according to claim 1 , wherein the Vhh2 is encoded by a nucleotidic sequence having at least a 90% identity to SEQ ID No 3.

6. A method for the detection of SARS-CoV-2, wherein a Vhh according to claim 1 is used for detecting the presence of the virus in a sample by an immunoassay.

7. The method according to claim 6 wherein the immunoassays is selected of ELISA, Immunoblotting, lateral flow assays, agglutination assay, Immunohistochemistry or Immunoprecipitation.

8. The method according to claim 7 wherein the immunoassays use Vhh1 and Vhh2.

9. A method for therapy of SARS-CoV-2, wherein a Vhh according to claim 1 is used as therapeutic measures against the virus.

10. A method, wherein a Vhh according to claim 1 is used as to bind the Nucleoprotein in the intracellular compartments.

1 1 . A method, wherein a Vhh according to claim 1 to 5 is used as to capture the Nucleoprotein to a diagnostic device o sensor. A method, wherein a Vhh according to claim 1 to 5 is used as to detect the Nucleoprotein in a diagnostic device o sensor. The single domain antibody, Vhh, according to claim 1 wherein the single domain antibody has been at least partly humanized. The antigen binding molecule Vhh, of claim 1, wherein the antigen binding molecule is or comprises an immunoconjugate. A method of diagnosing or monitoring a SARS-CoV-2 virus infection and/or a disease, disorder or condition associated therewith in a subject, said method including the step of contacting the subject and/or a biological sample from the subject with the single domain antibody, Vhh, of any one of claims 1 to 5.