Single domain vhh antibodies against hantavirus

Abstract

The present invention provides alpaca derived VHHs, therapeutic agents, compositions and methods for the prevention, amelioration and treatment of Hantavirus infections. The inventors obtained two neutralizing single domain VHH antibodies, VHH1 and VHH2 that specifically recognize hantavirus glycoproteins Gn / Gc presented on Viral-like Particles (VLPs). The invention also provides a method for the detection of Hantavirus in a sample and a method for the neutralization of Hantavirus, with VHH1 and VHH2 where the in vivo neutralization of the virus allows to control the disease in an individual.

Description

Single domain VHH antibodies against Hantavirus DESCRIPTION Field of the invention The present disclosure relates to the field of alpaca derived VHHs, therapeutic agents, compositions and methods for the prevention, amelioration and treatment of Hantavirus infections. Hantaviruses and hantavirus disease Hantaviruses (family Hantaviridae) are zoonotic viruses that belong to the Bunyavirales order. Most of the yet known human pathogenic hantaviruses are rodent-borne from the Orthohantavirus genus. The greatest global public health significance of hantavirus infections is due to two types of severe hantavirus disease when the virus is transmitted to humans: haemorrhagic fever with renal syndrome (HFRS), with a case fatality rate up to 12%, and hantavirus pulmonary syndrome (HPS), with a case fatality rate of up to 50% (Schmaljohn, C.S. A Brief History of Bunyaviral Family Hantaviridae. Diseases 2023, 11, 38. https: / / doi.org / 10.3390 / diseases11010038). The 11th revision of WHO’s International Classification of Diseases suggested to collectively group all these diseases under the term “hantavirus disease” subcode 1D62.0 (World Health Organization. ICD-11. International Classification of Diseases 11th Revision.2022. Available online: https: / / icd.who.int / en, accessed on 22 February 2023). 38 orthohantavirus species have been registered by the International Committee on Taxonomy of Viruses (http: / / www.ictvonline.org / virusTaxonomy.asp; Virus Taxonomy: 2021 Release). Among them, Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava-Belgrade virus (DOBV), and Puumala virus (PUUV) are the prototype viruses of HFRS in Europe and Asia. Orthohantaviruses such as Andes virus (ANDV), Sin Nombre virus (SNV), Bayou virus, Black Creek Cannal virus, CañoDelgadito virus, Choclo virus, El Moro Canyon virus, Laguna Negra virus, Maporal virus, Montano virus, and several other related viruses are known as causative agents of HPS in the Americas (Figueiredo, L. T., Souza, W. M., Ferres, M. & Enria, D. A. Hantaviruses and cardiopulmonary syndrome in South America. Virus Res 187, 43-54, doi:10.1016 / j.virusres.2014.01.015 S0168- 1702(14)00029-X (2014); Watson, D. C. et al. Epidemiology of Hantavirus infections in humans: a comprehensive, global overview. Crit Rev Microbiol 40, 261-272, doi:10.3109 / 1040841X.2013.783555 (2014). Human infections by hantaviruses occur from exposure to contaminated excreta and secretions of rodents, and agriculture and forestry activities are involved in most cases (Jonsson, C. B., Figueiredo, L. T. & Vapalahti, O. A global perspective on hantavirus ecology, epidemiology, and disease. Clin Microbiol Rev 23, 412-441, doi:10.1128 / CMR.00062-09 (2010)). However, person-to-person transmission of ANDV has been described in Argentina (Padula, P. J. et al. Hantavirus pulmonary syndrome outbreak in Argentina: molecular evidence for person- to-person transmission of Andes virus. Virology 241, 323-330, doi:S0042-6822(97)98976-5 10.1006 / viro.1997.8976 (1998); Martínez et al., "Super-Spreaders" and Person-to-Person Transmission of Andes Virus in Argentina N Engl J Med 383(23):2230-2241 (2020) doi: 10.1056 / NEJMoa2009040) and in Chile (Ferres, M. et al. Prospective evaluation of household contacts of persons with hantavirus cardiopulmonary syndrome in chile. J Infect Dis 195, 1563-1571, doi:JID3746610.1086 / 516786 (2007); Martinez-Valdebenito, C. et al. Person-to-person household and nosocomial transmission of Andes hantavirus, Southern Chile, 2011. Emerg Infect Dis 20, 1629-1636, doi:10.3201 / eid2010.140353 (2014)). The mean incubation period from exposure to illness onset is 18.5 days (range 7–42 days), Vial, P. A. et al. Incubation period of hantavirus cardiopulmonary syndrome. Emerg Infect Dis 12, 1271-1273, doi:10.3201 / eid1208.051127 (2006). In addition, it is important to perform a strict clinical follow-up of contacts to reduce the lethality of infected individuals. Currently, primary health care facilities perform rapid initial screenings for presumptive hantavirus diagnosis using commercial assays, but the results must be confirmed by reference laboratories.Single domain VHH antibodies The target-specific VHH derived from heavy-chain antibodies (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 360 nt long. VHH sequences are cloned in a Bacterial display vector, thus, after the transformation of the 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 and quantitative biochemical parameters such as ITC. 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 disulfide bonds. Several milligrams can be isolated from one litter 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. Immune invisibility VHH can be used as therapeutic bullets against pathogens such as viruses, as well as against other acute and chronic diseases. However, treatment with antibodies or other foreign proteins can trigger immune response and subsequent clearance. In the case of VHH, their 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 have 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 tend 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. Some VHH can even cross the blood-brain barrier. Finally, due to the small size compared to conventional antibodies, VHH show an enhanced accessibility in tissues and into surface epitopes of macromolecules. 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 to stop viral outbreaks is the earlier detection of infected individuals and opportune isolation and treatment. Thus, instant detection of viral antigen based on VHH is one of the key tools to fight spreading of emerging infectious diseases. Therapies Several VHH have been developed in the context of different experimental therapeutic applications against different viruses: human immunodeficiency virus-1, hepatitis B virus, influenza virus, respiratory syncytial virus, rabies virus, FMDV, poliovirus, rotavirus, and PERVs. Remarkably, VHHcan 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 multispecific VHH, or combined with other VHH or circulating proteins such as albumin to increase their turnover and therapeutic effectiveness. Rabies virus causes lethal brain infection in people. Immediately after exposure, anti-rabies prophylaxis is provided with plasma- derived immunoglobulins, in addition to an inactivated-virus based vaccine. 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. Palivizumab, a humanized IgG1 antibody is approved to in neonates to prevent and treat RSV infections. Also, a trivalent VHH-based therapy is in phase II clinical trials. The absolute novelty of the RSV therapy developed by Ablynx, 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 titer by 10.000 times. VHH are also used for immunotherapies against cancer.BRIEF DESCRIPTION OF THE FIGURES Figure 1. Alpacas’ polyclonal serum before and after immunization with Hantavirus VLPs. Neutralizing assay against ANDV using sera from alpacas immunized with VLPs. Infectious ANDV, at a MOI 0.1 was pre-incubated for 1 h at 37°C with different dilutions of polyclonal sera obtained from immunized alpacas with ANDV VLPs. The antibody-virus mixture was subsequently added to Vero E6 cells for 1 h at 37°C. The viral infection was detected 16 h later by flow cytometry, quantifying the presence of the viral nucleoprotein staining with an anti-N (clone 7B3 / F7) antibody as a marker for ANDV infection. Figure 2. Selection of best clone candidates by bacterial display A. Gating strategy for cell sorting of E. coli expressing single domain VHH antibodies derived from an alpaca immunized with ANDV-VLPs. The bacterial cells were incubated with Strep-tag II-labelled ANDV VLPs and later stained with streptavidin-phycoerythrin (PE) for the selection of the clones that presented the highest affinity to ANDV glycoproteins on VLPs represented by highest PE fluorescence. These bacterial cells were sorted, isolated and subsequently cultured overnight to repeat the process several times until the culture was enriched with cells that present high affinity to viral glycoproteins. B. Flow cytometry histograms showing PE fluorescence of VHH-1 and VHH-2 selected clones compared with the initial bacterial cell culture as negative control; displacement to the right indicates binding to Hantavirus VLPs. Figure 3. Binding of purified VHH antibodies to ANDV glycoproteins A. Binding of VHH antibodies to VLPs using dot blot. VLPs of ANDV were added to a nitrocellulose membrane under non-denaturating conditions. Subsequently, the membrane was incubated with the VHH antibodies conjugated with myc-tag, using a secondary mouse anti-myc-tag as secondaryantibody and an anti-mouse HRP as tertiary antibody to evaluate its reactivity by chemiluminescence. The monoclonal antibody 7A6 / C7 wa used as negative control. B. Binding of VHH antibodies to transfected cells expressing glycoproteins of Hantavirus. Fixed and unpermeabilised HEK293FT cells were transfected with a plasmid coding for Hantavirus GPC and further incubated with a control, VHH-1 or VHH-2 antibodies. Subsequently, the cells were fixated and incubated with a mouse anti-myc-tag as secondary antibody and an anti-mouse Alexa-Fluor 488 as tertiary antibody to evaluate its reactivity using flow cytometry. Figure 4. Neutralization of ANDV with purified VHH antibodies. A. Dose-dependent neutralization of ANDV with VHH molecules. ANDV at a MOI 0.1 was pre- incubated for 1 h at 37°C with different dilutions of VHH antibodies to subsequently be adsorbed to Vero E6 cells for 1 h at 37°C. The infection was detected 16 h later by flow cytometry, quantifying the presence of the viral nucleoprotein using the anti-N (clone 7B3 / F7) antibody as a marker of infection. nnVHH is a non-neutralizing VHH antibody used as negative control. B. Summary of the concentration of 50% inhibition (IC50) of each neutralizing VHH derived from experiment shown in panel (A).BRIEF DESCRIPTION OF THE SEQUENCES

[0001] SEQ ID NO: 1: Nucleotide sequence coding for to the VHH-1 molecule.

[0002] SEQ ID NO: 2: Amino acid sequence corresponding to the VHH-1 molecule, comprehending its three CDR sequences in SEQ ID No.3, 4 & 5.

[0003] SEQ ID NO: 3: Amino acid sequence corresponding to CDR1 of the VHH-1 molecule.

[0004] SEQ ID NO: 4: Amino acid sequence corresponding to CDR2 of the VHH-1 molecule.

[0005] SEQ ID NO: 4: Amino acid sequence corresponding to CDR3 of the VHH-1 molecule.

[0006] SEQ ID NO: 6: Nucleotide sequence coding for to the VHH-2 molecule.

[0007] SEQ ID NO: 7: Amino acid sequence corresponding to the VHH-2 molecule, comprehending its three CDR sequences in SEQ ID No.3, 4 & 5.

[0008] SEQ ID NO: 8: Amino acid sequence corresponding to CDR1 of the VHH-2 molecule.

[0009] SEQ ID NO: 9: Amino acid sequence corresponding to CDR2 of the VHH-2 molecule.

[0010] SEQ ID NO: 10: Amino acid sequence corresponding to CDR3 of the VHH-2 molecule.DETAILED DESCRIPTION OF THE INVENTION The invention provides neutralising single domain VHH antibodies against Hantavirus. The inventors obtained two neutralizing single domain VHH antibodies that specifically recognize hantavirus glycoproteins Gn / Gc presented on Viral-like Particles (VLPs). Hantavirus VLPs are viral particles that resemble those of the native hantaviruses in both structural and antigenic terms. This type of particle consists of a lipid bilayer membrane in which Gn / Gc glycoproteins are anchored. The VLPs used here lack other viral proteins and viral RNA and were prepared as described in Chilean patent application CL 201101085. The invention was developed using the purified virus like particles of Hantavirus as the purified antigen. An alpaca was immunized and further monoclonal single domain VHH antibodies were cloned from peripheral lymphocytes and further characterised. For the VHH-1 molecule, the nucleotide sequence is detailed in SEQ ID No.1 and corresponding amino acid sequences are indicated in SEQ ID No. 2, and the amino acid sequences of its CDR sequences are detailed in SEQ ID No.3, 4 & 5. For the VHH-2 molecule, the nucleotide sequence is specified in SEQ ID No. 6 and corresponding amino acid sequences are SEQ ID No 7, comprehending amino acid sequences of its CDR sequences No 8, 9 &10. The VHH-1 against the Hantavirus according to the invention comprises 3 CDR having at least a 90% identity with the aminoacidic sequence according to SEQ ID No 3, 4 and 5. The VHH-2 against the Hantavirus 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 an embodiment of the invention, the VHH-1 is encoded by a nucleotide sequence having at least a 90% identity to SEQ ID No 1. In an embodiment of the invention, the VHH-1 has an aminoacidic sequence having at least a 90% identity with SEQ ID No 2. In a further embodiment of the invention, the VHH-2 is encoded by a nucleotide sequence having at least a 90% identity to SEQ ID No 6. In a further embodiment of the invention, the VHH-2 has an aminoacidic sequence having at least a 90% identity with SEQ ID No 7. For the experts working in this area, it will be evident that a VHH, such as VHH-1 and / or VHH-2 that neutralize the live infective hantavirus can be useful in a prophylactic and treatment therapy against the Hantavirus pulmonary syndrome. In a particular embodiment, the VHH such as VHH-1 and / or VHH-2 are useful in prophylactic and treatment therapy against orthohantaviruses leading to Hantavirus Pulmonary Syndrome such as Andes virus, Caño Delgadito virus, Choclo virus, Laguna negra virus, Maporal virus and other viruses that cause hantavirus disease. In therapy, a VHH that recognizes Hantavirus surface proteins can be used to neutralize the virus and to control the Hantavirus Pulmonary Syndrome in an individual. The individual can be an animal, a mammal, a human. Single domain VHH antibodies can be produced as fusion protein with the Fc domain of human antibodies, to simulate human antibodies. This allows the link between the singledomain VHH antibodies and the human immune system to enhance recognition of the virus by the human immune system. Therefore, the invention also provides a method for the neutralization of Hantavirus leading to Hantavirus Cardiopulmonary Syndrome, where a VHH according to the invention is used to neutralize the virus. The in vivo neutralization of the virus allows to control the Hantavirus Cardiopulmonary Syndrome disease in an individual. In a preferred embodiment of the invention, the VHH of the invention VHH-1 and / or VHH-2 used in the neutralization method has been at least partly humanized. In another embodiment of the invention, the VHH is bound to a carrier molecule, such as antibodies and or VHH fusions, polyethylene glycol, sialic acid polymers, beta carboxyterminal peptides, albumin or albumin binding peptides and others. Optionally, the one or more VHH according are bound to one human Fc fragment to increase circulatory time and link the immune response of the host. In another embodiment of the invention VHH-1 and / or VHH-2 are useful in a method for the detection of Hantavirus, wherein the VHH-1 and / or VHH-2 is / are used for detecting the presence of the virus in a sample by an immunoassay. Wherein immunoassay is selected from ELISA, Immunoblotting, Immunohistochemistry or Immunoprecipitation among others. For the experts working in this area, it will be evident that a VHH, such as VHH-1 and / or VHH-2 that recognizes hantaviruses can be useful in diagnostics. In diagnosis or virus detection in humans and animals, VHH-1 and / or VHH-2, that recognizes Hantavirus, can be used in any technic available, for example in ELISA, Immunoblotting,Immunohistochemistry, Immunoprecipitation, lateral flow test, agglutination in latex, cytometry bases studies, cytometric Bead Array (CBA), radiotracer, live imaging, and others. 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, identifying the 3 CDRs of the VHH-1 sequences SEQ ID No.3, 4 and 5 and the VHH-2 sequences SEQ ID 8, 9 and 10 would be a routine procedure for an expert in the field. It will also be evident that since the specificity of VHH is given by their CDRs, the VHH from this invention, VHH-1 and / or VHH-2, 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 of SEQ ID NOs: 3, 4, 5 or 8, 9 &10. Equally, these VHH could be defined as a VHH with at least a 90% identity with the 3 CDR amino acid sequences of a VHH selected from the group contained in SEQ ID NOs: 3, 4, 5 or 8, 9 &10. Additionally, the invention comprises structures formed by linking the scFv and / or the VHH of the invention, VHH-1 and / or VHH-2, to the Fc fragment of the desired species, keeping their specificity, binding properties, and activity. For example, VHH-1 and / or VHH-2, is bound to a human Fc fragment.Examples Example 1. VLPs Immunisation and neutralizing VHHs libraries First, the inventors obtained VLPs according to previous reported procedures(Acuña R, Cifuentes- Muñoz N, Márquez CL, Bulling M, Klingström J, Mancini R, Lozach PY, Tischler ND. 2014. Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles.. J Virol. 88(4):2344-8. doi: 10.1128 / JVI.03118-13) by expressing ANDV Gn / Gc glycoproteins in 293FT cells and purifying them from the supernatant of transfected cells by ultracentrifugation. A male alpaca (Vicugna pacos) called “Martin” was immunized 4 times with ~250 µg of Hantavirus VLPs. A day before its first immunization, 5 ml of blood was collected for pre-immune serum tests. Therefore, the VLPs were dissolved in 2 ml adjuvant (Veterinary Vaccine Adjuvant, GERBU FAMA) diluted 1:1 in sterile water and injected subcutaneously in the alpaca. The entire alpaca immunization process followed the protocol "Animal use in research" generated by the Bioethics Committee of the Austral University of Chile. A total volume of 4 ml was injected in four different locations. A 5 ml blood sample was collected 3 days after the last immunization. The neutralizing antibody responses of the alpaca was next assayed by testing the neutralizing activity of the sera. Neutralizing assay against ANDV using sera from alpacas immunized with VLPs. Infectious ANDV, at a MOI 0.1 was pre-incubated for 1 h at 37°C with different dilutions of polyclonal sera obtained from immunized alpacas with ANDV VLPs. The antibody-virus mixture was subsequently added to Vero E6 cells for 1 h at 37°C. The viral infection was detected 16 h later by flow cytometry, quantifying the presence of the viral nucleoprotein staining with an anti-N (clone 7B3 / F7) antibody as a marker for ANDV infection. A strong virus neutralization activity of the polyclonal sera was detected, with 50% of viral inhibition at a dilution of approximately 1:50.000 compared to pre-immune sera that did not show virus neutralizing activity (Figure 1). This result shows, that the alpaca immunized with VLPs included high-levels of neutralizing antibodies circulating in peripheral blood and was hence in perfect conditions for PBMC preparation and RNA extraction.On day 14, the alpaca was immunized again with 250 µg VLPs, 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 × rpm, 80 min, RT), the PBMC fraction was recovered from the interphase, washed twice in HBSS by centrifugation (3.500 × rpm, 10 min), resuspended in 4 ml of sterile PBS 1x (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 µl of each synthesized cDNA was used as a template in a total PCR reaction volume of 50 µl 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, Frère JM, Kinne J, Wyns L, Muyldermans S. Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the camelidae. Antimicrob Agents Chemother.2001 Oct;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 SfiI and NotI (Thermo Scientific) restriction enzymes and ligated into the same sites of purified vector pNeae2 (Salema V, López-Guajardo A, Gutierrez C, Mencía M, Fernández LÁ. 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 achievinga library size of ∼1 × 107individual clones, as determined by plating on LB-Chloramphenicol agar plates with 2% w / v glucose incubated at 30ºC. 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 stored at -80ºC in LB broth with 30% glycerol. Once the library was obtained, the inventors applied a cell sorter procedure consisting in the isolation of bacteria expressing VHH capable to bind to phycoerythrin (PE)-labelled ANDV VLPs (Figure 2a). The bacteria binding to the colours Hantavirus VLPs where enriched after 4 rounds of sorting round and finally plated onto chloramphenicol containing LB plates (Figure 2a). Single colonies were isolated and tested individually for its capacity to bind PE-labelled hantavirus VLPs (Figure 2b). As can be seen, the clones VHH1 and VHH2 showed a high reactivity with PE-labelled VLPs in this assay. Example 2. Binding of VHH to the surface proteins Gn / Gc of Hantavirus The Individual colonies were further sequenced giving rise to the VHH-1 SEQ ID No.1 and SEQ ID No.2 or VHH-2 SEQ ID No.6 and SEQ ID No.7, respectively were further subcloned into a pHen6 bacterial expression vector tagged with a myc and a His tag, and further the VHH-1 and VHH-2 were affinity purified. The binding of VHH-1 and VHH-2 to the surface proteins of ANDV Gn / Gc was determined by slot blot of VLPs to a nitrocellulose membrane and subsequent incubation with VHHs and detection through mouse anti-myc antibody followed by an anti-mouse HRP-conjugate and chemiluminescence substrate (SuperSignal West Dura, Thermo) (Figure 3a). The VHH-1 (SEQ ID No.1 and SEQ ID No.2); and VHH-2 (SEQ ID No.6 and SEQ ID No.7) showed a strong binding capability for full-length Gc / Gn in the slot blot analysis, unveiling diagnostic and therapeutic capabilities. Additionally, binding assays to mammalian cells expressing the ANDV Gn / Gc spike proteins at the cell surface. The non-neutralizing anti-Gn / Gc mouse monoclonal antibody was used to set the reactivity to 100%. Interestingly, the VHH-1 and VHH-2 show strong binding capabilities tothe Gn / Gc proteins of hantaviruses which were 3 to 5 times higher compared to the anti-Gn / Gc mouse mAb (Figure 3b). Example 3. In vitro neutralization of Hantavirus Neutralization studies were performed using infectious life ANDV Orthohantavirus. The infective virus at a mean of infection (MOI) of 0.1 was left untreated or pre-incubated for 1 h at 37°C with VHH-1, VHH-2 or the combination of VHH-1 and VHH2 antibodies. The nnVHH was used as negative control antibody. Subsequently, the control virus without antibody or the virus-antibody mixture was added to Vero E6 cells and incubated for 1 h at 37°C to allow of the viral infection, and further incubated for 16 h. Subsequently, the cells were trypsinized, resuspended, fixed with 4% PFA and permeabilized with Triton X-100. For flow cytometry studies, the cells were incubated with anti-N 7B3 / F7 antibody, using Alexa Fluor 488-conjugated anti-mouse IgG immunoglobulin (Life Technologies) as secondary antibody. ≥ 5,000 cells were analysed using flow cytometry (Barriga et al. A rapid method for infectivity titration of Andes hantavirus using flow cytometry J Virol Methods 193(2):291-4 (2013) doi: 10.1016 / j.jviromet.2013.06.022). The percentage of neutralization was established by setting uninfected cells to 100 % neutralization, infected cells without VHH to 0% neutralization and the IC50 of each VHH was calculated performing non-linear regression in Graph Pad Prism 7. Both, the VHH- 1 and VHH-2 antibodies showed a high capacity to block ANDV infection of cells while the negative control nnVHH did not show inhibitory effects, highlighting the high specificity of the ANDV neutralizing VHH-1 and VHH-2 antibodies (Figure 4a). The concentration of 50% inhibition (IC50) was calculated and showed inhibitory concentrations in the nanomolar range (Figure 4b). Importantly, the mixture of the VHH-1 and VHH-2 antibodies gave the best result of virus neutralization.

Claims

Claims 1. A VHH against the surface Gn / Gc glycoproteins of hantaviruses responsible for Hantavirus disease such as Hantavirus pulmonary syndrome, wherein the VHH comprises 3 CDR having at least a 90% identity with the aminoacidic sequence according to SEQ ID No 3, 4 and 5.

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

3. The VHH according to claim 2, wherein the VHH is encoded by a nucleotide sequence having at least a 90% identity to SEQ ID No 1.

4. The VHH according to claim 1, wherein the VHH comprises 3 CDR having the aminoacidic sequence according to SEQ ID No 3, 4 and 5.

5. A VHH against the surface proteins of Hantavirus Gn / Gc, Gc responsible for the Hantavirus cardiopulmonary syndrome wherein the VHH comprises 3 CDR having at least a 90% identity with the aminoacidic sequence according to SEQ ID No 8, 9 and 10.

6. The VHH according to claim 5, wherein the CDR are contained in a VHH with an aminoacidic sequence having at least a 90% identity with SEQ ID No 7.

7. The VHH according to claim 6, wherein the VHH is encoded by a nucleotide sequence having at least a 90% identity to SEQ ID No 6.

8. The VHH according to claim 5, wherein the VHH comprises 3 CDR having the aminoacidic sequence according to SEQ ID No 8, 9 and 10.

9. A method for the detection of Hantavirus, wherein a VHH according to claim 1 is used for detecting the presence of the virus in a sample by an immunoassay.

10. The method according to claim 9, wherein the immunoassay is selected from ELISA, Immunoblotting, Immunohistochemistry or Immunoprecipitation.

11. A method for the neutralization of Hantavirus, wherein a VHH according to claim 1 is used to neutralize the virus.

112. The method according to claim 11, wherein the in vivo neutralization of the virus allows to control the disease in an individual.

13. The method according to claim 11, wherein the single domain antibody of any one of the preceding claims, wherein the single domain antibody has been at least partly humanized.

14. The method according to claim 11, wherein the VHH is bound to a carrier molecule.

15. The method according to claim 11, wherein the one or more VHH according claim 1 are bound to a human Fc fragment.

16. A method for the detection of Hantavirus, wherein a VHH according to claim 5 is used for detecting the presence of the virus in a sample by an immunoassay.

17. The method according to claim 16, wherein the immunoassay is selected from ELISA, Immunoblotting, Immunohistochemistry or Immunoprecipitation.

18. A method for the neutralization of Hanta virus, wherein a VHH according to claim 5 is used to neutralize the virus.

19. The method according to claim 18, wherein the in vivo neutralization of the virus allows to control the disease in an individual.

20. The method according to claim 18, wherein the VHH is humanized.

21. The method according to claim 18, wherein the VHH is bound to a carrier molecule.

22. The method according to claim 18, wherein the VHH according claim 4are bound to a human Fc fragment.

23. A method according to claim 12 and claim 19, a combination of VHH-1 SEQ ID No 1 and 2 identified by the 3 CDR sequences SEQ ID No 3, 4 and 5 can be combine with VHH-2 SEQ ID No 6 and 7 identified by the 3 CDR sequences SEQ ID No 8, 9 and 10, wherein the in vivo neutralization of the virus allows to control the disease in an individual. 2

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