Vhh and methods for the detection of hantavirus and for the neutralization of the hanta virus

Description

1 / 18 “HHV AND METHODS FOR THE DETECTION OF HANTAVIRUS AND FOR THE NEUTRALIZATION OF HANTA VIRUS” DESCRIPTION Field of invention

[001] This disclosure relates to the field of alpaca-derived VHHs, therapeutic agents, compositions and methods for the prevention, improvement and treatment of Hantavirus infections. Hantavirus and hantavirus disease

[002] Hantaviruses (family Hantaviridae) are zoonotic viruses belonging to the order Bunyavirales. Most known human pathogenic hantaviruses are transmitted by rodents of the genus Orthohantavirus. The major global importance of hantavirus infections to public health stems from two types of severe diseases caused by the virus when it is transmitted to humans: hemorrhagic fever with renal syndrome (HFRS), with a mortality rate of up to 12%, and hantavirus pulmonary syndrome (HPS), with a mortality rate of up to 50%. (Schmaljohn, CS A Brief History of Bunyaviral Family Hantaviridae. Diseases 2023, 11, 38. https: / / doi.org / 10.3390 / diseases11010038). The 11th revision of The WHO International Classification of Diseases has suggested collectively grouping 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 February 22, 2023).

[003] The International Committee on Taxonomy of Viruses has registered 38 species of orthohantaviruses (http: / / www.ictvonline.org / virusTaxonomy.asp; Taxonomy of Viruses: 2021 version). Among them, Hantaan virus (HTNV), Seoul virus (SEOV), Dobrava-Belgrade virus (DOBV), and Puumala virus (PUUV) are the prototype viruses of the HFRS in Europe and Asia. Orthoviruses such as Andes virus Petition 870250089842, dated 02 / 10 / 2025, page 43 / 69 2 / 18 (ANDV), Sin Nombre virus (SNV), Bayou virus, Black Creek Canal virus, Cano Delgadito 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, DA 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, DC 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 due to exposure to contaminated excrement and secretions from rodents, and agricultural and forestry activities are involved in most cases (Jonsson, CB, 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, PJ et al. The Hantavirus Pulmonary Syndrome Outbreak in Argentina: Molecular Evidence for Person-to-Person Transmission of Andes Virus. Virology 241, 323330, 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 people with hantavirus cardiopulmonary syndrome in Chile. J Infect Dis 195, 1563-1571, doi:JID37466 10.1086 / 516786 (2007); Martinez-Valdebenito, C. et al. House-to-person and nosocomial transmission of hantavirus from the Andes, southern Chile, 2011. Emerg Infect Dis 20, 1629-1636, doi: 10.3201 / eid2010.140353 (2014)). The average incubation period between exposure and onset of illness is 18.5 days (range 7 to 42 days). Petition 870250089842, dated 02 / 10 / 2025, p. 44 / 69 3 / 18 (Vial, PA et al. Incubation period of hantavirus cardiopulmonary syndrome. Emerg Infect Dis 12, 1271-1273, doi:10.3201 / eid1208.051127 (2006). Furthermore, it is important to conduct rigorous clinical follow-up of contacts to reduce the lethality of infected individuals. Currently, primary health care units perform rapid initial screenings for presumptive diagnosis of Hantavirus using commercial tests, but the results must be confirmed by reference laboratories. Single-domain VHH antibodies

[004] Target-specific VHH derived from camelid heavy chain antibodies (HCAbs) is generally obtained rapidly after immunization with the target protein plus adjuvant. Analysis of the VHH structure reveals how hypervariable regions are designed in loops outside the central structure. To isolate target-specific VHH genomic sequences, it is first necessary to obtain peripheral B lymphocytes to isolate total RNA, followed by cDNA preparation to finally amplify the VHH region of the repertoire gene V. The fragment encoding VHH in gene V is only 360 nt long. VHH sequences are cloned into a bacterial display vector, thus, after transformation of competent bacteria, bacterial display technology allows VHH to be expressed on the surfaces of the bacteria and therefore expressing the VHH of interest, allowing affinity purification.The final isolated VHHs are recombinantly expressed in bacteria, and their binding abilities can be characterized by ELISA and quantitative biochemical parameters such as ITC. VHHs are then produced in a renewable and economical manner. Other advantages of VHHs are their small size, the possibility of humanization, their stable structure and behavior in aqueous solutions, their specific and high-affinity binding to a single target protein, and their natural production by camelids. Therefore, VHHs are the best tools available. Petition 870250089842, dated 02 / 10 / 2025, pp. 45 / 69 4 / 18 currently for affinity-based diagnoses and therapies. Advantages of single-domain HHV antibodies

[005] Here, we summarize in detail the advantages and uses of VHH. Purification

[006] VHH purification is simple compared to any other antibody source. They are frequently expressed bound 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 suitable disulfide bonds. Several milligrams can be isolated from a liter of culture, and the isolated recombinant VHH can be further isolated by standard biochemical techniques. Stability

[007] VHHs are small, compact polypeptides, frequently expressed in the periplasm of bacteria. They are very stable at high temperatures, from 6°C, compared to human VHs, and are also resistant to denaturing chemical agents. Immunological invisibility

[008] VHHs can be used as a therapeutic weapon against pathogens, such as viruses, as well as against other acute and chronic diseases. However, treatment with antibodies or other external proteins can trigger an immune response and subsequent elimination. In the case of VHHs, their small size, rapid elimination from the blood, and high homology with the human variable region of the VH heavy chain make them poorly immunogenic. Only a few amino acids differ between VHH and human VH, and the substitution of camelid amino acids with human amino acids has been used to humanize camelid VHHs and make them even safer for Petition 870250089842, dated 02 / 10 / 2025, pp. 46 / 69 5 / 18 therapies. Accessibility

[009] VHHs are strict monomers, and their affinity for substrates depends on the projection of the three hypervariable loops. Consequently, VHHs tend to interact with cavities in the spatial structure of polypeptides, but not efficiently with peptides. For example, several identified VHHs directly block enzymatic active sites. Some VHHs can even cross the blood-brain barrier. Finally, due to their small size compared to conventional antibodies, VHHs exhibit enhanced accessibility in tissues and to macromolecule surface epitopes. Use of single-domain HHV antibodies Diagnostics

[0010] Single-domain VHH antibodies are a superior tool for diagnostics. Their unlimited in vitro production capability makes them more reliable than conventional antibodies and independent of batch preparation limitations or animal serum. VHH can be produced as a fused protein with peptides or reporter proteins for direct staining or visualization, including affinity markers (Flag, HA, V5, and cMyc), fluorescent proteins (GFP, RFP, etc.), and enzymes for colorimetric measurements, such as horseradish peroxidase (HRP). For example, ELISA assays can be enhanced using VHH for specific immobilization or detection using a specific VHH coupled to horseradish peroxidase (HRP).

[0011] Today, the best chances of preventing viral outbreaks are early detection of infected individuals and timely isolation and treatment. Thus, instant detection of the viral antigen based on HHV is one of the main tools to combat the spread of disease. Petition 870250089842, dated 02 / 10 / 2025, pp. 47 / 69 6 / 18 emerging infectious diseases. Therapies

[0012] Several HHVs 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. Notably, HHVs can neutralize HIV infection; cell-to-cell spread was inhibited using HIV isolated from patients. Due to the low immunoreactivity of HHVs in humans, they can be injected into patients with very few or no side effects. To make them more efficient and specific, HHVs can be linked to produce bivalent, multivalent, and / or multispecific HHVs, or combined with other HHVs or circulating proteins, such as albumin, to increase their turnover and therapeutic efficacy. Rabies virus causes lethal brain infection in humans.Immediately after exposure, rabies prophylaxis is provided with plasma-derived immunoglobulins, in addition to an inactivated virus-based vaccine. This often occurs soon after an attack by an animal that may be infected. Rabies HVH can significantly prolong survival or even completely cure the disease in animal models. Respiratory syncytial virus (RSV) is a leading cause of hospitalization in children. Every year, more than 1.9 million children under one year of age are infected, and more than 0.3 million children under five years of age are hospitalized. Palivizumab, a humanized IgG1 antibody, is approved for use in newborns to prevent and treat RSV infections. In addition, a trivalent HVH-based therapy is in phase II clinical trials.The groundbreaking innovation of the RSV therapy developed by Ablynx, ALX-0171, is the direct neutralization of the virus in the lungs of infected laboratory animals. VHH is administered via nebulization and... Petition 870250089842, dated 02 / 10 / 2025, pp. 48 / 69 7 / 18 reduces the viral load by 10,000 times. HHVs are also used for immunotherapies against cancer. BRIEF DESCRIPTION OF THE FIGURES

[0013] Figure 1. Polyclonal serum of alpacas before and after immunization with Hantavirus VLPs, Neutralization assay against ANDV using sera from alpacas immunized with VLPs. Infectious ANDV, at an MOI of 0.1, was pre-incubated for 1 ha at 37°C with different dilutions of polyclonal sera obtained from alpacas immunized with ANDV VLPs. The antibody-virus mixture was subsequently added to Vero E6 cells for 1 ha at 37°C. Viral infection was detected 16 h later by flow cytometry, quantifying the presence of viral nuclear protein stained with an anti-N antibody (clone 7B3 / F7) as a marker of ANDV infection.

[0014] Figure 2. Selection of the best clone candidates by bacterial display, A. Screening strategy for cell classification of E. coli expressing single-domain VHH antibodies derived from an alpaca immunized with ANDV-VLPs. Bacterial cells were incubated with Strep-tag II labeled ANDV VLPs and subsequently stained with streptavidin-phycoerythrin (PE) to select clones exhibiting the highest affinity for ANDV glycoproteins in VLPs, represented by the highest PE fluorescence. These bacterial cells were classified, isolated, and subsequently cultured overnight to repeat the process several times until the culture was enriched with cells exhibiting high affinity for viral glycoproteins; B. Flow cytometry histograms showing the PE fluorescence of selected clones VHH-1 and VHH-2 compared to the initial bacterial cell culture as a negative control; shift to the right. Petition 870250089842, dated 02 / 10 / 2025, pp. 49 / 69 8 / 18 indicates connection to Hantavirus VLPs.

[0015] Figure 3. Binding of purified VHH antibodies to ANDV glycoproteins, A. Binding of VHH antibodies to VLPs using dot blot. ANDV VLPs were added to a nitrocellulose membrane under non-denaturing conditions. Subsequently, the membrane was incubated with VHH antibodies conjugated with myc-tag, using a mouse anti-myc-tag secondary antibody as the secondary antibody and an anti-mouse HRP antibody as the tertiary antibody to assess their reactivity by chemiluminescence. The monoclonal antibody 7A6 / C7 was used as a negative control; B. Binding of VHH antibodies to transfected cells expressing Hantavirus glycoproteins. Fixed and non-permeabilized HEK293FT cells were transfected with a plasmid encoding Hantavirus GPC and subsequently incubated with a control, VHH-1 or VHH-2 antibodies. The cells were then fixed and incubated with a mouse anti-myc-tag antibody as a secondary antibody and an Alexa-Fluor 488 anti-mouse antibody as a tertiary antibody to assess their reactivity using flow cytometry.

[0016] Figure 4. Neutralization of ANDV with purified VHH antibodies, A. Dose-dependent neutralization of ANDV with VHH molecules. ANDV at an MOI of 0.1 was pre-incubated for 1 ha at 37°C with different dilutions of VHH antibodies and subsequently adsorbed onto Vero E6 cells for 1 ha at 37°C. Infection was detected 16 h later by flow cytometry, quantifying the presence of the viral nucleoprotein using anti-N antibody (clone 7B3 / F7) as an infection marker. nnVHH is a non-neutralizing VHH antibody used as a negative control. Petition 870250089842, dated 02 / 10 / 2025, pp. 50 / 69 9 / 18 B. Summary of the 50% inhibition concentration (IC50) of each neutralizing VHH derived from the experiment shown in panel (A). BRIEF DESCRIPTION OF THE SEQUENCES

[0017] SEQ ID No: 1: Nucleotide sequence that codes for the VHH-1 molecule.

[0018] SEQ ID No: 2: Amino acid sequence corresponding to the VHH-1 molecule, comprising its three CDR sequences in SEQ ID No 3, 4 and 5.

[0019] SEQ ID No: 3: Amino acid sequence corresponding to CDR1 of the VHH-1 molecule.

[0020] SEQ ID No: 4: Amino acid sequence corresponding to CDR2 of the VHH-1 molecule.

[0021] SEQ ID No: 4: Amino acid sequence corresponding to CDR3 of the VHH-1 molecule.

[0022] SEQ ID No: 6: Nucleotide sequence that codes for the VHH-2 molecule.

[0023] SEQ ID No: 7: Amino acid sequence corresponding to the VHH-2 molecule, comprising its three CDR sequences in SEQ ID No 3, 4 and 5.

[0024] SEQ ID No: 8: Amino acid sequence corresponding to CDR1 of the VHH-2 molecule.

[0025] SEQ ID No: 9: Amino acid sequence corresponding to CDR2 of the VHH-2 molecule.

[0026] SEQ ID No: 10: Amino acid sequence corresponding to CDR3 of the VHH-2 molecule. DETAILED DESCRIPTION OF THE INVENTION

[0027] The invention provides neutralizing single-domain VHH antibodies against Hantavirus. The inventors obtained two VHH antibodies. Petition 870250089842, dated 02 / 10 / 2025, pp. 51 / 69 10 / 18 single-domain neutralizing agents that specifically recognize Hantavirus Gn / Gc glycoproteins presented on virus-like particles (VLPs). Hantavirus VLPs are viral particles that resemble those of native Hantaviruses, both structurally and antigenically. This type of particle consists of a lipid bilayer membrane to which the Gn / Gc glycoproteins are anchored. The VLPs used here do not contain other viral proteins or viral RNA and were prepared as described in Chilean patent application CL 201101085.

[0028] The invention was developed using purified particles similar to Hantavirus as a purified antigen. An alpaca was immunized and, subsequently, single-domain monoclonal antibodies to HHV were cloned from peripheral lymphocytes and characterized.

[0029] For the VHH-1 molecule, the nucleotide sequence is detailed in SEQ ID No. 1 and the 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 and 5. For the VHH2 molecule, the nucleotide sequence is specified in SEQ ID No. 6 and the corresponding amino acid sequences are in SEQ ID No. 7, comprising the amino acid sequences of its CDR sequences No. 8, 9 and 10.

[0030] VHH-1 against Hantavirus, according to the invention, comprises 3 CDRs with at least 90% identity to the amino acid sequence according to SEQ ID No. 3, 4 and 5.

[0031] VHH-2 against Hantavirus, according to the invention, comprises 3 CDRs with at least 90% identity to the amino acid sequence according to SEQ ID No. 8, 9 and 10.

[0032] In one embodiment of the invention, VHH-1 is encoded by a nucleotide sequence with at least 90% identity to SEQ ID No. 1. Petition 870250089842, dated 02 / 10 / 2025, pp. 52 / 69 11 / 18

[0033] In one embodiment of the invention, VHH-1 has an amino acid sequence with at least 90% identity to SEQ ID No. 2.

[0034] In another embodiment of the invention, VHH2 is encoded by a nucleotide sequence with at least 90% identity to SEQ ID No. 6.

[0035] In another embodiment of the invention, VHH2 has an amino acid sequence with at least 90% identity to SEQ ID No. 7.

[0036] For specialists working in this area, it will be evident that a VHH, such as VHH-1 and / or VHH-2, which neutralizes live infectious Hantavirus, can be useful in prophylactic therapy and in the treatment of Hantavirus pulmonary syndrome.

[0037] In a specific embodiment, VHH, such as VHH-1 and / or VHH-2, is useful in prophylactic and treatment therapy against orthohantaviruses that cause Hantavirus Pulmonary Syndrome, such as Andes virus, Cano Delgadito virus, Choclo virus, Laguna Negra virus, Maporal virus, and other viruses that cause Hantavirus disease.

[0038] In therapy, a VHH that recognizes Hantavirus surface proteins can be used to neutralize the virus and control Hantavirus Pulmonary Syndrome in an individual. The individual can be an animal, a mammal, or a human. Single-domain VHH antibodies can be produced as a fusion protein with the Fc domain of human antibodies, to mimic human antibodies. This allows the binding between single-domain VHH antibodies and the human immune system to enhance recognition of the virus by the human immune system.

[0039] Therefore, the invention also provides a method for neutralizing the Hantavirus that leads to Cardiopulmonary Syndrome by Petition 870250089842, dated 02 / 10 / 2025, pp. 53 / 69 12 / 18 Hantavirus, where a VHH according to the invention is used to neutralize the virus. In vivo neutralization of the virus allows for the control of Hantavirus Cardiopulmonary Syndrome disease in an individual.

[0040] 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 partially humanized. In another embodiment of the invention, the VHH is linked to a carrier molecule, such as antibodies and / or VHH fusions, polyethylene glycol, sialic acid polymers, beta carboxy-terminal peptides, albumin or albumin-binding peptides, and others. Optionally, one or more VHHs according to the invention are linked to a human Fc fragment to increase circulation time and trigger the host immune response.

[0041] In another embodiment of the invention, VHH-1 and / or VHH-2 are useful in a method for detecting Hantavirus, wherein VHH1 and / or VHH-2 are used to detect the presence of the virus in a sample by means of an immunoassay. Wherein the immunoassay is selected from ELISA, immunoblotting, immunohistochemistry or immunoprecipitation, among others.

[0042] For specialists working in this area, it will be evident that a VHH, such as VHH-1 and / or VHH-2, which recognizes Hantavirus, can be useful in diagnosis.

[0043] In the diagnosis or detection of viruses in humans and animals, VHH-1 and / or VHH-2, which recognizes Hantavirus, can be used in any available technique, for example, in ELISA, immunoblotting, immunohistochemistry, immunoprecipitation, lateral flow test, latex agglutination, cytometry-based studies, Cytometric Bead Array (CBA), radiotracer, live imaging and others.

[0044] The specificity of an antibody and a VHH is determined by the structural complementarity between the binding site of Petition 870250089842, dated 02 / 10 / 2025, pp. 54 / 69 13 / 18 antibody and the antigenic determinant. Antibody binding sites are hypervariable regions, also known as complementarity-determining regions (CDRs). VHHs have three CDRs, therefore the specificity of each VHH produced by this invention is determined by its three CDRs. Typically, CDRs can be identified by analyzing the DNA or protein sequence of the antibody or VHH in a suitable computational system, and several state-of-the-art systems are available. Therefore, identifying the 3 CDRs of the SEQ ID sequences No. 3, 4 and 5 of VHH-1 and the SEQ ID sequences No. 8, 9 and 10 of VHH-2 would be a routine procedure for a specialist in the field.

[0045] It will also be evident that, since the specificity of VHH is given by its CDRs, the VHH of this invention, VHH-1 and / or VHH-2, may show alterations in its “structural 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 as SEQ ID No. 3, 4, 5 or 8, 9 and 10 of the VHH. Similarly, these VHHs can be defined as a VHH with at least 90% identity with the 3 CDR amino acid sequences of a VHH selected from the group contained in SEQ ID No. 3, 4, 5 or 8, 9 and 10.

[0046] In addition, the invention comprises structures formed by linking the scFv and / or VHH of the invention, VHH-1 and / or VHH-2, to the Fc fragment of the desired species, maintaining its specificity, binding properties and activity. For example, VHH-1 and / or VHH-2 are linked to a human Fc fragment. Examples Example 1. Immunization with VLPs and neutralizing VHH libraries.

[0047] First, the inventors obtained VLPs according to previously reported procedures (Acuna R, Cifuentes-Munoz N, Petition 870250089842, dated 02 / 10 / 2025, pp. 55 / 69 14 / 18 Márquez CL, Bulling M, Klingstrom 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) expressing ANDV Gn / Gc glycoproteins in 293FT cells and purifying them from the supernatant of transfected cells by ultracentrifugation. A male alpaca (Vicugna pacos) named “Martin” was immunized 4 times with ~250 μg of Hantavirus VLPs. One day before his first immunization, 5 ml of blood were collected for pre-immune serum testing.

[0048] Thus, the VLPs were dissolved in 2 ml of adjuvant (Veterinary Vaccine Adjuvant, GERBU FAMA) diluted 1:1 in sterile water and injected subcutaneously into the alpaca. The entire alpaca immunization process followed the “Use of animals in research” protocol developed by the Bioethics Committee of the Austral University of Chile. A total volume of 4 ml was injected at four different sites. A 5 ml blood sample was collected 3 days after the last immunization. The alpaca's neutralizing antibody responses were then tested through the neutralizing activity of the sera.

[0049] Neutralization assay against ANDV using sera from alpacas immunized with VLPs. Infectious ANDV, at an MOI of 0.1, was preincubated for 1 ha at 37°C with different dilutions of polyclonal sera obtained from alpacas immunized with ANDV VLPs. The antibody-virus mixture was subsequently added to Vero E6 cells for 1 ha at 37°C. Viral infection was detected 16 h later by flow cytometry, quantifying the presence of viral nuclear protein stained with an anti-N antibody (clone 7B3 / F7) as a marker of ANDV infection. Strong viral neutralization activity of the polyclonal sera was detected, with 50% viral inhibition at a dilution of approximately 1:50,000 compared to preimmune sera that showed no viral neutralization activity (Figure 1). This Petition 870250089842, dated 02 / 10 / 2025, pp. 56 / 69 15 / 18 results show that the alpaca immunized with VLPs had high levels of neutralizing antibodies circulating in the peripheral blood and was therefore in perfect condition for PBMC preparation and RNA extraction.

[0050] On day 14, the alpaca was immunized again with 250 μg of VLPs and, on day 15, a 120 ml blood sample was collected from the jugular vein in tubes containing 3.8% sodium citrate as an anticoagulant. The unclotted blood sample was mixed with the same volume of calcium-free HBSS medium (Gibco), divided into 10 ml aliquots, and 5 ml of Ficoll-Paque Premium (GE Healthcare) was added on top of each aliquot in sterile 15 ml Falcon tubes. After centrifugation (1200 rpm, 80 min, RT), the PBMC fraction was recovered from the interface, washed twice in HBSS by centrifugation (3500 rpm, 10 min), and resuspended in 4 ml of sterile 1x PBS (Gibco phosphate-buffered saline). RNA extraction and cDNA production were performed using the commercial RNeasy Mini Kit (Qiagen) and the QuantiTect Reverse Transcription Kit (Qiagen), respectively.Approximately 2 μl of each synthesized cDNA were used as templates in a total PCR reaction volume of 50 μl with CALL001 (5'-GTC CTG GCT CTC TTC TAC AAG G-3') and CALL002 (5'-GGTACGTGCTGTTGAACTGTTCC-3') oligonucleotides (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 camelidae. The amplified fragments of ~0.6 kb, corresponding to the VHH-CH2 domains, and ~0.9 kb, corresponding to the conventional VH-CH1-CH2 domains, were separated on a 1.2% (w / v) low-melting-point agarose gel and the band of Approximately 0.6 kb was purified (QIAEX II Gel Extraction kit, Qiagen). This fragment was used as a template in a second PCR reaction with VHH-Sfi2 oligonucleotides (5'GTC CTC GCA ACT GCG GCC CAG CCGGCC ATG GCT CAG GTG CAG CTG). Petition 870250089842, dated 02 / 10 / 2025, pp. 57 / 69 16 / 18 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 the VHH domains. The amplified VHH fragments were digested with Sfi I and Not I restriction enzymes (Thermo Scientific) and ligated at the same sites as the purified pNeae2 vector (Salema V, López-Guajardo A, Gutierrez C, Mencía M, Fernández LÁ. Characterization of human fibrinogen-binding nanobodies selected by displaying E. coli. J Biotechnol. 2016 Sep 20;234:5865). Ligations were electroporated into E. coli DH10B-T1 R cells, achieving a library size of ~1 χ 107 individual clones, as determined by plating on LB-chloramphenicol agar plates with 2% w / v glucose incubated at 30°C. Less than 0.7% of religated vectors were estimated from a control ligation performed in parallel without DNA insertion.The transformed bacteria were scraped from the plates and stored at -80°C in LB broth with 30% glycerol.

[0051] Once the library was obtained, the inventors applied a cell screening procedure that consisted of isolating bacteria expressing VHH capable of binding to phycoerythrin (PE)-labeled ANDV VLPs (Figure 2a). Bacteria that bound to the Hantavirus VLP were enriched after 4 rounds of screening and finally placed on LB plates containing chloramphenicol (Figure 2a). Single colonies were isolated and individually tested for their ability to bind to PE-labeled Hantavirus VLPs (Figure 2b). As can be seen, clones VHH1 and VHH2 showed high reactivity with PE-labeled VLPs in this assay. Example 2. Binding of VHH to the Gn / Gc surface proteins of Hantavirus.

[0052] Individual colonies were subsequently sequenced, giving rise to VHH-1 SEQ ID No. 1 and SEQ ID No. 2 or VHH-2 SEQ ID No. 6 and SEQ ID No. 7, respectively, which were subsequently subcloned into a Petition 870250089842, dated 02 / 10 / 2025, pp. 58 / 69 17 / 18 bacterial expression vector pHen6 labeled with a myc tag and a His tag, and subsequently VHH-1 and VHH-2 were purified by affinity.

[0053] The binding of VHH-1 and VHH-2 to ANDV Gn / Gc surface proteins was determined by slot blotting of VLPs on a nitrocellulose membrane and subsequent incubation with VHHs and detection via mouse anti-myc antibody, followed by an anti-mouse HRP conjugate and chemiluminescence substrate (SuperSignal West Dura, Thermo) (Figure 3a). VHH-1 (SEQ ID No. 1 and SEQ ID No. 2) and VHH-2 (SEQ ID No. 6 and SEQ ID No. 7) showed strong binding capacity to full-length Gc / Gn in slot blot analysis, revealing diagnostic and therapeutic capabilities. Furthermore, binding assays were performed on mammalian cells expressing ANDV Gn / Gc spike proteins on the cell surface. Non-neutralizing mouse anti-Gn / Gc monoclonal antibody was used to define reactivity at 100%.Interestingly, VHH-1 and VHH-2 show strong binding capabilities to Hantavirus Gn / Gc proteins, which were 3 to 5 times greater compared to the anti-Gn / Gc mouse mAb (Figure 3b). Example 3. In vitro neutralization of Hantavirus

[0054] Neutralization studies were performed using infectious Orthohantavirus ANDV. Infectious virus with a median infectivity index (MOI) of 0.1 was left untreated or pre-incubated for 1 ha at 37°C with VHH-1, VHH-2, or a combination of VHH-1 and VHH-2 antibodies. nnVHH was used as a negative control antibody. Subsequently, the unantibody-free control virus or the virus-antibody mixture was added to Vero E6 cells and incubated for 1 ha at 37°C to allow viral infection, and subsequently incubated for 16 h. Cells were then trypsinized, resuspended, fixed with 4% PFA, and permeabilized with Triton X-100. For studies of Petition 870250089842, dated 02 / 10 / 2025, pp. 59 / 69 18 / 18 flow cytometry, cells were incubated with anti-N 7B3 / F7 antibody, using anti-mouse IgG immunoglobulin conjugated with Alexa Fluor 488 (Life Technologies) as a secondary antibody. > 5,000 cells were analyzed 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 defining uninfected cells as 100% neutralization, infected cells without VHH as 0% neutralization, and the IC50 of each VHH was calculated by performing non-linear regression in the GraphPad Prism 7 program. VHH-1 and VHH-2 antibodies showed a high capacity to block ANDV infection of cells, while the nnVHH negative control showed no inhibitory effects, highlighting the high specificity of the ANDV-neutralizing VHH-1 and VHH-2 antibodies (Figure 4a).The 50% inhibition concentration (IC50) was calculated and showed inhibitory concentrations in the nanomolar range (Figure 4b). It is important to highlight that the mixture of VHH-1 and VHH-2 antibodies showed the best result in neutralizing the virus. Petition 870250089842, dated 02 / 10 / 2025, pages 60 / 69

Claims

1 / 3 Claims 1. VHH against the surface Gn / Gc glycoproteins of hantaviruses responsible for Hantavirus disease, such as Hantavirus pulmonary syndrome, characterized by VHH comprising 3 CDRs with at least 90% identity to the amino acid sequence according to SEQ IDs No. 3, 4 and 5.

2. VHH according to claim 1, characterized in that the CDRs are contained in a VHH with an amino acid sequence having at least 90% identity with SEQ ID No.

2.

3. VHH according to claim 2, characterized in that VHH is encoded by a nucleotide sequence with at least 90% identity to SEQ ID No.

1.

4. VHH according to claim 1, characterized by the VHH comprising 3 CDRs with the amino acid sequence according to SEQ ID No. 3, 4 and 5.

5. VHH against hantavirus surface proteins Gn / Gc, Gc responsible for Hantavirus cardiopulmonary syndrome, characterized by VHH comprising 3 CDRs with at least 90% identity to the amino acid sequence according to SEQ ID No. 8, 9 and 10.

6. VHH according to claim 5, characterized in that the CDRs are contained in a VHH with an amino acid sequence having at least 90% identity with SEQ ID No.

7.

7. VHH according to claim 6, characterized in that VHH is encoded by a nucleotide sequence with at least 90% identity to SEQ ID No.

6.

8. VHH according to claim 5, characterized by the VHH comprising 3 CDRs with the amino acid sequence according to SEQ ID Nos. 8, 9 and 10. Petition 870250089842, dated 10 / 02 / 2025, pp. 61 / 69 2 / 3 9. METHOD FOR THE DETECTION OF HANTAVIRUS, characterized by a VHH according to claim 1 being used to detect the presence of the virus in a sample by means of an immunoassay.

10. METHOD according to claim 9, characterized by the immunoassay being selected from ELISA, immunoblotting, immunohistochemistry or immunoprecipitation.

11. METHOD FOR NEUTRALIZING HANTAVIRUS, characterized by a VHH according to claim 1 being used to neutralize the virus.

12. METHOD according to claim 11, characterized by the in vivo neutralization of the virus enabling control of the disease in an individual.

13. METHOD according to claim 11, characterized in that VHH is at least partially humanized.

14. METHOD according to claim 11, characterized in that VHH is linked to a carrier molecule.

15. METHOD according to claim 11, characterized by the VHH according to claim 1 being linked to a human Fc fragment.

16. METHOD FOR THE DETECTION OF HANTAVIRUS, characterized by a VHH according to claim 5 being used to detect the presence of the virus in a sample by means of an immunoassay.

17. METHOD according to claim 16, characterized in that the immunoassay is selected from ELISA, immunoblotting, immunohistochemistry or immunoprecipitation.

18. METHOD FOR NEUTRALIZING HANTA VIRUS, characterized by a VHH according to claim 5 being used to neutralize the virus.

19. METHOD according to claim 18, characterized by the in vivo neutralization of the virus to allow control of the disease in an individual.

20. METHOD according to claim 18, characterized in that VHH is at least partially humanized.

21. METHOD according to claim 18, characterized in that VHH is linked to a carrier molecule.

22. METHOD according to claim 18, characterized in that VHH, according to claim 5, is linked to a human Fc fragment.

23. METHOD according to claim 12 and claim 19, characterized by a combination of VHH-1 SEQ ID Nos. 1 and 2 identified by the 3 CDR sequences SEQ ID Nos. 3, 4 and 5 being able to be combined with VHH-2 SEQ ID Nos. 6 and 7 identified by the 3 CDR sequences SEQ ID Nos. 8, 9 and 10, wherein in vivo neutralization of the virus allows control of the disease in an individual. Petition 870250089842, dated 02 / 10 / 2025, pp. 63 / 69

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