NANO antibody against herpes simplex virus type 1

Abstract

The present invention describes a nano antibody specific against herpes simplex virus type 1, with inhibiting effect against the virus, and the use thereof both to diagnose and to treat the infection caused by the virus. A method for producing the nano antibody is also described.

Description

[0001] NANOANTIBODY AGAINST HERPES SIMPLEX VIRUS TYPE 1

[0002] DESCRIPTION

[0003] TECHNICAL FIELD OF INVENTION

[0004] The present invention falls within the fields of medicine, immunology, biotechnology, and virology, and more specifically, relates to the treatment of infectious diseases transmitted by viruses using immunology techniques. Specifically, it describes a nanobody specific to the Herpes simplex virus type 1 for use in both the diagnosis and treatment of the infection caused by said virus.

[0005] STATE OF THE PRIOR ART

[0006] The herpes simplex virus (or “HSV”) is one of the most common infections worldwide. It is divided into two types: herpes simplex type 1 (HSV-1) and herpes simplex type 2 (HSV-2). HSV-1 is primarily transmitted through oral contact and causes oral and cold sores, and less commonly, it can also cause genital herpes. HSV-2 is transmitted through sexual contact and causes genital herpes. Both oral and genital herpes are mostly asymptomatic, but can cause painful blisters or ulcers. The infection lasts a lifetime, and symptoms may reappear after many years.

[0007] According to the latest data, an estimated 67% of the world's population is infected with HSV-1, many of which are acquired during childhood. Furthermore, it is estimated that in the United States alone, this number will increase by more than 600,000 new infections per year by 2050.

[0008] HSV-1 infection can cause complications in immunosuppressed individuals, where the virus can cause more severe symptoms and viral reactivation is more common. Furthermore, there are cases in which HSV-1 infection can even cause serious problems such as encephalitis or keratitis.

[0009] HSV belongs to the Herpesviridae family, the Alphaherpesvirinae subfamily, and the Simplexvirus genus. It consists of a core containing linear, double-stranded DNA. This genome is encapsulated within an icosahedral capsid, or nucleocapsid, which is surrounded by a layer of proteins called the tegument, which are involved in viral replication in the host cell. The outermost envelope contains four important membrane proteins: the trimeric glycoprotein B (gB), the receptor-binding glycoproteins C and D (gC and gD), and the gH / gL heterodimer, which mediate host cell entry through interaction with the host cell's surface proteins. Unlike other viruses, herpesviruses encode receptor-binding and membrane-fusion functions using different proteins.

[0010] gB is a trimeric transmembrane protein with five extracellular domains. gB is the viral fusion protein, responsible for insertion into the host cell membrane and driving the fusion of the viral envelope and the cell membrane.

[0011] HSV-1 infection involves two main stages: first, the virus binds to the cell surface, and second, it fuses with the plasma membrane. Binding to the cell surface occurs through specific cellular receptors on the viral envelope, such as gB and gC, which bind to the cell surface proteoglycan "heparan sulfate." gD, for its part, specifically binds to at least one of the three known entry receptors: herpesvirus entry mediator (HVEM), nectin-1, or sulfated heparan sulfate 3-0. The subsequent stage of fusion of the viral envelope with the cell plasma membrane involves the glycoproteins gB, gD, gH, and gL.

[0012] Therefore, it can be said that in HSV-1 the complex formed by the gD dimer, the gH / gL heterodimer and the gB trimer are necessary and sufficient for the entry of the virus into the cell. The binding of gD to the entry receptor displaces the carboxyl terminus of the gD ectodomain and transmits a signal to the gH / gL complex which in turn activates the gB fusion protein to insert hydrophobic fusion loops into the cell membrane [1, 2]

[0013] In addition to the glycoproteins involved in entry, other virion component proteins are involved in the development of the infection. These include: vhs (unique long region 41 or U L 41) involved in inducing the inhibition of host protein synthesis, VP16 which prevents the degradation of viral mRNAs, or protein kinase U L 13 (single long region 13) whose absence blocks infection.

[0014] Currently, there are various treatments that help reduce the severity and frequency of symptoms caused by HSV-1, but these treatments do not cure the infection, so people infected with HSV-1 are forced to adapt to living with the infection. The main drugs used are the antivirals acyclovir (the drug of choice), famciclovir, and valacyclovir. All of them belong to the group of nucleoside analogues. These analogues are an important class of antiviral agents frequently used against infections with various viruses, including human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and herpes simplex virus (HSV). Nucleoside analogues are involved in DNA replication and are utilized by viral polymerases, but not by human ones.Acyclovir is phosphorylated intracellularly by viral kinases, and the resulting triphosphate competes with guanosine for incorporation into viral DNA, blocking viral DNA polymerase activity. Acyclovir was approved for use in the treatment of herpes simplex virus infections in 1982. It can be administered topically or systemically. The recommended oral dose is 200 to 800 mg three or five times daily for 5 to 10 days, and the typical intravenous dose for severe infections is 5 to 10 mg / kg every 8 hours for 5 to 10 days.

[0015] The World Health Organization currently has programs to increase awareness of herpes simplex virus infection and improve access to antiviral medications. It also supports research to develop new infection prevention and control strategies, demonstrating the importance and need for new alternatives to existing treatments.

[0016] Antibodies or immunoglobulins are molecules composed of two heavy chains (H) and two light chains (L). In 1993, Professor Raymond Hamers published that species of the Camelidae family contain a fraction of functional antibodies composed solely of heavy chains (Heavy Chain Antibodies or “HcAbs”). These HcAbs are homodimers where each monomer has an antigen-binding fragment (Fab fragment) reduced to a single variable domain (VHH or nanobody). These antibody fragments, derived from HcAbs, are also called single-chain antibodies, nanobodies, nanobodies, single-domain antibodies, VHH antibodies or “nanobodies” in English. In this specification, we will refer to them as nanoantibodies.

[0017] The H chain of nanoantibodies is composed of three globular domains: the VHH, a hinge region, and two constant Fe domains (CH2 and CH3). These Fe domains are highly homologous to the Fe domains of conventional antibodies. The organization of the variable domain of the H chain of conventional antibodies (VH domain) and the VHH domain is similar: both are composed of four conserved sequences, the framework regions (FR), flanking three hypervariable regions (HV) or complementarity-determining regions (CDRs).

[0018] However, there are some clear differences in the alignment of the amino acid sequences of the VH and VHH domains: the CDR1 and CDR3 regions of VHH are wider than those of VH; the VHH sequence carries major substitutions of highly conserved amino acids located in the framework-2 (FR2) region; the hydrophobic residues Val42, Gly49, Leu50 and Trp52 in VH of conventional antibodies are replaced in the VHH of single-chain antibodies by the hydrophilic residues Phe / Tyr42, Glu49, Arg / Cys50 and Leu / Gly52. The presence of more hydrophilic amino acids in VHH confers greater solubility to these antibodies.

[0019] Nanobodies are the smallest known antigen-binding fragments (2.5 nm in diameter and 4 nm in length) generated through recombinant DNA technology. Their isolation paved the way for generating fully active, stable, soluble, specific, high-affinity, non-immunogenic, and humanizable single-chain antibodies.

[0020] Nanobodies have unique properties such as the ability to recognize epitopes unrecognized by other antibodies and their enhanced stability. Furthermore, they have been successfully used in therapeutic approaches as blockers of receptors associated with different diseases or pathogenic viruses. For human administration, it is necessary to humanize these nanobodies, as has been done for Caplacizumab, the first approved drug developed with a nanobody [3].

[0021] DESCRIPTION OF THE INVENTION

[0022] This invention describes an invention based on the development of a specific nanoantibody for use in the diagnosis and treatment of infection caused by HSV-1.

[0023] The nanoantibody of the invention binds to the virus's gB, promoting the neutralization of the HSV-1 viral infection. gB plays a relevant role as part of the virus's infection machinery, as previously indicated, given that virus infection is closely linked to the presence of said protein. The binding between the nanoantibody of the invention and gB affects the insertion of hydrophobic fusion loops in the cell membrane, limiting the infective capacity of HSV-1. Therefore, the mechanism of action on which the inhibition of HSV-1 infection is based differs completely from the target on which the drugs currently available on the market for the treatment of HSV-1 infections act, such as those previously mentioned: acyclovir, famciclovir and valacyclovir.

[0024] It is demonstrated in the examples of this document that this nanoantibody has an in vitro antiviral activity against HSV-1, neutralizing the infection of Vero cell cultures at concentrations similar to those of acyclovir, the reference compound used as a control, presenting different mechanisms of action as already indicated.

[0025] Furthermore, the nanoantibody is selective, as demonstrated by immunofluorescence assays. It was observed that this nanoantibody labels proteins expressed on the surface of Vero cells when infected with HSV-1, while uninfected cells do not fluoresce, demonstrating its specificity. Therefore, it can be used for diagnosing HSV-1 infections.

[0026] An important aspect is that the nanoantibody is nontoxic to host cells, which makes it an excellent candidate for use in a safe treatment method for HSV-1.

[0027] This document also describes the procedure for obtaining said nanoantibody.

[0028] The NbHSV69B nanoantibody, hereinafter “nanoantibody of the invention”, in the context of the present invention, is defined as the protein with the amino acid sequence SEQ ID NO: 2, where SEQ ID NO: 2 is:

[0029] MAQVQLQESGGGSVQAGGSLRLSCAASIDNFRGYSVGWFRQAPGKEREWVSGIEAGY EWPVYADSVKGRFTLSRDNVKNVVYLQMNSLKPEDTAIYYCAAGITYGAYNYWGQGTQ VTVSSAAAYPYDVPDYGSH HHHHH .

[0030] The NbHSV69B nanoantibody is also defined by the nucleic acid molecule that constitutes the coding sequence of the protein collected in SEQ. ID NO: 2, and which would comprise various variants from: a) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID. NO: 2, b) nucleic acid molecules whose complementary chain hybridizes with the polynucleotide sequence of a), c) nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code, d) nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 90%, 95%, 98% or 99% with SEQ. ID NO: 2, and in which the polypeptide encoded by said nucleic acids has the activity and the structural characteristics of the NbHSV69B nanoantibody.

[0031] Among such nucleic acid molecules is the one picked up in the sequence herein referred to as the SEQ. ID NO: 1:

[0032] ATGGCCCAGGTGCAGCTGCAGGAGTCTGGGGGAGGCTCGGTGCAGGCTGGAGGGT CTCTGAGACTCTTCCTGTGCAGCCTCTATAGACAACTTCAGAGGTATTCGGTGGGCT GGTTCCGCCAGGCTCCAGGGAAGGAACGTGAGTGGGTGTCAGGTATTGAGGCTGGT TATGAATGGCCAGTTTACGCCGACTCCGTGAAGGGCCGATTCACCCTCTCCCGAGA

[0033] CAACGTCAAGAACGTGGTGTACCTACAAATGAACAGCCCTGAAACCTGAGGACACGG CCATCTATTACTGTGCGGCCGGCATAACATACGGCGCGTATATTGGGGCCAGG GGACCCAGGTCACC

[0034] The terms “polynucleotide” and “nucleic acid” are used here interchangeably, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA) and deoxyribonucleotides (DNA).

[0035] The terms “amino acid sequence,” “peptide,” “oligopeptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.

[0036] Therefore, a first aspect of the invention relates to the isolated nanoantibody NbHSV69B of amino acid sequence SEQ. ID NO: 2, or isolated protein with an identity of at least: a) 90% with SEQ. ID NO: 2, b) 95% with SEQ. ID NO: 2, c) 99% with SEQ. ID NO: 2, and which has the activity and structural characteristics of the nanoantibody NbHSV69B or nanoantibody of the invention.

[0037] A second aspect of the invention relates to the isolated nucleic acid of amino acid sequence SEQ ID NO: 1 that encodes the nanoantibody NbHSV69B.

[0038] A third aspect of the invention relates to a vector comprising the isolated nucleic acid of amino acid sequence SEQ ID NO: 1 encoding the nanoantibody NbHSV69B

[0039] A fourth aspect of the invention relates to a host cell comprising the nucleic acid of amino acid sequence SEQ ID NO: 1 or the vector comprising it.

[0040] A fifth aspect of the invention relates to a composition comprising the nanoantibody of the invention, the nucleic acid of amino acid sequence SEQ ID NO: 1, the vector comprising said nucleic acid or the aforementioned host cell, hereinafter "composition of the invention". Preferably, the composition of the invention is a pharmaceutical composition. More preferably, it also comprises a pharmaceutically acceptable carrier and / or pharmaceutically acceptable excipients. In a preferred embodiment of the composition of the invention, it also comprises another active ingredient.

[0041] A sixth aspect of the invention relates to the nanoantibody of the invention, the nucleic acid of amino acid sequence SEQ ID NO: 1, the vector comprising said nucleic acid, the aforementioned host cell or the composition of the invention in any of its embodiments for use in the preparation of a medicament for the treatment of infection caused by HSV, preferably type 1.

[0042] A seventh aspect of the invention relates to the nanoantibody of the invention for use in a method of diagnosing HSV-1 virus infection.

[0043] An eighth aspect of the invention relates to the method for diagnosing HSV-1 infection carried out on a previously isolated tissue sample that presents lesions compatible with the infection caused by the HSV-1 virus and which comprises: a) infecting a cell culture, preferably Vero cells, with said isolated sample; b) incubating the infected cell culture, preferably Vero cells, together with the nanoantibody of the invention; c) carrying out a test that allows detecting the nanoantibody of the invention bound to the cells of the cell culture, where appropriate Vero cells, preferably by immunofluorescence.

[0044] Preferably, the immunofluorescence test is carried out as described below in Example 5. The presence of fluorescence in the Vero cell culture is indicative of infection by the HSV-1 virus, regardless of the level of fluorescence.

[0045] A ninth aspect of the invention relates to a diagnostic kit comprising nanoantibody of the invention and means for detecting the specific binding of the nanoantibody to the HSV-1 virus.

[0046] A final aspect of the invention describes the method for obtaining the nanoantibody of the invention, which comprises: a. isolating mRNA from peripheral blood mononuclear cells extracted from camelids previously immunized with inactive HSV-1 virus, b. retrotranscribing the RNA to cDNA, c. amplifying the VHH sequences or camelid nanoantibodies by PCR, d. cloning and amplifying the nanoantibodies ligated into a phagemid vector in Escherichia coli, e. isolating the phage particles obtained, f. exposing the phage particles to HSV-1, g. selecting the phage particles specifically bound to the HSV-1 virus, h. amplifying the selected phage particles in E. coli, and i. selecting nanoantibodies with high binding affinity.

[0047] Throughout the description and claims, the word "comprise" and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and drawings are provided for illustrative purposes only and are not intended to limit the present invention.

[0048] BRIEF DESCRIPTION OF THE FIGURES

[0049] Figure 1: Immunization efficiency with inactivated HSV-1 antigen (A) Table of Ag+ / Ag- ratio from ELISA absorbance values. It shows values ​​for 14-fold serial dilutions of camel serum before immunization (Pre-Serum) and 14-fold serial dilutions of camel serum after immunization (Post-Serum). (B) Graph of the values ​​in the table above. An Ag+ / Ag- ratio greater than 2.5 indicates good immunization and the presence of specific nanobodies.

[0050] Figure 2: Data from the Biopanning rounds performed and the candidate selection process for the different rounds; 190 candidates were selected from Biopanning 1 (95 A and 95 B) and 95 from Biopanning 2 (C).

[0051] Figure 3: Calculation of the 50% infecting viral dose (TCID50): the percentage of cell viability in Vero cells at each HSV-1 dilution. The line labeled 50 marks 50% cell viability. The trend line marks the cutoff point between 50% cell viability and the required HSV-1 dilution or dose. Mean values ​​with standard deviation.

[0052] Figure 4: 100TCID50 HSV-1 neutralization assays in Vero cells. Percentage of cell viability from the different assays performed, with different concentrations of the inventive nanoantibody NbHSV69B, and acyclovir, used as a positive control. The line labeled 50 marks 50% cell viability of the uninfected and untreated cell control, in addition to the minimum neutralizing concentration of acyclovir or the inventive nanoantibody using 100TCID50 HSV-1. Mean values ​​with their standard deviation.

[0053] Figure 5: Cytotoxicity assay in uninfected Vero cells. Cell viability under decreasing concentrations of acyclovir (green), DMSO (yellow) as a solvent control for acyclovir, and the inventive NbHSV69B nanobody (blue). Mean values ​​with standard deviation.

[0054] Figure 6: Immunofluorescence assay. Images obtained by fluorescence microscope. (A) HSV-1-infected Vero cell in the presence of the NbHSV69B nanobody plus primary anti-hemagglutinin (HA) antibodies (Biolegend, purified anti HA.11 Epitope Tag Antibody) and secondary anti-mouse conjugated with Alexa 488 (A11001, Life Technologies), detecting the virus surface proteins on the cell membrane (in green). In blue, cell nuclei stained with 4',6-diamidino-2-phenylindole (DAPI). (B) HSV-1-infected Vero cells without NbHSV69B with primary and secondary antibodies. (C) Vero cell not infected with HSV-1 in the presence of the NbHSV69B nanobody plus primary and secondary antibodies. (D) HSV-1 uninfected Vero cells without NbHSV69B with primary and secondary antibodies.

[0055] Figure 7: Analysis of data generated by immunoprecipitation. (A) Venn diagram table showing the interrelation of all proteins detected in each replicate of HSV-1 immunoprecipitation with the nanoantibody of the invention. (B) Venn diagram figure of Table A.

[0056] Figure 8: Map of the pMES4 phagemid and sequence landmarks (GenBank: GQ907248.1). Lac UV5 promoter: bases 143–198; RBS (ribosome binding site): bases 211–215; pelB (signal peptide): bases 225–290; Multiple cloning site (Pst I, Xba I, Bst Eli): bases 305–325; His-tag (six tandem histidines): bases 340–357; HA (hemagglutinin) tag: bases 361–385; bacteriophage M13 gene III: bases 391–1623; ampicillin resistance: bases 2701–3558.

[0057] DETAILED DESCRIPTION OF THE INVENTION

[0058] Culture of cells and viruses used for the

[0059] Vero cells (African green monkey kidney cells) were maintained in DMEN (Dulbecco's Modified Eagle Medium) High Glucose medium (Biowest, L0103), supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (Sigma Aldrich, 10270106), 100U / ml Penicillin, 100ug / ml Streptomycin (Sigma Aldrich, P0781) and 1% L-Glutamine (Sigma Aldrich, G8540) in a humidified environment at 37°C and 5% CO2.

[0060] HSV-1 virus was purchased in two formats: inactivated virus (Ref: 229-20255, RayBiotech) and active virus VR-1493™ from American Type Culture Collection (ATCC), Rockville, MD, USA, which were propagated in Vero cells in a laboratory with a biosafety level 2.

[0061] Example 1: Generation, expression and purification of the nanoantibody of the invention.

[0062] Immunization and isolation of lymphocytes. A camel was immunized with 0.6 mg of inactivated HSV-1 (229-20255) (RayBiotech) as antigen, inoculating at 0.1 mg per week for six weeks.

[0063] A serum sample was collected before starting immunization to check the immune response to the antigen.

[0064] Peripheral blood collected after immunization was serially diluted v in sterile PBS and peripheral blood mononuclear cells (PMBC) were isolated.

[0065] To verify the efficiency of immunization and continue with the generation of the nanoantibody library, an antibody ELISA test was performed with the sera collected before (pre) and after (post) immunization.

[0066] In Nunc-lmmuno™ MicroWell™ 96-well flat-bottom plates (Sigma Aldrich, M5785), seven wells in two separate rows were immobilized with HSV-1 antigen (229-20255, RayBiotech) and incubated at 4°C overnight, leaving the row below each well for negative control of pre- and post-immunization serum.

[0067] The following day, 1-fold serial dilutions of the pre- and post-immunization serum were made in 2% milk in a 96-well microtiter plate (Deltalab, 900011). The pre- and post-immunization serum dilutions were added to the ELISA plate as the primary antibody with the immobilized antigen and pre-washed with PBST (phosphate-buffered saline with a low concentration of detergent solution). After 1 hour of incubation, an anti-llama antibody (Thermo Fisher, A16060) conjugated with peroxidase (HRP) was added as the secondary antibody under the same conditions as the previous antibody. After 1 hour of incubation at room temperature, the plate was washed again with PBST. To reveal the experiment, ABTS (2,2'-azino-di-(3-ethylbenzothiazoline-6-sulfonate substrate solution) / H2O2 of HRP (Roche ABTS tablets, REF 11112422001) (100 pl / well) was added and the reading was performed at 405 nm in a spectrophotometer (Infinite M200PRO).

[0068] The Antigen positive / No antigen (Ag+ / Ag-) ratio of the ELISA test absorbance values ​​post immunization, greater than 2.5 [4], show good immunization and presence of specific nanoantibodies (Figure 1A and 1 B).

[0069] Generation of a phage nanoantibody library

[0070] For the construction of the HSV-1 nanobody library, the protocol described below was followed [5], - mRNA extraction and reverse transcription; total mRNA extracted from PBMCs was reverse transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo) and Oligo primers (dT^. The parameters for reverse transcription were: 1 cycle at 42°C for 60 minutes and 1 cycle at 70°C for 20 minutes.

[0071] - Amplification of VHH sequences; From the purified cDNA, gene fragments encoding the variable regions of the heavy chains of all camelid immunoglobulins were amplified. The oligonucleotides used were: CALL001: 5'-GTCCTGGCTGCTCTTCTACAAGG-3' (SEQ. ID. NO: 3) and CALL002: 5'- GGTACGTGCTGTTGAACTGTTCC-3' (SEQ. ID. NO: 4). The polymerase chain reaction (PCR) was performed with Hot Start Taq DNA polymerase (5 U / ml, Roche) and the PCR parameters were: 95°C, 15 minutes to activate the polymerase and subsequently, 32 cycles at (94°C, 1 minute; 55°C, 1 minute; 72°C, 1 minute) and finally an extension step of 72°C, 10 minutes. The PCR product was purified and used as a template in a second PCR. To amplify the VHH sequences, the nested oligonucleotides were used: A6E: 5'-GATGTGCAGCTGCAGGAGTCTGGRGGAGG-3' (SEQ. ID. NO: 5) and 38: 5'-GGACTAGTGCGGCCGCTGGAGACGGTGACCTGGGT-3' (SEQ. ID.NO: 6) containing the target sequences of the Pstl and BstEII enzymes at their ends for subsequent cloning of the inserts. The amplification parameters were: 95°C, 15 minutes, 18 cycles (94°C, 45 seconds; 55°C, 45 seconds; 72°C, 45 seconds) and 72°C, 10 minutes.

[0072] - Cloning of the nanoantibody repertoire into a phagemid vector; the amplified VHH fragment repertoire and the pMES4 phagemid (GenBank: GQ907248.1) (Figure 8) were digested for subsequent overnight ligation with T4 DNA ligase, which catalyzes the formation of a phosphodiester bond between the 5'-terminal phosphate and the juxtaposed 3'-terminal hydroxyl for the union of the two desired DNA molecules (insert and vector) into a plasmid.

[0073] The ligation product was transformed into competent E. coli TG1 strain cells (Biosearch Technologies), which were infected with the helper phage VCSM 13 (Agilent) and incubated in Luria Bertani (LB) selective medium containing the antibiotics Ampicillin (Sigma Aldrich) and Kanamycin (Sigma Aldrich) to arrange the library into phage particles. After incubation, phage particles were isolated by precipitation through polyethylene glycol 6000 centrifugation (Sigma Aldrich).

[0074] Enrichment of HSV-1 Specific Clones: Selection Rounds or Biopanninq One ​​well of a 96-well flat-bottom ELISA plate was coated with a given amount of HSV-1 antigen. After a timed incubation, it was blocked with 2% milk powder in phosphate-buffered saline (1X PBS: 137 mM NaCl (Scharlau), 2.7 mM KCl (Sigma Aldrich), 10 mM Na2HPO4 (Sigma Aldrich), and 1.8 mM KH2PO4 (Sigma Aldrich)) for 2 hours at room temperature.

[0075] Then 10 were added 11 phage particles and incubated to allow specific binding. Antigen-specific phage particles were eluted with 100 mM triethylamine (TEA) (pH 11.0), transferred to a sterile tube, and neutralized with 100 µl of 1.0 M Tris-HCl (pH 7.4).

[0076] The same procedure was performed simultaneously in a well without antigen to determine the number of nonspecifically bound phage particles.

[0077] Phage particles eluted from the antigen-containing well (Ag+) were identified as phage particles from the first round of panning.

[0078] These were amplified by infecting E. coli TG1 cells in exponential phase in the presence of glucose to repeat the same process in successive rounds of screening until a significant enrichment greater than 300 of the well with antigen to that without antigen was determined.

[0079] Two biopannings were performed with three rounds of enrichment each. From the third round, with an enrichment greater than 300, 190 candidates were selected from the first biopanning (95A and 95B), and likewise, another 95 candidates were selected from the second biopanning (C), obtaining a total of 285 candidates that were stored at -80°C in sterile 96-well plates in culture medium (Figure 2).

[0080] Selection of specific clones against HSV-1.

[0081] In 96-well Nunc-lmmuno™ MicroWell™ flat-bottom plates (Sigma Aldrich, M5785), all wells in a row were coated with HSV-1 antigen (229-20255, RayBiotech), leaving the bottom row for the negative control of each candidate without antigen.

[0082] On the other hand, in 2.2 ml storage plates (Thermo Fisher, AB0932), the 285 candidates were grown in 2XTY / ampicillin 100 pg / ml / 1% glucose medium at 37 ° C until reaching an optical density (OD) at 600 nm of «1 and were induced with 1 mM of isopropyl-pD-1-thiogalactopyranoside (IPTG). Subsequently, they were centrifuged at 3000g for 20 minutes and resuspended in PBS to add each candidate to the ELISA plate in a position with antigen (Ag +) and without antigen (Ag -).

[0083] Specific nanoantibody detection was performed using mouse anti-hemagglutinin (HA) monoclonal antibody (Biolegend, purified anti HA.11 Epitope Tag Antibody) in 2% milk powder in PBS incubated for 1 hour. It was washed with PBST (phosphate-buffered saline with a low concentration of detergent solution), and anti-mouse immunoglobulin G secondary antibody conjugated with peroxidase (HRP) (Promega, W402B) was added under the same conditions as the previous antibody.

[0084] To reveal the experiment, ABTS (2,2'-azino-di-(3-ethylbenzothiazoline-6-sulfonate substrate solution) / H2O2 of HRP (Roche ABTS tablets, REF 11112422001) (100 pl / well) was added and the reading was performed at 405 nm in a spectrophotometer (Infinite M200PRO). All candidates with an Ag+ / Ag- ratio greater than or equal to 2.5 were considered positive [4]. 13 that did not meet this condition were discarded, leaving 272 candidates.

[0085] Sequencing and characterization of VHHs.

[0086] The 272 nanoantibodies with the highest affinity were sequenced by the Sanger method using the oligonucleotide MP57: TTATGCTTCCGGCTCGTATG (SEQ. ID. NO: 7), specific to the phagemid, as a primer.

[0087] All DNA sequences were translated into proteins and then aligned and organized according to their percentage of similarity. Eight groups with different amino acid sequences were obtained from all the sequences. One nanoantibody was randomly selected from each group, and the VH and VHH sequences were determined following the criteria previously described for the ImMunoGeneTics-IMGT Information System Numbering http: / / www.imgt.org corresponding to positions 37, 44, 45, and 47 in Kabat numbering, http: / / www.kabatdatabase.com).

[0088] Expression and purification of nanoantibodies in E. coli.

[0089] The expression and purification of the 8 nanobodies of different sequences that were obtained was carried out following the protocol [6] used by the E. coli strain\N .Q.

[0090] In 1 liter of TB / ampicillin / glucose medium, 3 ml of the nanoantibody preculture in WK6 cells grown the previous night were added. It was incubated at 37°C with shaking until the OD at 600 nm reached the exponential phase ~ 0.8. Then, 1 mM isopropyl pD-1-thiogalactopyranoside (IPTG) (Thermo Scientific™) was added to induce nanoantibody expression. Subsequently, the pellet was centrifuged at 4°C for 8 minutes at 9000g. The supernatant was discarded and the pellet was resuspended in TES buffer (C6H14NNaO6S) and incubated at 4°C for 3 hours. Subsequently, 24 ml of TES / 4 (14x dilution of TES buffer) was added and incubated overnight at 4°C on a horizontal shaker. 210 mM MgCl2 was added and centrifuged at 9000g and 4°C for 30 minutes. The supernatant, containing the nanobodies, was equilibrated with the binding buffer (20 mM phosphate buffer, 0.5 M NaCl, 30 mM imidazole pH 7.4) and purified by fast protein liquid chromatography (FPLC) using the AKTA™ protein purification system (GE Healthcare Life Sciences), [7] with 1 ml His-Trap™ HP columns. Protein concentration was quantified by the Bradford method.

[0091] Example 2: Determination of the TCID50 of HSV-1

[0092] An assay was performed to determine the quantity of infectious HSV-1 particles required to reach 50% infection of the culture (TCID50) following the Spearman-Karber method as previously described by [8, 9]. Briefly, 10 4 Vero cells / well in 96-well plates minus column 12 and incubated for 24 hours. Serial dilutions (10') were made 1 — 10' 7) of the virus stock in Minimal Essential Medium (MEM) (Biowest, L0430), with 2% inactivated Fetal Bovine Serum (Sigma Aldrich, 10270106) and 50U of Penicillin, 50 pg of Streptomycin (Sigma Aldrich, P0781) and 100 pl of each dilution was added to each well of the 96-well plate. Additionally, wells with uninfected cells were placed as a cell control (CC) in column 11 and column 12 as a developer control. The plates were incubated at 37°C and 5% CO2 for 2 days.

[0093] Cell viability was determined by staining with the Neutral Red developer (Sigma Aldrich, N4638) following the protocol [9] using 80% acetone (Scharlau, AC03141000) in DPBS (Sigma Aldrich, D8537) as a fixative.

[0094] The TCID50 titer of the virus was 4.22x10 5 / ml and was calculated according to the Spearman-Karber method [8] as shown in Figure 3.

[0095] Example 3: Viral neutralization assay.

[0096] Vero cells were seeded in 96-well plates at 10 4cells / well, leaving column 12 empty and incubated overnight at 37°C and 5% CO2. The nanoantibody of the invention NbHSV69B and acyclovir (Sigma Aldrich, PHR1254), as a positive neutralization control, were diluted in Minimum Essential Medium (MEM) (Biowest, L0430), 2% heat-inactivated Fetal Bovine Serum (Sigma Aldrich, 10270106) and 100 ll / ml of Penicillin, 100 pg / ml of Streptomycin (Sigma Aldrich, P0781) at an initial concentration of 40 pM making serial dilutions 1 in a final volume of 50 pl. In column 9, eight wells were used as a virus infection control (100TCID50 / well) in 100 pl. In column 10, a virus titer control was performed by making 1 / 10 dilutions of the previously titrated viruses in 100 µl. In column 11, eight wells were used for QC in 100 µl.Dilutions of the nanoantibody and acyclovir were mixed with an equal volume of HSV-1 virus (50 µl) diluted to produce 100 TCID50 / well and incubated for 1 hour in a humidified environment at 37°C and 5% CO2. After incubation, the virus and nanoantibody or virus and acyclovir mixtures were transferred to the cells preseeded and washed with DPBS (Sigma Aldrich, D8537). They were incubated for 48 hours at 37°C and 5% CO2.

[0097] Cell viability was determined by staining with Neutral Red (Sigma Aldrich, N4638) following the same protocol described for determining the TCID50. Wells with a viability equal to or greater than 50% of the control cell mean were defined as neutralizing. The results of the assay are shown in Figure 4. The 50 line marks 50% of cell viability calculated using CC. Above this line, cell viability is high, and therefore, virus infection of cells is low.

[0098] In this test it was determined that the nanoantibody of the invention (NbHSV69B) is capable of neutralizing the infection of cells by HSV-1, showing concentrations that resemble those of acyclovir, the treatment of choice currently used, as can be seen in the following Table 1, where it is observed that there are no statistically significant differences between the two.

[0099] Table 1: Comparison between the cell viability results of cells infected with HSV-1 and subjected to treatment with nanoantibody of the invention (NbHSV69B) and acyclovir at various concentrations. The P values ​​obtained with a parametric t Test performed in GraphPad Prism version 9. Significant differences are considered p values ​​<0.01.

[0100] Example 4: Cytotoxicity assay.

[0101] A cytotoxicity assay was performed at different concentrations of the nanoantibody of the invention NbHSV69B, acyclovir and the vehicle or solvent of the latter (DMSO), in Vero cells. Vero cells were seeded in 96-well plates at 10 4cells / well, leaving column 12 empty and incubated overnight at 37°C and 5% CO2. The nanoantibody of the invention NbHSV69B, acyclovir (Sigma Aldrich, PHR1254) and DMSO (Sigma Aldrich, D8418), were diluted in Minimum Essential Medium (MEM) (Biowest, L0430), added with 2% heat-inactivated Fetal Bovine Serum (Sigma Aldrich, 10270106) and 100 U / ml of Penicillin, 100 pg / ml of Streptomycin (Sigma Aldrich, P0781) at an initial concentration of 75 pM making 1-fold dilutions in a final volume of 50 pl. Nanoantibody dilutions, acyclovir and DMSO were transferred to pre-seeded cells washed with DPBS (Sigma Aldrich, D8537). They were incubated for 48 hours at 37°C and 5% CO2 and cell viability was determined by staining with Neutral Red (Sigma Aldrich, N4638) following protocol [9] using 80% acetone (Scharlau, AC03141000) in DPBS (Sigma Aldrich, D8537) as a fixative.

[0102] The results of the cytotoxicity assay confirmed that the NbHSV69B nanobody did not significantly decrease cell viability at any concentration, which was even greater than the concentrations used in the neutralization assay and which are effective against neutralizing infection (Figure 5).

[0103] Unlike acyclovir, at concentrations greater than 40 pM, the NbHSV69B nanobody is not toxic to cells. Acyclovir reduces cell viability to below 80% at concentrations greater than 40 pM, although dimethyl sulfoxide (DMSO) as its solvent and vehicle is excluded from this effect.

[0104] Example 5: Immunofluorescence.

[0105] Vero cells were seeded on 13 mm circular coverslips placed in 24-well plates and incubated overnight for adhesion. Cells were infected with 0.5 ml of the dilution that had generated 50% cell viability in a previous assay, and this viability was checked and confirmed by microscopic observation of the cultures. This dilution corresponded to the 10' dilutions. 3from the same batch of amplified and titrated viruses for neutralization assays. 0.5 ml of virus-free medium was added to the uninfected cell control. They were incubated for 24 hours in a humidified environment at 37°C and 5% CO2. Twenty-four hours post-infection, the medium was removed and the cells were washed 3 times with DPBS and then fixed with 4% (v / v) PFA for 10 minutes at room temperature. The fixative was removed, washed 3 times with DPBS and blocked with 10% bovine serum albumin (BSA) (A7906, Sigma Aldrich) for 60 minutes at room temperature.

[0106] After incubation of infected and uninfected cells, they were washed with DPBS and both were incubated with the NbHSV69B nanoantibody of the invention. 200 pl / well and a final amount of nanoantibody of 2.64 pg were added to each well. The incubation was carried out at 4°C overnight (ON). Wells to which no nanoantibody was added were incubated with 200 pl of dilution buffer. After incubation, 3 washes were performed.

[0107] The mouse anti-hemagglutinin (HA) monoclonal antibody (Biolegend, purified anti HA.11 Epitope Tag Antibody) was used as the primary antibody at a 1 / 500 dilution in dilution buffer. The antibodies were incubated for 1 hour at room temperature and, after washing, were incubated with the secondary antibody (anti-mouse conjugated to Alexa 488) (A11001, Life Technologies) at a 1 / 500 dilution in dilution buffer for 30 minutes at 4°C in the dark.

[0108] The test conditions were as follows: infected cells plus NbHSV69B plus primary and secondary antibodies, infected cells without NbHSV69B with primary and secondary antibodies, and these same two conditions with uninfected cells.

[0109] Finally, all coverslips were mounted on slides (J2800AMNZ, Thermo Fisher Scientific) with Vectashield (H-1200, Vector), including DAPI for nuclear counterstaining.

[0110] Images were taken on a Zeiss Axio Imager A.1 fluorescence microscope with a 63x immersion objective. Images (Figure 6) were captured from randomly selected fields of view.

[0111] It is observed that the NbHSV69B nanobody labels the proteins expressed on the surface of Vero cells when infected by HSV-1, while uninfected cells do not show fluorescence, which shows its specificity.

[0112] Example 6: Immunoprecipitation. For immunoprecipitation, 500 pg of inactivated HSV-1 (229-20255, RayBiotech) was solubilized in RIPA buffer at a 1:1 ratio. Viruses were incubated on ice for 10 minutes and then vortexed for 30 seconds. The solution was mixed with the NbHSV69B nanobody at a concentration of 2 pg / 100 pl and incubated overnight at 4°C.

[0113] Next, 7 pg of anti-hemagglutinin (HA) antibody (Biolegend, purified anti HA.11 Epitope Tag Antibody) was added and incubated at room temperature for 3 hours, followed by an additional 2 hours incubation with Pierce™ Protein A / G Magnetic Beads (Thermo Scientific). This procedure was performed three times at different times, obtaining 4 different replicates.

[0114] HSV-1 immunoprecipitation was also performed with different antibodies to rule out nonspecific binding. These antibodies were the nanobody-specific anti-hemagglutinin (HA) (Biolegend, purified anti HA.11 Epitope Tag Antibody), an anti-IgG (12-370, Sigma Aldrich), and a nanobody that specifically recognizes a membrane protein of the parasitic protozoan Trypanosoma brucei. The samples were washed and eluted following the manufacturer's recommendations. Protein identification was performed by liquid chromatography-mass spectrometry in the proteomics service of the University of Córdoba.

[0115] Negative controls were used to eliminate all proteins detected nonspecifically or as contaminants. Bioinformatics analysis was then performed using a Venn diagram that correlated all proteins detected in the four replicates.

[0116] After verifying that only three proteins were repeated in the 4 replicates (gB, ICP4 and MCP), it was determined that only gB was a viral membrane protein and is also found in the membrane of HSV-1-infected cells, so it can be detected by immunofluorescence as in the previous assay. The other two, ICP4 and MCP, are described as proteins that in HSV-1-infected cells are found in the nucleus or cytoplasm, not in the plasma membrane, so they could be nonspecific bindings of the assay. The location of the proteins of interest was determined using the protein database www.uniprot.org. This showed that the NbHSV69B nanoantibody of the invention is binding to gB of the viral envelope (Figure 7). Furthermore, it is the protein that has been detected the most times and with a higher percentage of peptide coverage of this protein in all replicates. This protein is involved in the infection process.The binding of the NbHSV69B nanoantibody and the gB would justify the results that have been obtained in the virus neutralization assays.

[0117] References

[0118] 1. Cairns, Tina M., and Sarah A. Connolly. 2021. 'Entry of alphaherpesviruses'. Current Issues in Molecular Biology 41:63-124. doi: 10.21775 / cimb.041.063.

[0119] [ PMC free article ] [ PubMed ] [ Cross Ref ] 2. Karasneh GA, Shukla D. Herpes simplex virus infects most cell types in vitro: clues to its success. Virol J. 2011 Oct 26;8:481. doi: 10.1186 / 1743-422X-8-481. PMID: 22029482; PMCID: PMC3223518.

[0120] 3. Duggan S. Caplacizumab: First Global Approval. Drugs. 2018 Oct;78(15):1639-1642. doi: 10.1007 / S40265-018-0989-0. Error in: Drugs. 2018 Dec;78(18):1955. PMID: 30298461; PMCID: PMC6280848.

[0121] 4. Yan J, Li G, Hu Y, Ou W, Wan Y. Construction of a synthetic phage-displayed Nanobody library with CDR3 regions randomized by trinucleotide cassettes for diagnostic applications. J Transí Med. 2014 Dec 10; 12:343. doi: 10.1186 / s12967-014-0343-6.

[0122] 5. Romáo, Ema, Vianney Poignavent, Cécile Vincke, Christophe Ritzenthaler, Serge Muyldermans, and Baptiste Monsion. 2018. ‘Construction of High-Quality Camel Immune Antibody Libraries’. Pp. 169-87 in Methods in Molecular Biology. Vol. 1701. Humana Press Inc.

[0123] 6. Vincke, Cécile, Carlos Gutiérrez, Ulrich Wernery, Nick Devoogdt, Gholamreza Hassanzadeh-Ghassabeh, and Serge Muyldermans. 2012. ‘Generation of Single Domain Antibody Fragments Derived from Camelids and Generation of Manifold Constructs’. Methods in Molecular Biology 907:145-76. doi: 10.1007 / 978-1 -61779-974-7_8.

[0124] 7. Gotesman M, Wang Y, Madasu SC, Mitchell CA. 2022. ‘Purification of Cytoskeletal Proteins by Fast Protein Liquid Chromatography (FPLC) Using an ÁKTA Start System’. Pp. 237-49 in Cytoskeleton .

[0125] 8. Ramakrishnan, Muthannan Andavar. 2016. ‘Determination of 50% Endpoint Titer Using a Simple Formula’. World Journal of Virology 5(2):85. doi: 10.5501 / wjv.v5.i2.85.

[0126] 9. Repetto, Guillermo, Ana del Peso, and Jorge L. Zurita. 2008. ‘Neutral Red Uptake Assay for the Estimation of Cell Viability / Cytotoxicity’. Nature Protocols 3(7):1125-31. doi: 10.1038 / nprot.2008.75.

Claims

CLAIMS 1. Isolated NBHSV69B nanoantibody of amino acid sequence SEQ ID NO: 2, or isolated protein with an identity of at least 90%, or 95%, or 99% with SEQ ID NO: 2 and that has the activity and structural characteristics of the NBHSV69B nanoantibody.

2. Isolated nucleic acid of sequence SEQ ID NO: 1 encoding the NBHSV69B nanoantibody.

3. Vector comprising the nucleic acid according to the preceding claim.

4. Host cell comprising the nucleic acid according to claim 2 or the vector according to claim 3.

5. Composition comprising the nanoantibody according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3 or the host cell according to claim 4.

6. The composition according to the preceding claim which is a pharmaceutical composition.

7. The composition according to the preceding claim, further comprising a pharmaceutically acceptable carrier and / or pharmaceutically acceptable excipients.

8. The composition according to the preceding claim which further comprises another active ingredient.

9. The nanoantibody according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3 or the host cell according to claim 4, or the composition according to any of claims 5 to 8 for use in the treatment of infection caused by Herpes simplex virus type 1.

10. Use of the nanoantibody according to claim 1, the nucleic acid according to claim 2, the vector according to claim 3 or the host cell according to claim 4, or the composition according to any of claims 5 to 8 for the development of a drug for the treatment of infection caused by the Herpes simplex virus type 1.

11. The nanoantibody according to claim 1 for use in an in vitro method for diagnosing Herpes simplex virus type 1 infection.

12. In vitro method for diagnosing infection by the Herpes simplex virus type 1, which is carried out on a previously isolated tissue sample that presents lesions compatible with the infection caused by the Herpes simplex virus type 1 and which comprises: a) infecting a cell culture with said isolated sample, b) incubating the infected cell culture together with the nanoantibody according to claim 1, and c) carrying out a test that allows detecting the nanoantibody according to claim 1 bound to the cells of the cell culture.

13. In vitro diagnostic kit comprising nanoantibody according to claim 1 and means for detecting the specific binding of the nanoantibody to the Herpes simplex virus type 1.

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