Neutralizing nanoantibody AUR39 against human adenovirus type 4 Hexon protein and its application

Abstract

The present invention relates to a nanobody directed against the human adenovirus type 4 Hexon protein and its use. The nanobody has a unique CDR region, can bind to the adenovirus type 4 Hexon protein with high affinity, and can neutralize adenovirus type 4 pseudovirus with high neutralizing activity in virus neutralization tests. The present invention also provides the use of the nanobody in the preparation of a drug for treating and / or preventing diseases caused by human adenovirus type 4, as well as a diagnostic kit.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to an alpaca-derived nanobody and its application, and more specifically to an alpaca-derived nanobody that binds to adenovirus type 4 Hexon protein, a polynucleotide encoding the same, an expression vector comprising the polynucleotide, a transformed cell, and its application in drugs for preventing, treating and / or detecting adenovirus type 4 infection. Background Art

[0002] Human adenovirus (HAdV) is a DNA virus that often causes respiratory tract infections. It is divided into seven subgenera, AG, of which B, C, and E can cause respiratory diseases such as upper respiratory tract infections, lower respiratory tract infections, and bronchitis.

[0003] Adenovirus infection is widespread worldwide, prone to occurring in spring and winter, spreading rapidly and infecting a wide range of age groups, especially children. Children with adenovirus and those who are latently infected are the main sources of infection, and outbreaks often occur in closed or crowded places. In large-scale epidemics, most patients present with mild, self-limiting infections. Some cases are also accompanied by gastrointestinal symptoms such as diarrhea, vomiting, and abdominal pain. Severe cases can lead to pneumonia or even death. Among them, pneumonia caused by adenovirus infection is particularly serious. The mortality rate of patients with untreated severe HAdV pneumonia or disseminated disease may exceed 50%. Patients with severe pneumonia not only show symptoms such as heart failure and respiratory failure, but also encephalitis, liver damage, myocarditis or myocardial damage, and may even die. In addition, the effect of intervention with relevant means is still poor.

[0004] Adenovirus virulence is composed of multiple major and minor capsid proteins, of which hexon, penton, and fiber are the three major capsid proteins. Hexon is the most abundant protein in the adenovirus capsid and constitutes the primary surface structure of the viral particle. All three major capsid proteins on the viral capsid surface possess immunogenicity and can stimulate the production of neutralizing antibodies. Hexon is the primary protein involved in the formation of the adenovirus capsid and the production of specific antibodies, inducing a strong immune response in the host, including the production of neutralizing antibodies and cellular immune responses.

[0005] In summary, there is currently no effective vaccine or drug for the prevention and treatment of HAdV pneumonia, especially pneumonia caused by group E type 4 adenovirus.

[0006] With the development of genetic engineering technology, alpaca-derived nanobodies are increasingly showing advantages over traditional antibodies. They not only possess strong antigen targeting and binding capabilities, but also overcome the shortcomings of traditional antibodies, such as large size, low stability, high immunogenicity, and slow clearance. Furthermore, the ability of nanobodies to enhance their functionality through bioconjugation with functional groups such as toxins, enzymes, radionucleotides, and fluorescent groups has given them potential application in the diagnosis and treatment of human and animal diseases. The present invention aims to provide an alpaca-derived nanobody capable of targeting adenovirus type 4, and furthermore, its use in the preparation of drugs for the prevention or treatment of adenoviruses. Summary of the Invention

[0007] Based on the above objectives, the present invention first provides a nanobody against human adenovirus type 4 Hexon protein. The nanobody described in the present invention is an antibody with only a heavy chain, wherein the heavy chain includes a variable region and a constant region. The variable region has three complementary determining regions (CDRs): CDR1, CDR2 and CDR3, which are highly variable and diverse. The sequence diversity of the CDR region determines the specificity and affinity of the antibody because they recognize and bind to specific antigenic determinants by interacting with the antigen. The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the nanobody against human adenovirus type 4 Hexon protein provided by the present invention are shown as amino acid residues 26-33, 51-57 and 96-113 of SEQ ID NO: 1, respectively.

[0008] In a preferred technical solution, the amino acid sequence of the heavy chain variable region of the Nanobody against human adenovirus type 4 Hexon protein is shown in SEQ ID NO: 1. In the present invention, the Nanobody having this heavy chain variable region is named "Nanobody AUR39".

[0009] Secondly, the present invention provides a polynucleotide encoding the above-mentioned anti-human adenovirus type 4 Hexon protein nanobody, and the sequence of the polynucleotide encoding the heavy chain variable region of the anti-human adenovirus type 4 Hexon protein nanobody is shown in SEQ ID NO: 2.

[0010] Third, the present invention provides a fusion protein containing the nanobody, in which the C-terminus of the nanobody is fused with a human immunoglobulin Fc segment as shown in SEQ ID NO: 4. The fusion protein of the nanobody and human immunoglobulin Fc provided by the present invention is a conventional modification of the nanobody in the application field, for example, to extend half-life, increase stability, enhance effector functions such as CDC and ADCC, and simplify preparation. Those skilled in the art can also choose other modification strategies based on the application purpose, such as fusion with albumin. These modified fusion proteins still maintain the specificity of the nanobody against human adenovirus type 4 Hexon protein provided by the present invention. Therefore, the above fusion protein modification strategy should not be regarded as a limitation of the nanobody against human adenovirus type 4 Hexon protein provided by the present invention.

[0011] Fourthly, the present invention provides a polynucleotide encoding the fusion protein, wherein the polynucleotide further contains a polynucleotide encoding a signal peptide, and the sequence of the signal peptide is shown in SEQ ID NO:3.

[0012] Fifth, the present invention provides a vector containing the polynucleotide encoding the aforementioned Nanobody against human adenovirus type 4 Hexon protein. The vector is used to clone and / or express the gene encoding the Nanobody against human adenovirus type 4 Hexon protein. In a specific embodiment of the present invention, the vector is pcDNA3.4. Other vectors known to those skilled in the art, in particular eukaryotic expression vectors, can also be used for cloning and expressing the encoding genes of the present invention.

[0013] Sixth, the present invention provides a host cell containing a vector containing a polynucleotide encoding the aforementioned anti-human adenovirus type 4 Hexon protein Nanobody. The host cell is used to express the aforementioned anti-human adenovirus type 4 Hexon protein Nanobody. In a specific embodiment of the present invention, the host cell is an Expi293F cell. Other host cells known to those skilled in the art, in particular eukaryotic host cells, can also be used to express the Nanobodies of the present invention.

[0014] Seventh, the present invention provides an mRNA comprising an mRNA fragment transcribed from the aforementioned polynucleotide. Using a carrier, such as a liposome nanoparticle, the mRNA provided herein can be delivered to specific target cells, thereby causing the target cells to express the nanobody against the human adenovirus type 4 Hexon protein described herein. In addition to the mRNA fragment transcribed from the aforementioned polynucleotide, the mRNA can be configured with a cap structure and a 5' UTR at the 5' end, and a 3' UTR and poly(A) at the 3' end, thereby achieving the aforementioned expression in target cells.

[0015] Eighth, the present invention provides the use of the above-mentioned anti-human adenovirus type 4 Hexon protein nanobody in the preparation of a drug for treating and / or preventing human adenovirus type 4 infectious diseases. The nanobody provided by the present invention has excellent affinity, binding activity and affinity activity with human adenovirus type 4 Hexon protein, and can specifically target human adenovirus type 4 Hexon protein. Therefore, these properties of the nanobody are used as a specific inhibitor of the binding of human adenovirus type 4 to host cells, or to specifically target therapeutic drugs to infected lesions or pathogens to play a clinical therapeutic or preventive role in infection. Therefore, the present invention provides the use of the above-mentioned anti-human adenovirus type 4 Hexon protein nanobody in the preparation of a drug for treating and / or preventing human adenovirus type 4 infectious diseases.

[0016] Finally, the present invention provides a detection kit containing the above-mentioned nanoantibody against human adenovirus type 4 Hexon protein. Based on the excellent affinity of the nanoantibody provided by the present invention to human adenovirus type 4 Hexon protein, it can be used to detect human adenovirus type 4 particles containing Hexon protein that may be present in a sample. The detection antigen is a single antibody detection, that is, the nanoantibody specifically binds to the pathogen as a primary antibody, and the binding is then detected by a secondary antibody; it can also be a double antibody combination detection, for example, combined with other antibodies targeting different antigenic determinants of the Hexon protein as a capture antibody and a detection antibody, to implement a double antibody sandwich immunoassay.

[0017] The present invention immunized alpacas with adenovirus type 4 Hexon protein, constructed an antibody library, and used phage display technology to screen for the nanoantibody AUR39 that specifically binds to adenovirus type 4 Hexon protein with high affinity. Surface plasmon resonance testing confirmed that the nanoantibody AUR39 of the present invention can bind to adenovirus type 4 Hexon protein with high affinity, with a KD value of 9.21×10 -10 M, E.C. 50 The value is 3.17ng / ml. And in the virus neutralization test, it can neutralize type 4 adenovirus pseudovirus with high neutralization activity, IC 50 The value was 1.31 ng / ml, indicating that the nanobody AUR39 is an alpaca-derived nanobody against adenovirus type 4 with high affinity and high neutralizing activity.

[0018] The nanoantibodies provided by the present invention have the advantages of small molecular weight (~15kDa), low immunogenicity, good solubility and stability, and a longer CDR3 region. They can be administered by aerosolization, can reach the lungs directly, and take effect faster, providing a potential treatment strategy for adenovirus type 4 or other subgenus adenovirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the SDS-PAGE identification results of the Nanobody AUR39 of Example 3 of the present invention;

[0020] Figure 2 This is a schematic diagram of the detection of the antigen binding activity of the nanobody AUR39 of Example 4 of the present invention;

[0021] Figure 3 This is a schematic diagram of the detection of the neutralization activity of the nanobody AUR39 and the antigen according to Example 5 of the present invention;

[0022] Figure 4 Schematic diagram of affinity detection between the nanobody AUR39 of Example 6 of the present invention and the antigen;

[0023] Figure 5 This is a schematic diagram of the thermal stability test of the nanobody AUR39 of Example 7 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Unless otherwise expressly stated, throughout the specification and claims, the term "including" or its variations such as "comprising" or "including" will be understood to include the stated elements or components, without excluding other elements or other components.

[0025] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following detailed description. It should be understood by those skilled in the art that the present invention can be practiced without certain specific details. In some embodiments, raw materials, components, methods and means well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0026] Hereinafter, the present invention will be described in detail.

[0027] definition

[0028] Nanobodies (Nbs), also known as heavy-chain single-domain antibodies, are a unique type of single-domain antibody found in camelids and sharks. This type of antibody contains only one heavy chain variable domain (VHH), and compared to other antibodies, the light chain is naturally absent.

[0029] Due to their own biophysical advantages, nanoantibodies can be easily atomized and delivered directly to the lungs through an inhaler to treat infections caused by respiratory viruses. They are considered to be very promising antibody drugs.

[0030] "Specific" binding, when referring to a ligand / receptor, antibody / antigen or other binding pair, refers to the determination of the presence or absence of said protein, e.g. the binding reaction of a Nanobody of the invention to adenovirus type 4 Hexon protein, in a heterogeneous population of proteins and / or other biologicals. Thus, under the specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and is not bound in significant amounts to other proteins present in the sample.

[0031] Chemical materials such as reagents, enzymes, culture media, and antibiotics used in the following examples of the present invention are all commercially available products, for example, TRIzo1 was purchased from Invitrogen, sfiI endonuclease was purchased from NEB, and RT-PCR kits were purchased from Novagen.

[0032] Some commonly used biological materials, such as competent cells, vectors, and helper phages, are also commercially available products. For example, Expi 293F cells were purchased from ATCC; electrocompetent XL1-Blue cells were purchased from Biomed; VCSM13 helper phage was purchased from StrataGene; and pcomb3X vector was purchased from Biomed.

[0033] Some synthetic biological materials, such as primers, sequences and other materials that require artificial synthesis, are commissioned to a synthesis company (Shanghai Bioengineering).

[0034] The adenovirus type 4 Hexon protein of the present invention is a recombinant protein (Genebank No. AY594253.1) constructed by the inventors in their laboratory through genetic engineering.

[0035] Example 1: Alpaca immunization and antibody library construction

[0036] Immunization was initiated after 3-5 days of observation with 5 mg of adenovirus type 4 Hexon protein from an adult alpaca aged 8 months or older. For each immunization, 0.5 mg of Hexon protein was diluted with PBS to a final volume of 1 mL, emulsified with 1 mL of complete Freund's adjuvant for 5 minutes, and injected subcutaneously at multiple sites. Immunizations were repeated every two weeks, using incomplete adjuvant to emulsify the Hexon protein. Twelve days after the third immunization, 50-60 mL of blood was collected and peripheral blood mononuclear cells (PBMCs) were isolated. The isolated PBMCs were added to 1 mL of TRIzo1 (purchased from Invitrogen), and RNA was extracted according to the manufacturer's instructions. cDNA was synthesized using the extracted RNA as a template using a reverse transcription kit (purchased from Novozymes). PCR was performed using the cDNA template using specific primers, and the 700 bp band was excised and recovered from the gel. The purified DNA was used as a template and nested PCR was performed using nested primers to amplify the nanobody (VHHs) sequence, and the purified VHHs sequence with a size of about 400 bp was recovered.

[0037] The recovered fragments and the pcomb3X vector were digested separately with sfiI. The fragments were purified directly using a gel extraction column, and the linearized pcomb3X vector was recovered using a gel extraction kit. The fragments were mixed with pcomb3X and ligated overnight at 4°C using T4 ligase. The purified ligation products were mixed with XL1-Blue competent medium on ice, aliquoted into a tube, and electroporated at 1800V. After electroporation, the mixture was transferred to 2YT-ATG medium containing ampicillin and tetracycline, and the volume was adjusted to 200 mL using 2YT-ATG. The libraries were then recovered by shaking at 37°C, 250 rpm, for 1 hour. This constituted the antibody bacterial library.

[0038] Example 2: Screening of specific nanobodies using phage display technology

[0039] The remaining library solution from Example 1 was incubated at 37°C and 250 rpm for 1 hour. VCSM13 helper phage was then added at a multiplicity of infection (MOI) of approximately 20 and allowed to infect for half an hour. The cells were then incubated at 37°C and 200 rpm for another hour. The pellet was collected by centrifugation and resuspended in 100 mL of 2YT-ATK medium (containing ampicillin, tetracycline, and kanamycin). Expression and amplification were carried out overnight at 30°C and 225 rpm. After overnight incubation, the supernatant was collected by centrifugation at 4000 rpm, filtered through a 0.22 μm membrane, and precipitated with 4% PEG800 and 3% NaCl in an ice bath. The precipitate was then centrifuged and dissolved in PBS (pH 7.4) and sterilized using a 0.2 μm syringe filter. The collected phage particles were then titered.

[0040] 2×10 11 Each of the collected phage was mixed with an equal volume of 5% (w / v) skim milk and added to a 96-well plate coated with adenovirus type 4 Hexon protein antigen. After incubation at room temperature for 1 hour, the specific phage was eluted with 0.2M glycine and neutralized with Tris-HCl (pH 9.1). These phage were then infected with XL1-Blue and amplified. A second round of panning was performed on a 96-well plate coated with adenovirus type 4 Hexon protein antigen to enrich for phage expressing specific nanobodies. Two rounds of panning were performed in total. After the second round of panning, different single colonies were randomly selected from the agar plates with colonies, cultured in a shaker at 37°C, and then VCSM13 helper phage was added for overnight culture. The culture medium was centrifuged the next day, and the phage supernatant was taken for ELISA experiment (using type 4 adenovirus Hexon protein as the coating antigen). When OD450nm>0.2, it was judged as a positive reaction. The corresponding clone was taken and the plasmid was sequenced using specific primers to obtain the sequence encoding VHHs in its plasmid. Through sequence determination, the core coding sequence of the nanobody AUR39 was obtained. The coding sequence of the heavy chain variable region of the nanobody AUR39 is shown in SEQ ID NO.2, the amino acid sequence is shown in SEQ ID NO.1, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region are shown in SEQ ID NO:1 at amino acid residues 26-33, 51-57, and 96-113, respectively.

[0041] Example 3: Expression and purification of nanobody AUR39

[0042] The signal peptide (MDAMKRGLCCVLLLCGAVFVSNS, SEQ ID NO. 3) was connected before the coding sequence of the nanobody AUR39 of the present application, and the coding sequence of the hFc (PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, SEQ ID NO. 4) tag and the translation termination codon TGA were connected thereto, and the restriction enzyme cutting site BamHI was used to construct the pCDNA3.4 vector and transfected into Expi After culturing 293F cells for 5 days, the supernatant was collected, centrifuged at 5000 rpm for 30 min, filtered through a 0.22 μm filter membrane, and purified by affinity chromatography using Protein A to obtain a relatively pure target protein. The target peak was identified by SDS-PAGE, such as Figure 1 As shown, a relatively pure nanobody AUR39 was obtained.

[0043] Example 4. ELISA detection of antibody binding activity

[0044] 1. The day before the experiment, coat a 96-well ELISA plate with 2 µg / ml Hexon protein, 100 µl per well, at 4°C overnight.

[0045] 2. Discard the coating solution, add 100 µl of blocking solution to each well, and incubate at 37°C for 1 hour.

[0046] 3. Discard the blocking solution and add 1µg / ml antibody in a 1:3 serial dilution. Incubate at 37°C for 1 hour.

[0047] 4. Discard the primary antibody and dilute HRP-labeled goat anti-human IgG secondary antibody (Abcam, ab97225) at 1:10,000 in diluent. Add 100 µl per well to the corresponding wells of the ELISA plate and incubate at 37° for 1 hour.

[0048] 5. Discard the secondary antibody and add 100 µl of TMB one-component colorimetric solution to each well. Develop the color for 6 minutes at room temperature in the dark. Then, add 50 µl of stop solution to each well to terminate the reaction.

[0049] 6. Detect the OD value at 450-630nm using a microplate reader and save and record the original data.

[0050] The results are as follows Figure 2 As shown, the EC of nanobody AUR39 50 The value is 3.17ng / ml.

[0051] Example 5. Detection of antibody neutralizing activity.

[0052] Neutralization levels were determined using HAdv-4 luciferase-containing pseudovirus (purchased from Promega, catalog number E1501).

[0053] 1. Add 50 μl of 100 μg / ml nanobody AUR39 to a 96-well plate, dilute it in a 1:3 gradient, add an equal volume of HAdV-4 luciferase, and incubate at 37° for 1 hour;

[0054] 2. After 1 hour, add 2×10 5 cells / ml of A549 cells in 100 μl and incubated at 37° for 24 h;

[0055] 3. After 24 hours, add 100 μl of lysis buffer and read the value.

[0056] The results are as follows Figure 3 As shown, the IC of nanobody AUR39 50 The value is 1.31ng / ml.

[0057] Example 6. SPR detection of antibody affinity

[0058] The affinity between the antibody and Hexon protein was detected using a Protein A chip using a Biacore T200 instrument. The determination method is as follows:

[0059] 1. Dilute the nanobody to 1 µg / mL and load it onto the Protein A chip at a flow rate of 10 µL / min for 120 s.

[0060] 2. Hexon protein was diluted from 100 nM to 6.125 nM in a 2-fold gradient and loaded onto the antibody-captured Protein A chip at a flow rate of 30 μL / min. Binding was performed for 120 s and dissociation for 900 s.

[0061] 3. Select 5 representative curves and calculate the affinity (KD) of the antibody using the instrument configuration analysis software.

[0062] The results are as follows Figure 4As shown, the KD value of the nanobody AUR39 is 9.21×10 -10 M.

[0063] Example 7. DSC detection of antibody thermal stability

[0064] Differential scanning calorimetry (DSC) studies transitions or processes caused by temperature changes, the most common of which is "thermal melting" caused by temperature increases. Therefore, DSC was used to study the thermal stability of the nanobody AUR39. The determination method is as follows:

[0065] 1. Turn on the instrument and DSC-RUN software in advance and preheat for 1 hour;

[0066] 2. Connect the cleaning device, 800ml of cleaning solution (5% decon90) and 1.5L of pure water;

[0067] 3. Place the 1.5ml centrifuge tube containing PBS and the antibody to be tested in a degasser with the lid open. Degas at 635Hg for 10 minutes and set aside.

[0068] 4. After the instrument is cleaned, use a pipette to slowly add 800 μL of PBS and the antibody sample to be tested to the reference well and sample well, respectively. Pipet and swirl repeatedly to remove any bubbles in the wells. Secure the reference well and sample well with a black plastic cap, then tighten the instrument cover.

[0069] 5. Observe whether the instrument's thermal value is stable. Once it is stable, start increasing the pressure to 3 atm.

[0070] 6. After the pressure rise is complete, set the temperature to 25-100°C, equilibrate for 600 seconds, and run the program.

[0071] 7. After the program reaches 100°C, the system automatically cools down and the program can be terminated. Once the temperature drops to approximately 30°C and the pressure drops to 0 atm, open the instrument lid, aspirate the sample, and clean the instrument.

[0072] The results are as follows Figure 5 As shown, the melting temperature of the heavy chain variable region of the nanobody AUR39 is 59.16°C, which has excellent thermal stability.

[0073] In summary, the alpaca-derived nanoantibody AUR39 of the present invention can be used as a candidate antibody drug for preventing, treating and detecting infections caused by adenovirus type 4 and other adenoviruses of the same genus.

[0074] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanobody against human adenovirus type 4 Hexon protein, characterized in that The amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the anti-human adenovirus type 4 Hexon protein nanobody are shown in amino acid residues 26-33, 51-57 and 96-113 of SEQ ID NO: 1, respectively.

2. The nanobody against human adenovirus type 4 Hexon protein according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the nanobody against human adenovirus type 4 Hexon protein is shown in SEQ ID NO:

1.

3. A polynucleotide encoding the nanobody against human adenovirus type 4 Hexon protein according to claim 1 or 2, characterized in that: The sequence of the polynucleotide encoding the heavy chain variable region of the nanobody against human adenovirus type 4 Hexon protein is shown in SEQ ID NO.

2.

4. A fusion protein comprising the Nanobody according to claim 1 or 2, characterized in that In the fusion protein, the C-terminus of the nanobody is fused with a human immunoglobulin Fc segment having a sequence as shown in SEQ ID NO:

4.

5. A polynucleotide encoding the fusion protein according to claim 4, characterized in that: The polynucleotide further contains a polynucleotide encoding a signal peptide, the sequence of which is shown in SEQ ID NO:

3. An expression vector comprising the polynucleotide of claim 3 .

7. A host cell containing the expression vector according to claim 6.

8. An mRNA, characterized in that The mRNA contains mRNA fragments obtained by transcription of the polynucleotide according to claim 3.

9. Use of the nanobody against human adenovirus type 4 Hexon protein according to claim 1 or 2 in the preparation of a drug for treating and / or preventing diseases caused by human adenovirus type 4.

10. Use of the nanobody against human adenovirus type 4 Hexon protein according to claim 1 or 2 in the preparation of a diagnostic kit for human adenovirus type 4 infectious diseases.

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