Research and development and application of nano antibody 2A5 for neutralizing respiratory syncytial virus in broad spectrum

Abstract

The invention relates to research and development and application of a nano antibody 2A5 for neutralizing a respiratory syncytial virus in a broad spectrum. In particular to a nano antibody which comprises a CDR1 shown as SEQ ID NO: 3, a CDR2 shown as SEQ ID NO: 4 and a CDR3 shown as SEQ ID NO: 5. The nano antibody provided by the invention has the characteristics of high affinity and high neutrality, and can effectively neutralize respiratory syncytial virus A2 and B strains, and the neutralizing capacity is superior to that of a marketed antibody Nirsevimab. Therefore, the nano antibody has a huge potential for further development of a candidate drug for preventing and treating the respiratory syncytial virus, and also provides a scientific basis for development of other antiviral treatment methods for the respiratory syncytial virus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the development and application of a nano antibody that can broadly neutralize respiratory syncytial virus. Background Art

[0002] Respiratory syncytial virus (RSV) is an important pathogen of acute lower respiratory tract infection in infants and young children worldwide. It can cause diseases such as bronchitis and pneumonia, and even death, posing a huge threat to public health. More than 95% of children under the age of 2 have been infected with RSV worldwide, and bronchiolitis caused by RSV infection is one of the main causes of hospitalization in children under the age of 2, and is also an important factor leading to rising infant mortality. In addition, the hospitalization and mortality caused by RSV in the elderly population are comparable to those of influenza. Despite the serious harm, there are currently very few safe and effective RSV vaccines worldwide for this special viral infection, and there is a lack of specific anti-RSV therapeutic drugs. Therefore, we urgently need to develop a new generation of RSV neutralizing antibodies that are safe, efficient, broad-spectrum and cost-effective.

[0003] RSV is an enveloped negative-strand RNA virus belonging to the genus Pneumovirus of the family Paramyxoviridae. The RSV genome is 15.2 kb long and contains 10 genes encoding 11 proteins. Among them, G and F proteins are two major glycoproteins on the surface of the virus, which play an important role in mediating the entry of the virus into cells. G protein mainly functions as an adsorption protein, which interacts with one or more molecules on the surface of the host cell to promote the binding of viral particles to target cells, but is not necessary for viral infection. The main function of F protein is to mediate the fusion of the viral envelope and the cell membrane, and it is the only protein responsible for membrane fusion, which is necessary for the entry and spread of the virus through the formation of syncytia. In addition, F protein and G protein are important antigens that stimulate the body to produce a protective immune response. They are the main targets of neutralizing antibodies and are also important targets for vaccine design and small molecule inhibitors. RSV strains are divided into two different subtypes (RSV A and RSV B) according to the difference in G protein. The amino acid homology of the two subtypes of G protein is only 50%; F protein is relatively conservative, with only 10% difference in sequence. Therefore, F protein is considered to be the most ideal target for the development of vaccines and drugs.

[0004] The development of safe, cost-effective vaccines and antibody drugs for respiratory syncytial virus (RSV) has always been a priority for the World Health Organization (WHO). Since the 1960s, many scientists have been developing drugs for the prevention and treatment of RSV. However, the number of RSV antibodies approved for marketing is still insufficient. The two RSV prevention antibodies currently on the market are Palivizumab and Nirsevimab®, but they are both used prophylactically. Palivizumab is limited to high-risk infants, and the cost of administration throughout the RSV season makes it unsuitable for all infants. Nirsevimab is a fully human monoclonal antibody optimized and modified from monoclonal antibody D25. It recognizes the Pre-F protein conformational epitope Ø and has a strong neutralizing effect on both subtypes A and B. Its neutralizing activity in clinical isolates is about 50 times higher than that of Palvizumab. The antibody crystallizable fragment (Fc) was optimized to form a single-dose long-acting antibody that is suitable for newborns and infants who are about to enter or were born in their first RSV infection season. It is expected to be used in China during the 2024-2025 RSV infection season.

[0005] In 1993, Belgian scientists first reported nanobodies in the journal Nature. The antibodies were derived from the variable domain of heavy chain antibodies (V H H), V H H retains complete antigen binding ability, and its molecular weight is only 15 KDa, which is 1 / 10 of traditional antibodies. It is also called nanoantibody. Compared with traditional antibodies, nanoantibodies have many advantages: 1) The molecular weight of nanoantibodies is about one-tenth of traditional antibodies, making it easier for nanoantibodies to penetrate into tissues; 2) Nanoantibodies have good specificity and strong affinity; 3) Nanoantibodies are easy to transform into multi-specific or multivalent antibodies; 4) Nanoantibodies have good stability and solubility, and stable physical and chemical properties; 5) Nanoantibodies can be highly expressed in prokaryotic systems, and the production cost is lower; 6) Since alpaca-derived nanoantibodies and human VH3 family sequences are highly homologous, they have low immunogenicity. In summary, nanoantibodies have great research potential and application value in basic biological research and medicine. Summary of the invention

[0006] Based on the above research background, it is urgent to solve the shortcomings of traditional RSV broad-spectrum neutralizing antibodies. The present invention aims to develop safe, broad-spectrum and cost-effective nanoantibodies to replace traditional monoclonal antibodies and improve the level of clinical treatment.

[0007] In order to solve the above problems, the inventors conducted scientific research and obtained a neutralizing nano antibody targeting the F protein of respiratory syncytial virus, which has an amino acid sequence of SEQ ID NO:1 and a corresponding nucleotide sequence of SEQ ID NO:2. The nano antibody has high binding activity and specificity with the F protein of respiratory syncytial virus. The nano antibody provided by the present invention has the characteristics of high affinity and high neutralization, can effectively neutralize RSV A2 and B strains, and its neutralization ability is better than that of the listed antibody Nirsevimab. Therefore, the nano antibody has great potential for further development as a candidate drug for the prevention and treatment of RSV, and also provides a scientific basis for the development of other antiviral therapies for RSV.

[0008] Specifically, the present invention relates to the following aspects:

[0009] On the one hand, the present invention relates to a Nanobody comprising a CDR1 as shown in SEQ ID NO:3, a CDR2 as shown in SEQ ID NO:4, and a CDR3 as shown in SEQ ID NO:5.

[0010] Wherein, the amino acid sequence of the above-mentioned nanobody is shown in SEQ ID NO:1.

[0011] Among them, the above-mentioned nanoantibody specifically binds to RSV F glycoprotein.

[0012] On the other hand, the present invention also relates to a polynucleotide encoding the above-mentioned Nanobody.

[0013] The polynucleotide is shown in SEQ ID NO:2.

[0014] In another aspect, the present invention also relates to an expression vector comprising the above-mentioned polynucleotide.

[0015] In another aspect, the present invention also relates to a host cell, which comprises the above-mentioned expression vector.

[0016] On the other hand, the present invention also relates to the use of the above-mentioned Nanobodies, polynucleotides or expression vectors in the preparation of drugs or kits for preventing or treating diseases associated with RSV infection in subjects.

[0017] Wherein, the subject is a mammal, preferably a human; the above-mentioned drug or kit comprises any one or more selected from the above-mentioned nanoantibodies, the above-mentioned polynucleotides and the above-mentioned expression vectors.

[0018] Wherein, the disease includes upper respiratory tract disease or lower respiratory tract infection, preferably, the lower respiratory tract infection includes pneumonia, bronchiolitis, long-term recurrent wheezing and asthma.

[0019] On the other hand, the present invention also relates to a method for preparing the above-mentioned Nanobody, which comprises the following steps:

[0020] (1) immunizing camels four times with 0.5 mg / time of RSV pre-F (DS Cav1 and DS2) protein, wherein the amino acid sequences of RSV pre-F (DS Cav1 and DS2) protein are shown in SEQ ID NO:6 and SEQ ID NO:7;

[0021] (2) Collect blood from the vein and separate PBMCs, and amplify V by PCR H H fragment, wherein the primers used for PCR are shown in SEQ ID NO:8 and SEQ ID NO:9;

[0022] (3) The amplified V H H was cloned into a phagemid to form a recombinant plasmid;

[0023] (4) The recombinant plasmid was introduced into the competent E. coli TG1 by electroporation to construct V H A phage library of H, wherein the step of freezing the phage library at -80°C can be further added as needed;

[0024] (5) Using phage display technology to display V H The H fragment was expressed on the coat protein on the surface of the phage, and KM13 was added to assist the phage in amplifying the V H The bacteriophage of H.

[0025] (6) performing two rounds of bio-panning to screen the nanobodies in the library;

[0026] (7) Obtain nanoantibodies with high DS2 binding activity.

[0027] More specifically, the present invention relates to the following research contents:

[0028] Research content 1: Construction of phage display nanoantibody library, screening of monoclonal positive phages, sequence alignment, and expression and purification of nanoantibodies;

[0029] (1) Construction of phage display library. Currently, phage display technology is a common technology for screening nanoantibodies. Studies have found that RSV F protein is considered to be the most ideal target for developing vaccines and drugs. Therefore, in order to construct a nanoantibody library against RSV F protein, we immunized camels with the highly pure antigenic proteins DS cav1 and DS2 (these two antigenic proteins are stable forms obtained after engineering modification of RSV pre F). We obtained the variable region sequence of the heavy chain antibody (V HH) Construction of phage display nanoantibody library.

[0030] (2) Screening and sequence comparison of monoclonal positive phages. We used the antigen protein DS2 to perform two rounds of panning on the library constructed above. Monoclonal clones were picked from the counting plates of the two rounds of panning and prepared into monoclonal phages. Then, positive monoclonal V was obtained through phage-ELISA binding experiment and gene sequencing. H The sequence information of H.

[0031] (3) V H H sequence alignment and expression and purification of nanobodies. H H contains FR region (conservative framework region) and CDR region (complementarity determining region). The FR region sequence is relatively conservative, while the differences between different antibodies are mainly located in the CDR region. After excluding repeated cloned sequences through sequence alignment, candidate nanobodies were obtained, and their sequences were cloned into the PTT5 vector, and expressed and purified by mammalian cells HEK 293F.

[0032] Research content 2: Detection of nano-antibody binding activity and neutralization activity;

[0033] (1) Nanobody binding activity (EC 50The binding activity of Nanobody (2A5) was evaluated by indirect ELISA. Three different antigen binding experiments were designed, namely pre-F (DS-Cav1) (for sequence, please refer to the literature: McLellan, JasonS et al. “Structure-based design of a fusion glycoprotein vaccine forrespiratory syncytial virus.” Science (New York, NY) vol. 342,6158 (2013):592-8.); pre-F (DS2) (for sequence, please refer to the literature: Joyce MG, Zhang B, Ou L, et al. Iterativestructure-based improvement of a fusion-glycoprotein vaccine against RSV. NatStruct Mol Biol. 2016;23(9):811-820.); post-F (for sequence, please refer to the literature: McLellan, JasonS et al. “Structure of respiratory syncytial virus fusion glycoprotein in thepostfusion conformation reveals preservation of neutralizing epitopes.” Journal of virology vol. 85,15 (2011): 7788-96), in which Nirsevimab (Wuhan Pujian Biotechnology Co., Ltd., DVV02802) (recognizes only the prefusion state, i.e., only pre-F) was used as a positive control neutralizing antibody. The ability of RSV neutralizing nanobodies to bind to different subtypes of F protein and whether they have a preference for pre-F protein and post-F protein were further explored.

[0034] (2) Neutralizing activity of nanoantibodies (IC 50 ) detection. Based on the RSV neutralization immunofluorescence experimental method, we plan to calculate the neutralizing activity IC of candidate nanoantibodies through immunofluorescence and high-content cell imaging analysis system. 50 The value is used to evaluate the ability of candidate nanoantibodies to neutralize viruses and provide a scientific basis for the subsequent screening of nanoantibodies.

[0035] Research content three: Evaluate the preventive and therapeutic effects of nanoantibodies in the RSV mouse infection model.

[0036] The mice were intraperitoneally injected with the candidate nanoantibodies one day before / after being infected with RSV via the nasal route. Four days after the infection, the serum was separated and the concentration of the nanoantibodies in the serum was detected. The number of viral RNA copies in the lung tissue and nasal turbinates of the mice was detected by real-time fluorescence quantitative PCR; and the viral titers in the lung tissue and nasal turbinates were determined by immunofluorescence.

[0037] The nanoantibody 2A5 screened in this study, whose amino acid sequence and the nucleotide sequence encoding it are shown in SEQ ID NO: 1 and 2, respectively, specifically targets the fusion F glycoprotein on the surface of respiratory syncytial virus, has excellent neutralization effect, can play an effective preventive and therapeutic role, and has obvious potential for clinical drug development.

[0038] The term "pre-F DS-Cav1" refers to the pre-fusion stable form of the RSV virus surface fusion F glycoprotein. Due to its secondary structure, RSV F is in a relatively unstable pre form before infecting cells, but after protein engineering, two mutations (S155C and S290C) were introduced at the C-terminus of the F protein to form a disulfide bond between them to increase the stability of the protein; in addition, modifications were made to the two grooves in the conformation (S190F and V207L) to fill them, obtain a more stable pre-F and name it DS-Cav1. Among them, the amino acid sequence of DS-Cav1 is shown in SEQ ID NO:6.

[0039] The term "pre-F DS2" is further optimized on the basis of DS-Cav1, through gene connection, deletion of fusion peptide, and stabilization of the movement between the protomers through additional disulfide bonds. It is called the second generation "DS2". DS2 immunogen is better, does not require furin protease cleavage and has enhanced antigen stability against heat inactivation. Among them, the amino acid sequence of DS2 is shown in SEQ ID NO:7.

[0040] The term "RSV post-F protein" refers to the RSV virus surface fusion F glycoprotein, which will form a more stable post-F subtype due to changes in secondary structure after infecting cells. Its amino acid sequence is shown in SEQ ID NO:8.

[0041] In the two subtypes of RSV F mentioned above, the pre-F protein is in a "lollipop" shape and unstable conformation before the RSV virus infects the host cell. When approaching the host, the fusion peptide pops out from the middle cavity of the protein trimer, and the heptad repeat sequence (HR) HRB-α helix and HRA-α helix form an HR hexamer (6HB) to undergo membrane fusion. The metastable pre-F undergoes drastic spatial conformational changes to form a "crutch"-shaped post-fusion conformation post-F, which connects the two membranes and allows the RSV genome to enter the cell.

[0042] The term "PBMC" refers to peripheral mononuclear cells, which include lymphocytes and mononuclear cells. In the embodiments of the present invention, it refers to camel lymphocytes.

[0043] The term "phagemid" refers to a plasmid containing the origin of replication of a filamentous bacteriophage.

[0044] The term "phage library" refers to the introduction of a specific target gene into a microorganism and storage in the microorganism. In the present invention, it specifically refers to the introduction of the above phagemid into the competent state of Escherichia coli TG1, so that all V H The gene of the H fragment can be stored in E. coli for a long time and used for subsequent screening.

[0045] The term "phage library bacteria" refers to a microorganism that stores the target gene. In the present invention, it specifically refers to a microorganism that stores all V H H fragment gene of Escherichia coli TG1.

[0046] The term "phage display technology" refers to inserting the gene sequence of a foreign protein into the appropriate position of the gene region encoding the coat protein of the phage, so that the foreign protein can be expressed on the coat protein on the surface of the phage as the phage proliferates.

[0047] The term "KM13 helper phage" refers to a special phage used to assist phagemid proliferation, and its own code does not affect the proliferation process of the target phage.

[0048] The term "neutralizing activity" refers to the ability of antibodies to block viruses from invading host cells. The detection of neutralizing activity generally requires the use of cell culture and corresponding pathogens such as viruses or bacteria, and is measured by observing the inhibition of the antibody on the infection of host cells by the pathogen, that is, the neutralizing antibody titer. Neutralizing antibodies have the property of binding antibodies, but binding antibodies do not necessarily have neutralizing activity. In other words, although antibodies can bind to viral surface glycoproteins, the binding location is not necessarily the key epitope that affects the virus's entry into cells.

[0049] The term "binding activity" refers to the ability of an antibody to specifically recognize a corresponding antigen. This binding can also occur in vitro and is generally determined by ELISA.

[0050] The term "IC 50 ” refers to the half inhibitory concentration. In the present invention, IC 50 The lower the value, the higher the neutralizing activity of the antibody.

[0051] The term “EC 50 ” refers to the half-maximal effect concentration. In this invention, EC 50 The lower the value, the higher the antibody-antigen binding activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Construction of RSV phage displayed nanoantibody library. Figure 1 A is the SDS-PAGE result of pre-F proteins (DS Cav1 and DS2). Figure 1 B is a serum sample collected after camel immunization, and the titer of RSV F-specific binding antibodies was detected by ELISA. Figure 1 C is the nanobody amplified by PCR with specific primers (V H H) Electrophoresis of gene fragment nucleic acid

[0053] Figure 2 This is the result of ELISA experiment on phages screened by DS2 protein.

[0054] Figure 3 The neutralization immunofluorescence results of 23 candidate nanoantibodies screened initially are shown in Figure 1. NC represents the normal cell blank control group without virus added.

[0055] Figure 4 To evaluate the RSV F protein neutralizing antibody binding activity using an indirect ELISA method. Where A is the EC of nanobody 2A5 binding to the antigen 50 Result figure; B EC of positive control neutralizing antibody Nirsevimab binding to antigen 50 Result graph.

[0056] Figure 5 In order to detect the neutralization effect of antibodies on viruses by indirect immunofluorescence, the fluorescence images taken by high-content instruments were analyzed and the data were exported to calculate the IC of nanoantibody 2A5 and positive control Nirsevimab. 50 Values ​​(n=3).

[0057] Figure 6 To evaluate the preventive and therapeutic effects of nanoantibody 2A5 in the RSV mouse infection model. Figure 6 AB are the body weights of all mice monitored before and after virus infection. Black asterisks indicate the significant difference between 2A5 and the control group PBS. Figure 6 CF is the results of RT-qPCR detection of the number of viral copies in the lung tissue and nasal concha of mice in the nanoantibody 2A5 prevention group and treatment group. Among them, CD represents the number of viral copies in the lung tissue and nasal concha of mice in the nanoantibody prevention group; EF represents the number of viral copies in the lung tissue and nasal concha of mice in the nanoantibody treatment group. The significant difference was calculated by paired t-test, the group with no difference was not marked, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

[0058] The present invention is studied through the following process: 1. Preparation of antigen protein; 2. Immunization of animals; 3. Establishment of nano antibody phage library; 4. Screening of specific nano antibodies by phage display technology; 5. Functional verification of nano antibodies; 6. Virus neutralization and animal protection experiments.

[0059] We immunized camels with antigens, collected blood after several rounds of immunization, and separated PBMC. We amplified the nanobody sequence using specific primers and cloned it into phagemids. We established a phage library using electroporation to display the nanobody sequence on the phage surface. We then screened out nanobodies that specifically bound to antigens using phage ELISA and DNA sequencing, and expressed candidate nanobodies through eukaryotic cells. Finally, we tested the neutralization and antiviral effects of nanobodies at the in vitro cell level and in vivo animal models.

[0060] The present invention is further described below in conjunction with the accompanying drawings (the methods used in the following embodiments are conventional methods unless otherwise specified, and the reagents used are commercially available reagents unless otherwise specified):

[0061] Example 1: Expression and purification of antigen protein

[0062] According to the amino acid sequences of RSV pre-F proteins (DS Cav1 and DS2) in the literature (SEQ ID NO:6 and SEQ ID NO:7), we synthesized the coding gene of RSV pre-F protein and constructed it into PTT5-his plasmid vector (Addgene, #52326). Next, polyethyleneimine (PEI, MACKLIN, P924174-1g) was used at 2.5×10 6 The target plasmid was transfected into suspended HEK 293F cells (Thermo Fisher Scientific, R70007) at a density of 10 cells / mL. The cells were cultured at (37 °C, 120 rpm, 5% CO2) and the cell supernatant was collected after 4 days. After the supernatant was filtered through a 0.22 μm filter membrane, the filtrate was passed through a nickel column to adsorb the target protein, and a 400 mM imidazole eluent was prepared. The protein specifically bound to the nickel column was eluted and concentrated by a protein purifier (Union-Biotech Co., Ltd; UEV 25D). Finally, the protein sample was subjected to sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis to confirm the purity and molecular weight of the target protein (DS Cav1 and DS2 protein).

[0063] The results are as follows Figure 1 As shown in the results of A, high-purity target protein bands (DS Cav1 and DS2) were obtained and used as RSV antigens to co-immunize camels.

[0064] Example 2: Immunization, library construction and screening

[0065] The antigens DS-Cav1 and DS2 obtained in Example 1 were used to immunize camels. The antigen protein was mixed with Freund's adjuvant in a ratio of 1:1 at a dose of 0.5 mg / time. After emulsification, it was injected on both sides of the lymph nodes. A total of 4 immunizations were performed, each with an interval of 30 days. Since complete Freund's adjuvant can stimulate the body to produce a stronger immune response. Therefore, except for the adjuvant used for the first immunization, which was complete Freund's adjuvant (containing mycobacteria), incomplete Freund's adjuvant (not containing mycobacteria) was used for the subsequent immunizations. Blood was drawn after each immunization to separate serum, and the antibody titer in the serum was determined by ELISA experiment. The results are shown in the figure. Figure 1 As shown in the results of B, when OD450 is 0.1, the corresponding serum dilution is greater than 10000. According to experimental experience, camel immunization is considered to be effective. In this embodiment, the serum dilution has reached more than 1000,00, indicating that the immunization effect is good.

[0066] After the antibody titer reaches the expected value, blood is collected and PBMC cells are isolated from it to start constructing V H The isolated PBMC cells were firstly cloned using EZNA ® Total RNA was extracted using Total RNA Kit I (OMEGA, #IR6834), reverse transcribed into cDNA (TAKARA, #6210A), and then expressed using specific V H H primers (the forward and reverse primer sequences are shown in SEQ ID NO:9 and SEQ ID NO:10), and V H H fragment, such as Figure 1 C results are shown. Then V H The H fragment was cloned into pR2 phagemid (BioVector, 67739, using NotI and NocI restriction sites) to form a recombinant vector, which was then transformed into competent E. coli TG1 (HonorGene, HG-VSW0286) by electroporation to establish a V H The final library capacity of the phage library containing H fragments was 3.7×10 7 .

[0067] The nanoantibody is displayed on the phage surface by phage display technology (wherein the phage display technology is a conventional technology known in the prior art, for example, see the literature: Jaroszewicz, Weronika et al. "Phage display and other peptide display technologies." FEMS microbiology reviews vol. 46,2(2022): fuab052.), and the nanoantibody specific to the antigen DS2 is screened out through two rounds of bio-panning, and finally the nanoantibody is enriched by the proliferation of phage. Screening is performed by monoclonal phage ELISA experiment, and the phage of the positive clone is sequenced by DNA to obtain the candidate nanoantibody sequence.

[0068] like Figure 2 The results showed that there were 86 different nanoantibody sequences among the 90 positive clones that finally passed the DS2 protein screening (screening criteria of OD 450nm>1). We preliminarily selected 23 candidate antibodies based on the sequence grouping. By setting 3 dilution gradients, the initial concentration was 5μg / mL, and the dilution factor was 100, the neutralization immunofluorescence experiment results were carried out, as shown in Figure 3 As shown in the results, 2A5 with the best effect was finally screened out as the nanobody of the present invention.

[0069] Example 3: Characterization of Nanobody 2A5

[0070] The 2A5 antibody nucleotide sequence (SEQ ID NO: 2) was constructed into the PTT5-Fc (Addgene, #52326) vector, transfected into HEK 293F (Thermo Fisher Scientific, #R70007) cells for eukaryotic expression, and cultured at 37 °C, 5% CO2, 120 rpm for 4 days. After 4 days, the cell supernatant was collected. After filtering through a 0.22 μm filter membrane, the filtrate was passed through a Protein A column, and a 0.1 M acetic acid eluent was prepared and the target protein was eluted by a protein purifier (Union-Biotech Co., Ltd; UEV25D).

[0071] The binding activity of the nanobody with the antigen was identified by ELISA experiment. Three RSV F proteins (DS-Cav1, DS2 and post-F) were coated on an ELISA plate (Corning, #3590) one day in advance. The antigen concentration was 2 μg / mL, 100 μL / well, and the nanobody was diluted from 2 μg / mL, 3-fold gradient dilution, 10 gradients were set, and incubated with the antigen for 1 hour. HRP-Rabbit Anti-Human IgG-Fc (Sino Biological, #10702-T16-H, 1:8000 dilution) was diluted as the secondary antibody and incubated at room temperature for 1 hour, and the color reading was performed. The binding curve was fitted using GraphPad Prism8.0, and the EC 50 Numeric value.

[0072] like Figure 4 The results show that Nirsevimab antibody (Nirsevimab antibody is an anti-RSV antibody known in the prior art, which serves as a positive control) presents the same data as the prior art paper, proving the accuracy of the 2A5 data under experimental conditions. The results show that 2A5 has much higher binding activity for pre-F than post-F, that is, 2A5 is more likely to bind to pre-F subtypes (DS-Cav1 and DS2). Studies have found that antibodies that preferentially recognize the pre-F epitope before fusion have stronger neutralizing activity than antibodies that target the post-F epitope after fusion, while antibodies that only bind to the post-fusion epitope have weak neutralizing or non-neutralizing effects (for example, see the literature: Rossey I, McLellan JS, Saelens X, Schepens B. 2018. Clinical potential of prefusion RSV F-specific antibodies. Trends Microbiol 26:209–219). In addition, recombinant protein vaccines designed based on pre-F protein are more effective than vaccines designed based on post-F protein (for example, see the literature: McLellan JS, Chen M, Joyce MG, et al. Structure-based design of afusion glycoprotein vaccine for respiratory syncytial virus [J]. Science, 2013, 342(6158): 592-8).

[0073] Example 4: Virus Neutralization Experiment

[0074] To further determine the neutralizing activity of candidate nanobodies, IC 50We used a conventional indirect immunofluorescence assay. One day before the experiment, Vero cells (BDBIO, #C5168) were plated at 1×10 4 The nanobodies were seeded at a density of 1 μg / mL into 96-well plates. We serially diluted the nanobodies starting from a concentration of 1 μg / mL. RSV A2 (NationalVirus Resource Center [NVRC]) and B strains (CH93(18)-18; ZeptoMetrix) (100 PFU per well) were mixed with the candidate nanobody dilutions and incubated at 37°C for 1 hour. The mixture was then added to the target cells (i.e., the Vero cells described above) and incubated at 37°C for approximately 2 hours. After being rinsed once with PBS, the Vero cells were cultured in fresh medium containing 2% FBS at 37°C for 3-4 days. Finally, the cells were fixed with 4% paraformaldehyde and stained with the RSV-specific detection antibody F-E2 (the amino acid sequence of the antibody F-E2 is shown in SEQ ID NO: 13), followed by incubation with the secondary antibody FITC-conjugated anti-human IgG (Proteintech, SA00003-12) at room temperature for 1 hour. Fluorescence was measured using a high-throughput imaging system (Moleculardevices, 76177-140) and quantified by comparing treated wells with pure virus controls and calculating the IC 50 value.

[0075] like Figure 5 The results showed that 2A5 could effectively neutralize RSV A2 (IC 50 =17.46 ng / ml) and B[CH93(18)-18;ZeptoMetrix] strain (IC 50 =42.33ng / ml, and its neutralization ability was better than Nirsevimab. It is worth noting that Nirsevimab can only neutralize RSV A2 (IC 50 =20.82 ng / ml) strain, but failed to neutralize the B [CH93(18)-18; ZeptoMetrix] strain.

[0076] Example 5: Animal protection experiment

[0077] 7-8 week old BALB / c female mice (Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.) were randomly divided into nano-antibody prevention group, nano-antibody treatment group, and model group (only challenged with virus but not administered with drug), with 5 mice in each group. The prevention group was administered with 2 mg / kg by intraperitoneal injection at -1 dpi (Day Post Infection, referring to the number of days after virus infection); the treatment group was administered with 8 mg / kg by intraperitoneal injection at 1 dpi. The control group mice were injected with an equal amount of solvent PBS. After being anesthetized with isoflurane, the mice were infected with RSV A2 virus (National Virus Resource Center [NVRC]) by intranasal route, with a titer greater than 1×10 6 pfu / mouse, 100 μL per mouse. The weight and status of mice were recorded every day. The experimental scheme is shown in the table below.

[0078] RSV Nanobody Drug In Vivo Experimental Program

[0079]

[0080] At 4 dpi, the mice were euthanized, and lung tissues and nasal turbinates were obtained by aseptic operation. Tissue RNA was extracted, and RT-qPCR (the detection kit was HiScript II One Step RT-qPCR SYBR Green Kit, Nanjing Novozymes Biotechnology Co., Ltd., #Q221-01) was performed using specific primers (forward and reverse primer sequences are shown in SEQ ID NO:11 and SEQ ID NO:12) to detect changes in RSV titers in tissues, thereby reflecting the antiviral effect of nanoantibodies in animals.

[0081] like Figure 6 AB results showed that in RSV-infected mice, nanobody 2A5 significantly improved the body weight of mice on the first day after infection compared with PBS in the prevention group and the control antibody Nirsevimab. In the treatment group, the body weight of mice in each group decreased significantly after infection. The body weight of mice in the 2A5 antibody group did not improve significantly.

[0082] like Figure 6The CF results showed that in the preventive medication, the viral copy numbers in the lungs and nasal turbinates of the 2A5 group and the Nirsevimab group were significantly lower than those of the model group (P < 0.01); in the therapeutic medication, the viral copy numbers in the lungs of the 2A5 and Nirsevimab groups did not decrease significantly, but the amount of virus in the nasal turbinates was significantly reduced. The above results show that the 2A5 nanobody has an effective effect in preventing RSV infection in the RSV mouse infection model, and the effect is better than Nirsevimab. In addition, 2A5 also has an excellent effect in treating RSV infection.

[0083] In summary, the present invention uses phage display technology to screen a neutralizing nanoantibody 2A5. Through molecular cloning, cell neutralization immunofluorescence experiments, ELISA experiments, and histological tests related to in vivo animal experiments, it is comprehensively analyzed and found that nanoantibody 2A5 has excellent neutralizing protection effect on respiratory syncytial virus, which is expected to bring new options and hope for clinical prevention and treatment of RSV.

[0084] Sequence Listing:

[0085] SEQ ID NO.1: 2A5 amino acid sequence

[0086] QLQLVESGGGLVQPGGSLRLSCTASGFTADDSHDMGWYRQAPGKKCELISTISSDGSTYYADSVKGRFTISRDNAKNTVYLQTNNLKPEDTAVYWCYRDCSEGYCYTGALTRVQAGATRERLCGVPGDFGYWGQGTQVTVSS

[0087] SEQ ID NO.2: 2A5 nucleotide sequence

[0088] CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTACAGCCTCTGGATTCACTGCGGATGATTCTCACATGGACATGGGCTGGTACCGCCAGGCTCCAGGAAAGAAGTGCGAGTTGATCTCAACTATCAGTAGTGATGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCCGAGACAACGCCAAGAACACGGTGTATCTGCAAACGAACAACCTGAAACCTGAGGACACGGCCGTGTATTGGTGTTACAGGGATTGTAGTGAGGGTTACTGTTACACCGGTGCCCTCACGCGGGTTCAGGCCGGTGCGACCCGAGAGCGACTTTGCGGGGTGCCGGGCGACTTTGGTTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA

[0089] SEQ ID NO.3: Amino acid sequence of 2A5 CDR1

[0090] DSHMDMG

[0091] SEQ ID NO.4: Amino acid sequence of 2A5 CDR2

[0092] TISSDGSTYYADSVKG

[0093] SEQ ID NO.5: Amino acid sequence of 2A5 CDR3

[0094] DCSEGYCYTGALTRVQAGATRERLCGVPGDFGY

[0095] SEQ ID NO.6: Amino acid sequence of antigen protein RSV pre-F DS Cav1

[0096] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0097] SEQ ID NO.7: Amino acid sequence of the antigenic protein RSV pre-F DS2

[0098] QNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0099] SEQ ID NO.8: Amino acid sequence of the antigen protein RSV post F

[0100] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKT NVTLSKKRKRRAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLI NDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIF NPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0101] SEQ ID NO.9: Amplified V H H fragment specific forward primer GCTGCACAGCCTGCTATGGCACAGKTGCAGCTCGTGGAGTCTGGGGG

[0102] SEQ ID NO.10: Amplified V H H fragment specific reverse primer

[0103] GAGTTTTTGTTCGGCTGCTGCTGAGGAGACGGTGACCTGGGTCCCC

[0104] SEQ ID NO.11: RT-qPCR forward primer

[0105] TTAGCAAAGTCAAGTTGAATGAT

[0106] SEQ ID NO.12: RT-qPCR reverse primer

[0107] ACATACCTATTAACCCAGTGAAT

[0108] SEQ ID NO.13: F-E2 amino acid sequence

[0109] QVQLVESGGGSVQPGGSLRLSCAAPGYIYSSGCMGWFRQAPGKEREGVAVRYIGGGNTYYFDLVKGRFTISQDSAKNTLYLQMNSLRPEDTAMYYCAADVDPVRCSLMGEPWQYNYRGQGTQVTVSS

[0110] SEQ ID NO.14: Nucleotide sequence encoding F-E2

[0111] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCCTGGATACATCTACAGTAGCGGGTGCATGGGTTGGTTCCGCCAGGCTCCAGGAAAAGAGCGCGAGGGGGTCGCAGTTAGATATATTGGTGGTGGTAACACATACTATTTCGACCTCG TGAAGGGCCGATTCACCATCTCCCAAGACAGCGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACTGCCATGTACTACTGTGCGGCAGACGTCGACCCCGTACGGTGCTCTCTAATGGGCGAGCCGTGGCAGTATAACTACAGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA

[0112] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nanobody comprising a CDR1 as shown in SEQ ID NO: 3, a CDR2 as shown in SEQ ID NO: 4, and a CDR3 as shown in SEQ ID NO:

5.

2. The Nanobody according to claim 1, wherein The amino acid sequence of the Nanobody is shown in SEQ ID NO:

1.

3. The Nanobody according to claim 1 or 2, wherein The nanobody recognizes and binds to the respiratory syncytial virus F protein.

4. A polynucleotide encoding the Nanobody according to any one of claims 1 to 3.

5. The polynucleotide according to claim 4, whose sequence is shown in SEQ ID NO:

2. An expression vector comprising the polynucleotide according to claim 4 or 5.

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

8. Use of the Nanobody according to any one of claims 1 to 3, the polynucleotide according to claim 4 or 5, or the expression vector according to claim 6 in the preparation of a medicament or kit for preventing or treating a disease associated with respiratory syncytial virus infection in a subject.

9. The use according to claim 8, wherein: The subject is a mammal, preferably a human.

10. The use according to claim 8 or 9, wherein: The diseases associated with respiratory syncytial virus infection include upper respiratory tract diseases or lower respiratory tract infections. Preferably, the lower respiratory tract infections include pneumonia, bronchiolitis, long-term recurrent wheezing and asthma.

Images