Research and development of potent neutralizing nano antibody 2B4 for preventing respiratory syncytial virus infection

Abstract

The invention relates to research and development of a potent neutralizing nano antibody 2B4 for preventing respiratory syncytial virus infection. 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, preferentially recognizes the pre-F subtype of the respiratory syncytial virus, and has excellent neutralizing activity on the pre-F; wide application prospects and important economic and social significance are realized in the aspects of development of kits or medicines for preventing and / or treating respiratory syncytial virus infection and research on the respiratory syncytial virus.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the research and development of a potent neutralizing nano-antibody for preventing respiratory syncytial virus infection. Background Art

[0002] Respiratory syncytial virus (RSV) is the main pathogen causing respiratory tract infections in infants, young children and the elderly. Clinical manifestations of RSV infection vary. Mild cases present with runny nose, nasal congestion and cough, while severe cases may present with high fever, dyspnea and even death. RSV has obvious seasonality, with the peak incidence in the cold months in temperate regions, and the main epidemic period is from November to March of the following year. RSV is usually transmitted through droplets or contact with contaminated hands and surfaces. Surveillance data from different regions around the world show that children under 5 years old are at high risk of RSV infection, among which children under 2 years old have the highest risk of infection and the heaviest disease burden. RSV poses a serious threat to the health and lives of children and the elderly around the world, and has imposed a heavy disease burden and medical burden on countries. Despite the serious harm, there are currently very few safe and effective RSV vaccines worldwide, 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] Human respiratory syncytial virus belongs to the genus Pneumovirus of the family Paramyxoviridae. It is an enveloped negative-strand RNA virus with a spherical shape and a diameter of 120~300 nm. The genome is 15.2 kb long, consists of 10 genes, and encodes a total of 11 proteins. Among them, G and F proteins are the two main glycoproteins on the surface of the virus, and play an important role in mediating the entry of the virus into cells. Among them, G protein mediates the attachment of the virus to the cell, but it is not necessary for infection. F protein plays a key role in binding to cell receptors and is the only protein responsible for membrane fusion, which is necessary for the entry of the virus and the spread through the formation of syncytia. In addition, F protein is more conserved than G protein. Therefore, it is considered to be the most ideal target for the development of vaccines and drugs.

[0004] In recent years, antibodies targeting different epitopes of RSV F protein have been developed one after another. The surface of F protein is almost all potential antibody targeting sites. It is currently reported that F protein has the following 6 antigenic epitopes, namely Site Ø, Site I, Site II, Site III, Site IV, and Site V. Among them, the neutralizing antibody targeting Site Ø epitope has the strongest neutralizing activity, and Site Ø and Site V only exist in the pre-F protein form. Currently, there are only two RSV neutralizing antibodies on the market, Palivizumab, which has been approved for marketing in China, and Nirsevimab, which has been approved for marketing in China, for the prevention of RSV infection in newborns and infants. Palivizumab is limited to high-risk infants, and the cost of administration throughout the RSV season makes it no longer suitable for all infants. Nirsevimab is a fully human monoclonal antibody optimized and modified from monoclonal antibody D25, which recognizes the Pre-F protein conformational epitope Ø and has a strong neutralizing effect on RSV A2 and B9320 strains. Its neutralizing activity in clinical isolates is about 50 times higher than that of Palvizumab. However, it is not recommended to inject Nirsevimab into RSV patients, as there is no evidence that it has a therapeutic effect on RSV infection. In addition, SiteØ is a dominant high-middle neutralizing epitope, but it has greater immune screening pressure and is prone to mutation. In clinical applications, resistant strains have appeared.

[0005] Antibodies are immunoglobulins. IgG antibodies in most animals and humans are Y-shaped structures composed of light chains and heavy chains, and their molecular weight is generally around 150 KDa. Nanobodies are heavy chain antibody variable regions that are naturally missing light chains and are found in camelids in 1989. They have a molecular weight of only 15 KDa, which is 1 / 10 of the molecular weight of conventional antibodies. They are the antibodies with the smallest known molecular weight, so they are called nanobodies. Compared with traditional antibodies, nanobodies have longer active binding regions, with up to 16 to 18 amino acids, and have stronger temperature stability and organic solvent tolerance. Some of them can also tolerate proteases and a wider range of acid and base (traditional antibodies can only tolerate pH 6 to 9, while nanobodies can tolerate pH 2 to 11). In addition, nanobodies are prepared by engineered bacterial expression, which is different from the hybridoma cell preparation method of traditional antibodies. Therefore, they also have the advantages of easy expression and easy genetic engineering. In short, nanobodies have advantages that traditional monoclonal antibodies cannot match due to their small size, strong tissue permeability, stable properties, and low immunogenicity. At present, nanobodies are widely used in basic research and medical fields. They can be quickly delivered to the respiratory tract, alveoli and other RSV-infected lesions through aerosolization, thereby effectively inhibiting viral infection. Therefore, nanobodies may become a new type of drug for the treatment of RSV infection. Summary of the invention

[0006] Based on the above research background, there is an urgent need to solve the shortage of potent neutralizing antibodies against RSV. The present invention aims to develop safe, efficient and economical broad-spectrum nanoantibodies to replace traditional monoclonal antibodies and improve the level of clinical treatment.

[0007] In order to solve the above problems, the inventors conducted a series of scientific studies and obtained a neutralizing nanobody specifically targeting the F protein (pre-F) 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 nanobody provided by the present invention has the characteristics of high affinity and high neutralization, preferentially recognizes the pre-F subtype of respiratory syncytial virus, and has excellent neutralizing activity against pre-F, and has broad application prospects and important economic and social significance in the development of kits or drugs for the prevention and / or treatment of respiratory syncytial virus infection and research on respiratory syncytial virus.

[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 (pre F) glycoprotein.

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

[0013] The sequence of 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 comprises upper respiratory tract disease or lower respiratory tract infection, preferably, the lower respiratory tract infection comprises 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 targeting RSV F protein, we immunized camels with the highly pure antigenic proteins DS-Cav1 and DS2 (these two antigenic proteins are stable forms of RSV pre F obtained after engineering modification). We obtained the variable region sequence of the heavy chain antibody (V H H) 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 50) detection. The binding activity of Nanobody (2B4) 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), of which Palvizumab (Wuhan Yipu Biotechnology Co., Ltd., #DVV02801, which can recognize both pre-fusion and post-fusion F proteins) and Nirsevimab (Wuhan Pujian Biotechnology Co., Ltd., DVV02802, which only recognizes the pre-fusion state) were used as 2 positive control neutralizing antibodies. The ability of candidate RSV neutralizing nanobodies to bind to different subtypes of F proteins 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] One day before / after mice were infected with RSV via the nasal route, the candidate nanoantibodies were injected intraperitoneally. Four days after the infection, the serum was separated and the concentration of the nanoantibody 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 virus titer in the lung tissue and nasal turbinates was determined by immunofluorescence.

[0037] The nanoantibody 2B4 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 of the conformation (S190F and V207L) to fill them, obtaining a more stable pre-F and naming it DS-Cav1. 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". The 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 Preparation of RSV antigenic proteins and immunization of camels. Figure 1 A is the SDS-PAGE result of pre-F proteins (DS Cav1 and DS2). Figure 1 B is a schematic diagram of camel immunization with antigenic proteins DS-Cav1 and DS2. Figure 1 C is a serum sample collected after camel immunization, and the titer of RSV F-specific binding antibodies was detected by ELISA.

[0053] Figure 2 For the construction of phage display nanoantibody library and antibody screening. Figure 2 A is the calculation of the library capacity of the nanobody library. According to the number of monoclonal clones on the plate, the library capacity is calculated to be 3.7×10 7 . Figure 2 B is the result of ELISA experiment of phage screened by DS2 protein.

[0054] Figure 3 The neutralization immunofluorescence results of the 11 candidate nanoantibodies that were initially screened. Cell represents the normal cell blank control group without virus, and Virus represents the cell positive control group with only virus but no antibody.

[0055] Figure 4 To evaluate the RSV F protein neutralizing antibody binding activity using an indirect ELISA method. Figure 4 A is the EC of nanobody 2B4 binding to antigen 50 Result graph; Figure 4 B and 4C are the EC values ​​of the positive control neutralizing antibodies Pavilizumab and Nirsevimab 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 2B4 and positive control Nirsevimab. 50 Values ​​(n=3).

[0057] Figure 6 To evaluate the preventive and therapeutic effects of nanoantibody 2B4 in the RSV mouse infection model. Figure 6 AB is RT-qPCR detection of the number of viral copies in the lung tissue and nasal nasal concha of mice prevented by nanoantibody 2B4. Figure 6 CD 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 groups without difference were not marked, * represents P < 0.05, ** represents P < 0.0 l, *** represents P < 0.001. DETAILED DESCRIPTION

[0058] The present invention is studied through the following process: 1. Preparation of antigen protein; 2. Immunization of camels; 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 6The 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, and 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 (DS Cav1 and DS2) bands were obtained. A camel was co-immunized as an antigen.

[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 specific immunization process was as follows: Figure 1 As shown in B. We mixed the antigen protein at a dose of 0.5 mg / time with Freund's adjuvant in a ratio of 1:1, and injected it on both sides of the lymph nodes after emulsification. A total of 4 immunizations were performed, with an interval of 30 days between each immunization. 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, complete Freund's adjuvant (containing Mycobacterium) was used, incomplete Freund's adjuvant (not containing Mycobacterium) was used for the subsequent immunizations. Blood was drawn after each immunization to separate the serum, and the ELISA experiment was used to determine the antibody titer in the serum. The results are shown in Figure 1 As shown in the results of C, 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 HThe H fragment then 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 H fragment phage library. Spread on a plate (Amp+) and culture for 12-16 hours. Calculate the library capacity based on the number of single clones on the plate. Figure 2 As shown in the results A, the final library capacity we obtained 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 As shown in the results of B, there are 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 10 candidate antibodies based on the sequence grouping. By setting 8 dilution gradients, the initial concentration was 1μg / mL, and the dilution factor was 3, the results of the neutralization immunofluorescence experiment were carried out, as shown in Figure 3 As shown in the results, 2B4 with the best effect was finally screened out as the nanobody of the present invention.

[0069] Example 3: Characterization of Nanobody 2B4

[0070] The 2B4 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 As shown in the results, Nirsevimab antibody (Nirsevimab antibody is an anti-RSV antibody known in the prior art, which, as a positive control, only binds to pre-F) and Palivizumab (Palivizumab antibody is an anti-RSV antibody known in the prior art, which, as a positive control, binds to both pre-F and Post-F) present the same data as the prior art papers, proving the accuracy of the 2B4 data under the experimental conditions. Figure 4The results showed that 2B4 has much higher binding activity for pre-F than post-F, that is, 2B4 is more likely to bind to pre-F subtypes (DS-Cav1 and DS2). Studies have found that antibodies that preferentially recognize the pre-fusion pre-F epitope have stronger neutralizing activity than antibodies targeting the post-fusion post-F epitope, 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 a fusion 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 50 We used conventional indirect immunofluorescence assay. One day before the experiment, Vero cells (BDBIO, #C5168) were plated at 1×10 4The nanobodies were seeded at a density of 1 μg / mL in a 96-well plate. We serially diluted the nanobodies starting from a concentration of 1 μg / mL. RSV A2 (National Virus 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 2B4 could effectively neutralize RSV A2 (IC 50 =15.79 ng / ml, with a neutralization capacity superior to that of Nirsevimab (IC 50 =19.98 ng / ml).

[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 5 mice in each group. The prevention group was intraperitoneally injected with 2 mg / kg at -1 dpi (Day Post Infection, referring to the number of days after virus infection); the treatment group was intraperitoneally injected with 8 mg / kg at 1 dpi. The control group mice were injected with an equal amount of IgG1. After being anesthetized with isoflurane, the mice were infected with RSV A2 virus (National Virus Resource Center [NVRC]) via intranasal drops, 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 The AB results showed that in the preventive medication, the number of viral copies in the lungs and nasal turbinates of the 2B4 group and the Nirsevimab group were significantly lower than those of the model group (P < 0.01); in the therapeutic medication, the number of viral copies in the lungs of the 2B4 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 2B4 nanobody has an effective effect in preventing RSV infection in the RSV mouse infection model, which is comparable to or even better than the best marketed antibody Nirsevimab. In addition, 2B4 also has an excellent effect in treating RSV infection.

[0082] In summary, the present invention uses phage display technology to screen a neutralizing nanoantibody 2B4. 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 2B4 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.

[0083] Sequence Listing:

[0084] SEQ ID NO.1:2B4 amino acid sequence

[0085] QLQLVESGGGSVHAGGSLKLSCVASGYIISNCAMDMGGWFRQAPGKERELVSHINCHGTTTYADSVKGRFTISQDNAKNTLYLQMNSLKIEDTAVYYCAADVAVTSAGWGVNYWGQGTQVTVSS

[0086] SEQ ID NO.2: 2B4 nucleotide sequence

[0087] CAGTTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCACGCTGGAGGGTCTCTGAAACTCTCCTGTGTAGCTTCTGGATACATCATCAGTAACTGCGCAATGGACGGAATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGCGAGTTGGTCTCGCATATTAATTGTCATGGTACCACAACATAT GCAGACTCCGTGAAGGGCCGATTCACCATCTCCCAAGACAATGCCAAGAACACGCTGTATCTGCAAATGAATAGCCTGAAAATTGAGGACACGGCCGTGTATTACTGTGCGGCAGATGTGGCCGTGACTAGTGCGGGTTGGGGTGTTAACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA

[0088] SEQ ID NO.3: 2B4 CDR1 amino acid sequence

[0089] NCAMDGM

[0090] SEQ ID NO.4: 2B4 CDR2 amino acid sequence

[0091] HINCHGTTTYADSVKG

[0092] SEQ ID NO.5: 2B4 CDR3 amino acid sequence

[0093] DVAVTSAGWGVNY

[0094] SEQ ID NO.6: Amino acid sequence of antigenic protein RSV pre-F DS Cav1

[0095] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0096] SEQ ID NO.7: Amino acid sequence of antigenic protein RSV pre-F DS2

[0097] QNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0098] SEQ ID NO.8: Amino acid sequence of the antigen protein RSV post F

[0099] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKT NVTLSKKRKRRAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLI NDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIF NPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0100] SEQ ID NO.9: Amplified V H H fragment specific forward primer GCTGCACAGCCTGCTATGGCACAGKTGCAGCTCGTGGAGTCTGGGGG

[0101] SEQ ID NO.10: Amplified V H H fragment specific reverse primer

[0102] GAGTTTTTGTTCGGCTGCTGCTGAGGAGACGGTGACCTGGGTCCCC

[0103] SEQ ID NO.11: RT-qPCR forward primer

[0104] TTAGCAAAGTCAAGTTGAATGAT

[0105] SEQ ID NO.12: RT-qPCR reverse primer

[0106] ACATACCTATTAACCCAGTGAAT

[0107] SEQ ID NO.13: F-E2 amino acid sequence

[0108] QVQLVESGGGSVQPGGSLRLSCAAPGYIYSSGCMGWFRQAPGKEREGVAVRYIGGGNTYYFDLVKGRFTISQDSAKNTLYLQMNSLRPEDTAMYYCAADVDPVRCSLMGEPWQYNYRGQGTQVTVSS

[0109] SEQ ID NO.14: Nucleotide sequence encoding F-E2

[0110] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTCGGTGCAGCCTGGAGGGTCTCTGAGACTCTCCTGTGCAGCCCTGGATACATCTACAGTAGCGGGTGCATGGGTTGGTTCCGCCAGGCTCCAGGAAAAGAGCGCGAGGGGGTCGCAGTTAGATATATTGGTGGTGGTAACACATACTATTTCGACCTCG TGAAGGGCCGATTCACCATCTCCCAAGACAGCGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACTGCCATGTACTACTGTGCGGCAGACGTCGACCCCGTACGGTGCTCTCTAATGGGCGAGCCGTGGCAGTATAACTACAGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA

[0111] 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.

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