Development and application of a broad-spectrum neutralizing nanobody 2a5 against respiratory syncytial virus

Abstract

The present application relates to the development and application of a kind of nanobody 2A5 for neutralizing broad-spectrum respiratory syncytial virus, and specifically relates to a kind of nanobody, which includes CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4, and CDR3 as shown in SEQ ID NO:5.The nanobody provided by the present application has the characteristics of high affinity and high neutralization, can effectively neutralize respiratory syncytial virus A2 and B strain, and the neutralization capacity is better than that of marketed antibody Nirsevimab.Therefore, the nanobody has great potential for further development as a candidate for preventing and treating respiratory syncytial virus, and also provides a scientific basis for the development of other antiviral therapies for respiratory syncytial virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a kind of nanobody development and application of broad-spectrum neutralization respiratory syncytial virus. BACKGROUND

[0002] Respiratory syncytial virus (RSV) is an important pathogen of acute lower respiratory tract infection in infants and young children worldwide, which can cause bronchitis and pneumonia and even death, causing great threat to public health. More than 95% of children under the age of 2 worldwide have been infected with RSV, and bronchiolitis caused by RSV infection is one of the main reasons for hospitalization of children under the age of 2, and is also an important factor for the increase of infant mortality. In addition, RSV virus causes hospitalization and mortality in the elderly population comparable to influenza. Although the harm is serious, there are 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 safe, efficient, broad-spectrum and cost-effective RSV neutralizing antibody.

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

[0004] The development of safe, cost-effective, and effective vaccines and antibody-based treatments for respiratory syncytial virus (RSV) has been a priority for the World Health Organization (WHO). Since the 1960s, numerous scientists have been researching and developing drugs for the prevention and treatment of RSV. However, the number of approved RSV antibodies is still insufficient. Currently, two marketed RSV preventive antibodies are palivizumab and nirsevimab®, but both are for prophylactic use. Palivizumab is limited to high-risk infants, and the cost of administering it throughout the RSV season makes it unsuitable for all infants. Nirsevimab, on the other hand, is a fully human monoclonal antibody derived from the optimized monoclonal antibody D25. It recognizes the conformational epitope Ø of the pre-F protein and has potent neutralizing activity against both subtypes A and B. Its neutralizing activity against clinical isolates is approximately 50-fold higher than that of palivizumab. After optimizing the antibody crystallizable fragment (Fc), a single-dose long-acting antibody was formed, which 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 nanobodies were derived from the variable domain of heavy chain antibodies (V H H), V H H retains full antigen-binding capacity, yet possesses a molecular weight of only 15 kDa, one-tenth the size of traditional antibodies. These antibodies are also known as nanobodies. Compared to traditional antibodies, nanobodies offer numerous advantages: 1) Their molecular weight is approximately one-tenth that of traditional antibodies, making them more easily penetrated into tissues; 2) they possess excellent specificity and strong affinity; 3) they are easily engineered into multispecific or multivalent antibodies; 4) they exhibit excellent stability and solubility, with stable physicochemical properties; 5) they can be highly expressed in prokaryotic systems, resulting in lower production costs; and 6) due to the high homology between alpaca-derived nanobodies and the human VH3 family sequences, they exhibit low immunogenicity. In summary, nanobodies hold enormous research potential and application value in both basic biological research and medicine. Summary of the Invention

[0006] Based on the above research background, there is an urgent need to address 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 nanobody targeting the respiratory syncytial virus F protein, which has the amino acid sequence of SEQ ID NO: 1 and the corresponding nucleotide sequence of SEQ ID NO: 2. The nanobody has high binding activity and specificity with the respiratory syncytial virus F protein. The nanobody 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 nanobody 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] In one aspect, the 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] In another aspect, the present invention also relates to polynucleotides encoding the above-mentioned Nanobodies.

[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 comprising 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 items 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) Camels were immunized 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) proteins 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 Escherichia coli TG1 competent cells by electroporation to construct V H The 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 is expressed on the coat protein on the surface of the phage, and KM13 is added to assist the phage in amplifying the V H bacteriophage of H;

[0025] (6) performing two rounds of biopanning 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-displayed nanoantibody library, screening of monoclonal positive phages, sequence alignment, and expression and purification of nanoantibodies;

[0029] (1) Construction of phage display library. Currently, the commonly used technology for screening nanoantibodies is phage display technology. 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 purified 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), phage display nanobody library was constructed.

[0030] (2) Screening of monoclonal positive phage and sequence alignment. We used the antigen protein DS2 to perform 2 rounds of panning on the library constructed above. Single clones were picked from the counting plate of the two rounds of panning, prepared into single clone phage, and through phage-ELISA combined experiments and gene sequencing, positive single clone V H H sequence information.

[0031] (3) Sequence alignment of V H H and expression and purification of nanobody. V H H contains FR region (conserved framework region) and CDR region (complementary determining region). The sequence of FR region is relatively conserved, while the difference between different antibodies is mainly located in CDR region. After sequence alignment to exclude duplicate clone sequences, candidate nanobody was obtained, and its sequence was cloned into PTT5 vector, and expressed and purified through mammalian cells HEK 293F.

[0032] Research content two: detection of nanobody binding activity and neutralizing 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), with Nirsevimab (Wuhan Pujian Biotechnology Co., Ltd., DVV02802) (which recognizes only the prefusion state, i.e., only pre-F) as a positive control neutralizing antibody. We further investigated the ability of RSV neutralizing nanobodies to bind to different F protein subtypes and whether they have a preference for pre-F versus post-F proteins.

[0034] (2) Neutralizing activity of nanoantibodies (IC 50 Based on the RSV neutralization immunofluorescence assay, we plan to calculate the IC value of candidate nanoantibodies’ neutralizing activity using immunofluorescence and high-content cell imaging analysis systems. 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 candidate nanoantibodies one day before / after being infected with RSV via the intranasal route. Four days after infection, the serum was separated and the concentration of nanoantibodies in the serum was detected; the number of viral RNA copies in the mouse lung tissue and nasal turbinates 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 2A5 screened in this study, whose amino acid sequence and 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. However, after protein engineering, two mutations (S155C and S290C) were introduced at the C-terminus of the F protein to form disulfide bonds 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, resulting in a more stable pre-F and named DS-Cav1. The amino acid sequence of DS-Cav1 is shown in SEQ ID NO:6.

[0039] The term "pre-F DS2" represents a further optimization of DS-Cav1 through genetic ligation, deletion of the fusion peptide, and stabilization of interprotomer movement through additional disulfide bonds. This second-generation DS2 is considered superior. The DS2 immunogen does not require furin cleavage and exhibits enhanced antigen stability against heat inactivation. 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 forms 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 both RSV F subtypes, the pre-F protein assumes a "lollipop" shape and exhibits an unstable conformation before RSV infects host cells. Upon approaching the host, the fusion peptide ejects from the central cavity of the protein trimer, and the heptad repeat (HR) HRB-α helix and HRA-α helix form an HR hexamer (6HB), undergoing membrane fusion. The metastable pre-F undergoes a dramatic spatial conformational shift, forming a "crutch-like" 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 E. coli TG1 so that all the V H The gene of the H fragment can be preserved 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 segment 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 encoding does not affect the proliferation process of the target phage.

[0048] The term "neutralizing activity" refers to the ability of an antibody to block viral invasion of host cells. Neutralizing activity is typically measured using cell culture and the corresponding viral or bacterial pathogen. Neutralizing antibody titer is determined by observing the antibody's ability to inhibit infection of host cells by the pathogen. Neutralizing antibodies possess the property of binding to antibodies, but binding does not necessarily confer neutralizing activity. This means that while antibodies can bind to viral surface glycoproteins, the binding site may not be at the critical epitope that influences viral entry into cells.

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

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

[0051] The term "EC 50 ” refers to the half-maximal effective 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 nanobody library. Figure 1 A is the SDS-PAGE results of pre-F proteins (DS Cav1 and DS2). Figure 1 B shows serum samples 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 nucleic acid fragments

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

[0054] Figure 3 Figure 2 shows the neutralization immunofluorescence results of 23 candidate nanobodies screened. NC represents the blank control group of normal cells without virus.

[0055] Figure 4 To evaluate the RSV F protein neutralizing antibody binding activity using an indirect ELISA method. Wherein, 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 against viruses by indirect immunofluorescence, fluorescence images were taken using a high-content instrument and analyzed. The data were exported to calculate the IC of nanoantibody 2A5 and the positive control Nirsevimab. 50 values ​​(n=3).

[0057] Figure 6 To evaluate the preventive and therapeutic effects of nanobody 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 PBS control group. Figure 6 Figures CF show the viral copy numbers in the lung tissues and nasal turbinates of mice in the nanobody 2A5 prevention and treatment groups as measured by RT-qPCR. CD represents the viral copy numbers in the lung tissues and nasal turbinates of mice in the nanobody prevention group; EF represents the viral copy numbers in the lung tissues and nasal turbinates of mice in the nanobody treatment group. Significant differences were calculated using paired t-tests. Groups with no significant differences are not indicated. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. DETAILED DESCRIPTION

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

[0059] We immunize camels with antigens, take blood after several rounds of immunization, and isolate PBMC. Nanobody sequences are amplified by specific primers, and then cloned into phagemids. The phage library is established by using electroporation method to display nanobody sequences on the surface of phages. Then, specific nanobodies that bind to antigens are screened by phage ELISA and DNA sequencing, and candidate nanobodies are expressed in eukaryotic cells. Finally, the neutralizing effect and antiviral effect of nanobodies are detected in vitro at the cell level and in vivo in animal models.

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

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

[0062] According to the amino acid sequences (SEQ ID NO: 6 and SEQ ID NO: 7) of RSV pre-F protein (DS Cav1 and DS2) in the literature, we synthesized the encoding gene of RSV pre-F protein and constructed it into the PTT5-his plasmid vector (Addgene, #52326). Next, according to the manufacturer's instructions, polyethyleneimine (PEI, MACKLIN, P924174-1g) was used to transfect the target plasmid into suspended HEK 293F cells (Thermo Fisher Scientific, R70007) at a density of 2.5 x 10 6 cells / mL. After incubation at (37 °C, 120 rpm, 5% CO2), 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 purification instrument (Union-Biotech Co., Ltd; UEV 25D) was used to elute and concentrate the protein specifically bound to the nickel column. Finally, the protein sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the purity and molecular weight of the target protein (DS Cav1 and DS2 protein).

[0063] The results are shown in Figure 1 As shown in A results, the band of high-purity target protein (DS Cav1 and DS2) was obtained. Camels were immunized with RSV antigens.

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

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

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

[0067] Nanobodies were displayed on the surface of phage using phage display technology (wherein the phage display technology is a conventional technique known in the art, for example, see the reference: Jaroszewicz, Weronika et al. "Phage display and other peptide display technologies." FEMS microbiology reviews vol. 46, 2(2022): fuab052.). Two rounds of biopanning were performed to screen for nanobodies specific for antigen DS2. Finally, the nanobodies were enriched through phage proliferation. Screening was performed using a monoclonal phage ELISA assay, and the positive clones were sequenced to obtain candidate nanobody sequences.

[0068] like Figure 2 The results showed that there were 86 different nanobody sequences among the 90 positive clones that finally passed the DS2 protein screening (screening criteria of OD 450nm>1). We initially selected 23 candidate antibodies based on the sequence grouping. By setting 3 dilution gradients, with an initial concentration of 5μg / mL and a dilution factor of 100, the neutralization immunofluorescence experiment results were as follows: 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 vector (Addgene, #52326) and transfected into HEK 293F (Thermo Fisher Scientific, #R70007) cells for eukaryotic expression. The cells were cultured at 37°C, 5% CO₂, and 120 rpm for 4 days. After 4 days, the cell supernatant was harvested. After filtration through a 0.22 μm filter, the filtrate was passed through a Protein A column, and the target protein was eluted with 0.1 M acetic acid in the eluent using a protein purifier (Union-Biotech Co., Ltd; UEV25D).

[0071] The binding activity of the nanobody to the antigen was identified by ELISA. Three RSV F proteins (DS-Cav1, DS2, and post-F) were coated on an enzyme-labeled 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 was set, 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. The color was developed and the reading was read. The binding curve was fitted using GraphPad Prism8.0, and the EC was calculated. 50 Numeric value.

[0072] like Figure 4 The results showed that nirsevimab (a known anti-RSV antibody in the prior art, used as a positive control) displayed data identical to those in prior art papers, confirming the accuracy of the 2A5 data under the experimental conditions. The results indicated that 2A5 had much higher binding activity against pre-F than post-F, meaning that 2A5 more readily bound to pre-F subtypes (DS-Cav1 and DS2). Studies have shown 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 or no neutralizing activity (e.g., see the reference: 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, i.e., 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 (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 aforementioned Vero cells) 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 (Molecular devices, 76177-140) and quantified by comparing treated wells with pure virus controls to calculate 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 weeks old BALB / c female mice (Jiangsu Huacheng Xinning Pharmaceutical Technology Co., Ltd.) were randomly divided into groups, namely nanobody prevention group, nanobody treatment group, model group (only infected without drug administration group), 5 mice in each group. The prevention group was administered by intraperitoneal injection at -1 dpi (Day Post Infection, referring to the number of days after infection), the dose was 2 mg / kg; the treatment group was administered by intraperitoneal injection at 1 dpi, the dose was 8 mg / kg. The control group of mice was injected with the same amount of solvent PBS. After the mice were anesthetized with isoflurane, they were infected with RSV A2 virus (National Virus Resource Center [NVRC]) through the nasal drop route, the titer was greater than 1×10 6 pfu / each, 100 μL per mouse was infected. The body weight and state of the mice were recorded every day. The experimental scheme is shown in the following table.

[0078] RSV nanobody drug in vivo experimental scheme

[0079]

[0080] The mice were euthanized at 4 dpi, and the lung tissue and nasal concha were obtained under sterile conditions, and the tissue RNA was extracted. RT-qPCR (reagent kit: HiScript II One Step RT-qPCR SYBR Green Kit, Nanjing Novozeno Biological Company, #Q221-01) was performed using specific primers (forward and reverse primer sequences are shown in SEQ ID NO: 11, SEQ ID NO: 12) to detect the change of RSV titer in the tissue, so as to reflect the antiviral effect of nanobody in the animal body.

[0081] As Figure 6 As shown in the results of A-B, in the RSV infected mice, compared with the PBS and the control antibody Nirsevimab in the prevention group, the nanobody 2A5 significantly improved the body weight of the mice at 1 dpi. In the treatment group, the body weight of the mice in each group decreased significantly after infection. The body weight of the mice in the 2A5 antibody group was not significantly improved.

[0082] As Figure 6CF results showed that during the preventive treatment, the viral copy numbers in the lungs and nasal turbinates of the 2A5 and nirsevimab groups were significantly lower than those in the model group (P < 0.01). During the therapeutic treatment, the viral copy numbers in the lungs of the 2A5 and nirsevimab groups did not decrease significantly, but the viral load in the nasal turbinates was significantly reduced. These results demonstrate that the 2A5 nanobody effectively prevents RSV infection in the RSV mouse infection model, outperforming nirsevimab. Furthermore, 2A5 also exhibits excellent therapeutic activity against 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, a comprehensive analysis found that nanoantibody 2A5 has excellent neutralizing protection against respiratory syncytial virus, which is expected to bring new options and hope for the 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.3:2A5 CDR1氨基酸序列

[0090] DSHMDMG

[0091] SEQ ID NO.4:2A5 CDR2氨基酸序列

[0092] TISSDGSTYYADSVKG

[0093] SEQ ID NO.5:2A5 CDR3氨基酸序列

[0094] DCSEGYCYTGALTRVQAGATRERLCGVPGDFGY

[0095] SEQ ID NO.6:抗原蛋白RSV pre-F DS Cav1的氨基酸序列

[0096] QNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

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

[0098] QNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0099] SEQ ID NO.8:抗原蛋白RSV post F的氨基酸序列

[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 are only specific embodiments of the present invention and are 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 that recognizes and binds to the respiratory syncytial virus F protein, 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. A polynucleotide encoding the Nanobody according to claim 1 or 2. The polynucleotide according to claim 3 , whose sequence is shown in SEQ ID NO:

2. An expression vector comprising the polynucleotide according to claim 3 or 4. A host cell comprising the expression vector according to claim 5 .

7. Use of the Nanobody according to any one of claims 1 to 2 in the preparation of a medicament for preventing or treating a disease associated with respiratory syncytial virus infection in a subject.

8. The use according to claim 7, wherein: The subject is a mammal.

9. The use according to claim 7, wherein: The subject is a human.

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