Development of a potent neutralizing nanobody 2B4 against respiratory syncytial virus infection

Abstract

The present application relates to the development of a potent neutralizing nanobody 2B4 for preventing respiratory syncytial virus infection. Specifically, it 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. The nanobody provided by the present application has the characteristics of high affinity and high neutralization, preferentially recognizes the pre-F subtype of respiratory syncytial virus, and has excellent neutralization activity against pre-F. It has a wide application prospect 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.

Description

Technical Field

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

[0002] Respiratory syncytial virus (RSV) is a major pathogen causing respiratory tract infections in infants, young children, and the elderly. RSV infection presents with a wide range of clinical manifestations. Mild cases present with runny nose, nasal congestion, and cough, while severe cases may present with high fever, dyspnea, and even death. RSV has a distinct seasonal pattern, with peak incidence occurring during the colder months in temperate regions, with the main epidemic occurring between November and March. RSV is typically transmitted through respiratory droplets or contact with contaminated hands and surfaces. Global surveillance data show that children under 5 years of age are at high risk for RSV infection, with children under 2 years of age experiencing the highest risk and the heaviest burden of disease. RSV poses a serious threat to the health and lives of children and the elderly worldwide, imposing a heavy burden of disease and healthcare on all countries. Despite its severity, there are currently very few safe and effective RSV vaccines available globally, as well as a lack of specific anti-RSV therapeutics. Therefore, there is an urgent need to develop a new generation of RSV neutralizing antibodies that are safe, highly effective, broad-spectrum, and cost-effective.

[0003] Human respiratory syncytial virus (HRSV) belongs to the genus Pneumovirus in 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 and consists of 10 genes encoding 11 proteins. The G and F proteins are the two major glycoproteins on the viral surface and play a crucial role in mediating viral entry into cells. The G protein mediates viral attachment to cells but is not essential for infection. The F protein plays a key role in binding to cell receptors and is the only protein responsible for membrane fusion, which is essential for viral entry and spread through syncytia formation. Furthermore, the F protein is more conserved than the G protein. Therefore, it is considered an ideal target for vaccine and drug development.

[0004] In recent years, antibodies targeting various epitopes on the RSV F protein have been developed. Almost every surface area of ​​the F protein is a potential target for antibodies. Currently, six epitopes have been reported on the F protein: Site Ø, Site I, Site II, Site III, Site IV, and Site V. Neutralizing antibodies targeting Site Ø have the strongest neutralizing activity, while Sites Ø and V are only present in the pre-F protein. Currently, two RSV neutralizing antibodies are commercially available for the prevention of RSV infection in newborns and infants. Palivizumab is limited to high-risk infants, and the cost of administering it throughout the RSV season makes it unsuitable for all infants. Nirsevimab, a fully human monoclonal antibody derived from the optimized monoclonal antibody D25, recognizes the conformational epitope Ø on the pre-F protein and exhibits potent neutralizing activity against RSV strains A2 and B9320. Its neutralizing activity against clinical isolates is approximately 50-fold higher than that of palvizumab. However, nirsevimab is not recommended for RSV patients, as there is no evidence that it is effective in treating RSV infection. Furthermore, SiteØ is a dominant high-neutralizing epitope, but it carries a greater immune screening burden and is prone to mutation. Resistant strains have emerged during clinical use.

[0005] Antibodies are immunoglobulins. IgG antibodies in most animals and humans have a Y-shaped structure composed of light and heavy chains, with a molecular mass generally around 150 kDa. Nanobodies, discovered in 1989, are a type of heavy chain antibody derived from camelids that naturally lacks the light chain variable region. With a molecular weight of only 15 kDa, this is one-tenth the molecular weight of conventional antibodies and the smallest known antibody, hence the name nanobody. Compared to traditional antibodies, nanobodies have a longer active binding region, containing 16 to 18 amino acids. They also exhibit greater temperature stability and organic solvent tolerance, and some are even protease-resistant. They can also tolerate a wider pH range (conventional antibodies are limited to pH 6-9, while nanobodies can tolerate pH 2-11). Furthermore, nanobodies are produced through bacterial expression, unlike traditional hybridoma cell production, offering advantages such as ease of expression and genetic engineering. In summary, nanobodies offer advantages over traditional monoclonal antibodies, including their small size, strong tissue penetration, stable properties, and low immunogenicity. Currently, nanobodies are widely used in basic research and medical fields. They can be rapidly delivered to RSV-infected lesions such as the respiratory tract and alveoli through aerosolization, 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, economical and broad-spectrum nanoantibodies to replace traditional monoclonal antibodies and improve the level of clinical treatment.

[0007] To address the above-mentioned issues, the inventors conducted a series of scientific studies and obtained a neutralizing nanobody that specifically targets the respiratory syncytial virus F protein (pre-F), which has the amino acid sequence of SEQ ID NO: 1 and the 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. It 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 in research on respiratory syncytial virus.

[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 nanoantibodies specifically bind to RSV F (pre F) glycoprotein.

[0012] In another aspect, the present invention also relates to polynucleotides encoding the above-mentioned Nanobodies.

[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 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 H H) Construction of phage-displayed nanobody 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 comprises the FR (conserved framework region) and CDR (complementarity determining region) regions. The FR sequences are relatively conserved, while the differences between different antibodies are primarily located in the CDR regions. After sequence alignment and exclusion of duplicate clones, candidate nanobodies were identified and cloned into the pTT5 vector for expression and purification in HEK 293F mammalian cells.

[0032] Research content 2: Detection of nanobody binding activity and neutralization activity;

[0033] (1) Nanobody binding activity (EC 50The 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), with Palvizumab (Wuhan Yipu Biotechnology Co., Ltd., #DVV02801, which recognizes both pre- and post-fusion F proteins) and Nirsevimab (Wuhan Pujian Biotechnology Co., Ltd., DVV02802, which recognizes only the pre-fusion state) serving as two positive control neutralizing antibodies. We further investigated the ability of candidate RSV neutralizing nanobodies to bind to different subtypes of F protein and whether they exhibited biases toward pre- and 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] Content 3: Evaluation of the preventive and therapeutic effects of the nanobodies in the RSV mouse infection model.

[0036] The mice were infected with RSV via the intranasal route one day before and after the intraperitoneal injection of the candidate nanobodies. Four days after the infection, the serum was isolated and the concentration of the nanobodies in the serum was detected. The viral RNA copy number in the lung tissue and the concha of the mice was detected by real-time fluorescent quantitative PCR. The viral titer in the lung tissue and the concha was determined by immunofluorescence.

[0037] The nanobody 2B4 screened in the present study, the amino acid sequence and the nucleotide sequence encoding the same are shown in SEQ ID NO: 1 and 2, respectively. The nanobody 2B4 specifically targets the fusion F glycoprotein on the surface of the respiratory syncytial virus and has excellent neutralizing effect, and can exert effective preventive and therapeutic effects, and has obvious clinical drug research and development potential.

[0038] The term "pre-F DS-Cav1" refers to the pre-fusion stabilized form of the RSV viral surface fusion F glycoprotein. Due to its secondary structure, the RSV F is in a relatively unstable pre-form before infecting the cell, but through protein engineering, two mutations (S155C and S290C) are 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 (S190F and V207L) are also made at the two concave groove sites to fill them and obtain a relatively stable pre-F, which is named as DS-Cav1. The amino acid sequence of the DS-Cav1 is shown in SEQ ID NO: 6.

[0039] The term "pre-F DS2" is further optimized on the basis of the DS-Cav1, and the intermolecular movement is stabilized by gene ligation, deletion of the fusion peptide, and additional disulfide bonds. It is called the second generation "DS2". The DS2 immunogen is more optimal, does not require furin cleavage, and has enhanced stability against thermal inactivation of the antigen. The amino acid sequence of the DS2 is shown in SEQ ID NO: 7.

[0040] The term "RSV post-F protein" refers to the RSV viral surface fusion F glycoprotein which, after infecting the cell, will form a post-F subtype with stronger stability due to the change in the secondary structure, and the 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 Preparation of RSV antigen protein and immunization of camels. Figure 1 A is the SDS-PAGE results 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 shows serum samples collected after camel immunization, and the titer of RSV F-specific binding antibodies was detected by ELISA.

[0053] Figure 2 Construction of phage-displayed nanoantibody library and antibody screening. Figure 2 A is the library capacity of the nanobody library. Based on 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 11 candidate nanoantibodies were initially screened. Cell represents the blank control group of normal cells without virus addition, and Virus represents the positive control group of cells with virus addition 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 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 2B4 and the positive control Nirsevimab. 50 values ​​(n=3).

[0057] Figure 6 To evaluate the prophylactic and therapeutic effects of Nanobody 2B4 in the RSV mouse infection model. Figure 6 A-B are the results of RT-qPCR detecting the viral copy number in the lung tissue and nasal turbinate of the mice prevented by Nanobody 2B4. Figure 6 C-D represent the viral copy number in the lung tissue and nasal turbinate of the mice treated by Nanobody. The significant difference is calculated by paired t-test, and no difference group is not marked, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001. DETAILED DESCRIPTION

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

[0059] We immunize camel with antigen, and take blood after several rounds of immunization, and isolate PBMC. Nanobody sequences are amplified by specific primers, and then cloned into phagemid. Phagemid library is established by using electroporation method, so that Nanobody sequences are displayed on the surface of phage. Then specific Nanobody binding to antigen is screened by phage ELISA and DNA sequencing, and candidate Nanobody is expressed in eukaryotic cells. Finally, the neutralization effect and antiviral effect of Nanobody are detected at the level of in vitro cells and in vivo animal model.

[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 PTT5-his plasmid vector (Addgene, #52326). Next, according to the manufacturer's instructions, polyethyleneimine (PEI, MACKLIN, P924174-1g) was used at 2.5 x 10 6The target plasmid was transfected into suspension 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 for 4 days. The cell supernatant was harvested after 4 days. The supernatant was filtered through a 0.22 μm filter and the target protein was adsorbed onto a nickel column. A 400 mM imidazole elution buffer was prepared and the protein specifically bound to the nickel column was eluted and concentrated using a protein purifier (Union-Biotech Co., Ltd.; UEV 25D). Finally, the protein samples were subjected to sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis to confirm the purity and molecular weight of the target proteins (DS Cav1 and DS2 proteins).

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

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

[0065] Camels were immunized with the antigens DS-Cav1 and DS2 obtained in Example 1. 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, emulsified it and injected it on both sides of the lymph nodes. A total of 4 immunizations were performed, with an interval of 30 days between each. 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, incomplete Freund's adjuvant (containing mycobacteria) was used for the subsequent immunizations. During the period, blood was drawn after each immunization to separate the serum, and the antibody titer in the serum was determined by ELISA test. The results are shown as follows. Figure 1 As shown in the results of C, 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. HThe H fragment then 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 H fragment phage library. Spread on a plate (Amp+) and incubate for 12-16 hours. Calculate the library capacity based on the number of single colonies on the plate. Figure 2 As shown in result A, the final library capacity we obtained was 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 As shown in result B, there are 86 different nanobody 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, with an initial concentration of 1μg / mL and a dilution factor of 3, the neutralization immunofluorescence experiment results are shown as follows: 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 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 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) presented the same data as the prior art paper, proving the accuracy of the 2B4 data under the experimental conditions. Figure 4The results showed that 2B4 has much higher binding activity against pre-F than post-F, meaning that 2B4 more readily binds 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 targeting the post-F epitope after fusion, while antibodies that only bind to the post-fusion epitope have weak or no neutralizing effects (e.g., 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, i.e., 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 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 2B4 could effectively neutralize RSV A2 (IC 50 =15.79 ng / ml, and its neutralization ability was better than 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 three groups: a nanobody prevention group, a nanobody treatment group, and a model group (a group that was challenged with the virus but not administered), with 5 mice in each group. The prevention group was administered with 2 mg / kg via intraperitoneal injection on -1 dpi (Day Post Infection, referring to the number of days after infection with the virus); the treatment group was administered with 8 mg / kg via intraperitoneal injection on 1 dpi. The control group was 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 injection with a titer greater than 1×10 6 Each mouse was infected with 100 μL of the virus (pfu / mouse). The body weight and condition of the mice were recorded daily. The experimental plan is shown in the table below.

[0078] RSV Nanobody Drug In Vivo Experimental Protocol

[0079]

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

[0081] like Figure 6 AB results showed that in the preventive treatment, the viral copy numbers in the lungs and nasal turbinates of the 2B4 and nirsevimab groups were significantly lower than those in the model group (P < 0.01). In the therapeutic treatment, the viral copy numbers in the lungs of the 2B4 and nirsevimab groups did not decrease significantly, but the viral load in the nasal turbinates was significantly reduced. These results demonstrate that the 2B4 nanobody effectively prevents RSV infection in the RSV mouse infection model, comparable to or even superior to the currently best marketed antibody, nirsevimab. Furthermore, 2B4 also exhibits excellent therapeutic effects against 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, a comprehensive analysis found that nanoantibody 2B4 has excellent neutralizing protection against 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:抗原蛋白RSV pre-F DS2的氨基酸序列

[0097] QNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGQSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL

[0098] SEQ ID NO.8:抗原蛋白RSV post F的氨基酸序列

[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 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 specifically targets the respiratory syncytial virus pre-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. The Nanobody according to claim 1 or 2, wherein The nanobody recognizes and binds to the respiratory syncytial virus pre-F protein.

4. A polynucleotide encoding the Nanobody of any one of claims 1 to 3. 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. 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 for preventing respiratory syncytial virus infection.

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