Use of adeno-associated virus vector in preparation of medicament for preventing and treating RSV infection and / or RSV-induced disease

Abstract

USE OF ADENO-ASSOCIATED VIRUS VECTOR IN PREPARATION OF MEDICAMENT FOR PREVENTING AND TREATING RSV INFECTION AND / OR RSV-INDUCED DISEASE For the first time, a recombinant adeno-associated virus vector is used to construct a medicament for preventing and treating RSV infection or a disease caused thereby. On this basis, a nucleic acid molecule is provided, comprising: (a1) a first region comprising a first adeno-associated virus inverted terminal repeat sequence, and (a2) a second region comprising a gene encoding a respiratory syncytial virus antigen. A recombinant adeno-associated virus comprising the nucleic acid molecule can produce a relatively good humoral immune effect and a relatively good cellular immune effect, and has a certain serum-neutralizing effect on both the subtype A strain and the subtype B strain of RSV. In particular, when the antigen is a fusion protein of the respiratory syncytial virus or a mutant of the fusion protein of the respiratory syncytial virus, the recombinant adenovirus vector comprising the nucleic acid molecule can successfully achieve the expression of the RSV pre-fusion F protein.

Description

Application of adeno-associated virus vectors in the preparation of drugs for the prevention and treatment of RSV infection and / or diseases caused by RSV Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of adeno-associated virus vectors in the preparation of drugs for the prevention and treatment of RSV infection and / or diseases caused by RSV. Background Technology

[0002] In 2020, the COVID-19 outbreak brought viral respiratory infections to the forefront of public attention. Currently, many types of viruses circulate globally, with respiratory syncytial virus (RSV) being a significant one. RSV is an enveloped, non-segmented, negative-sense RNA virus belonging to the Paramyxoviridae family. It is the leading cause of lower respiratory tract infections in pediatric patients worldwide, with approximately 33 million cases and 160,000-190,000 deaths annually, accounting for 6.7% of all infant deaths under one year old. Treatment for RSV infection or its associated diseases primarily involves supportive care for infected infants, such as oxygen therapy. Furthermore, palizumab is the only FDA-approved monoclonal antibody recommended by the American Academy of Pediatrics for the treatment of RSV infection in high-risk children (e.g., those with weakened immune systems, prematurity, congenital heart disease, or chronic lung disease). Natural immune protection against RSV is inefficient; therefore, an ideal and effective RSV vaccine should induce more durable humoral and cellular immunity than natural infection. However, regarding RSV infection prevention, only two recombinant protein RSV vaccines targeting individuals over 60 years of age were approved by the FDA in 2023: GSK's AREXVY and Pfizer's ABRYSVO. Therefore, there remains a significant demand and gap in the global RSV vaccine market, particularly in the development of new vaccine technologies. Society needs the development and market launch of novel RSV vaccines with broad-spectrum protective efficacy and long-term effectiveness.

[0003] The RSV viral genome encodes 11 proteins, among which the RSV glycoprotein (G) and fusion protein (F) induce neutralizing antibodies and immune responses in the host after infection. The RSV G protein is a type II glycoprotein that determines the RSV subtype (subtype A and subtype B), primarily mediates viral adsorption, and can be recognized by neutralizing antibodies produced by the host. The F protein is a type I transmembrane glycoprotein that elicits both humoral and cytotoxic T-cell immune responses in the host. Compared to the G protein, the F protein is relatively conserved across RSV strains, making it a more favorable candidate for vaccine antigens. However, the primary reason why an RSV vaccine will not be available until 2024 is that the viral F protein undergoes a conformational change during host cell invasion. This conformational change affects its main antigenic sites. The preconformation F protein before fusion with the host is lost. Therefore, the preconformation F protein (Pre-F) will induce a more potent neutralizing antibody as an effective antigen. Currently, several widely used Pre-F structures in research are DS-Cav1 (S155C-S290C-S190F-V207L), SC-DM (N67I-S215P), and SC-TM (N67I-S215P-E487Q). The RSV recombinant protein vaccine already approved by the FDA is developed based on the Pre-F structure of DS-Cav1.

[0004] Recombinant adeno-associated virus (rAAV) is an important gene therapy vector widely used globally. Existing studies have demonstrated that AAV does not cause any human disease. AAV is a replication-defective virus with low immunogenicity, broad tissue tropism, and non-pathogenicity. Summary of the Invention

[0005] The first aspect of the present invention is to provide a nucleic acid molecule.

[0006] A second aspect of the present invention is to provide a recombinant adeno-associated virus vector.

[0007] A third aspect of the present invention is to provide a host cell.

[0008] The fourth aspect of this invention is to provide a recombinant adeno-associated virus.

[0009] The fifth aspect of this invention is to provide a method for preparing recombinant adeno-associated virus, as described in the fourth aspect.

[0010] The sixth aspect of this invention aims to provide the application of the nucleic acid molecule of the first aspect, the recombinant adeno-associated virus vector of the second aspect, the host cell of the third aspect, and / or the recombinant adeno-associated virus of the fourth aspect.

[0011] The seventh aspect of this invention is to provide a medicine.

[0012] The object of the eighth aspect of the present invention is to provide a method.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] A first aspect of the present invention provides a nucleic acid molecule comprising:

[0015] (a1) First region: which contains the first adeno-associated virus (AAV) inverted terminal repeat (ITR); and

[0016] (a2) Second region: It contains the gene encoding respiratory syncytial virus (RSV) antigen;

[0017] The respiratory syncytial virus antigen is selected from one or more of the following: respiratory syncytial virus fusion protein (RSV F protein), respiratory syncytial virus fusion protein mutant (RSV Fm protein), and respiratory syncytial virus glycoprotein (RSV G protein).

[0018] The fusion protein mutant of the respiratory syncytial virus (RSV) has the following mutations compared to the RSV fusion protein: N42I and S190P.

[0019] In some embodiments of the present invention, the respiratory syncytial virus antigen is selected from any one of the respiratory syncytial virus fusion proteins, respiratory syncytial virus fusion protein mutants, and respiratory syncytial virus glycoproteins; further selected from any one of the respiratory syncytial virus fusion proteins and respiratory syncytial virus fusion protein mutants; and even further selected from respiratory syncytial virus fusion protein mutants.

[0020] In some embodiments of the present invention, the amino acid sequence of the respiratory syncytial virus fusion protein is the amino acid sequence from position 26 to 574 of the protein with accession number ACO83301.1.

[0021] In some embodiments of the present invention, the accession number of the respiratory syncytial virus glycoprotein is URP22622.1 (GenBank).

[0022] In some embodiments of the present invention, the nucleotide sequence of the gene encoding the fusion protein of respiratory syncytial virus is the nucleotide sequence of positions 852-2498 of SEQ ID NO:1.

[0023] In some embodiments of the present invention, the nucleotide sequence encoding the fusion protein mutant of respiratory syncytial virus is the nucleotide sequence of positions 852-2498 of SEQ ID NO:2.

[0024] In some embodiments of the present invention, the nucleotide sequence encoding the respiratory syncytial virus glycoprotein is the nucleotide sequence of positions 1921-2814 of SEQ ID NO:3.

[0025] In some embodiments of the present invention, the second region further includes a promoter.

[0026] In some embodiments of the present invention, the promoter is one or more of a constitutive promoter and an inducible promoter; more specifically, it is a constitutive promoter.

[0027] In some embodiments of the present invention, the constitutive promoter is selected from one or more of the following: CMV promoter, EF1A promoter, EFS promoter, CAG promoter, CBh promoter, SFFV promoter, MSCV promoter, SV40 promoter, mPGK promoter, hPGK promoter, and UBC promoter; and is further selected from any one of the following: CMV promoter and CAG promoter.

[0028] In some embodiments of the present invention, the inducible promoter is selected from one or more of the following: tetracycline-regulated promoter, alcohol-regulated promoter, steroid-regulated promoter, metal-regulated promoter, pathogenicity-regulated promoter, temperature / thermal-induced promoter, photo-regulated promoter, and IPTG-induced promoter.

[0029] In some embodiments of the present invention, when the respiratory syncytial virus antigen is a fusion protein of respiratory syncytial virus or a mutant fusion protein of respiratory syncytial virus, the promoter is a CMV promoter (the sequence of which is preferably the nucleotide sequence of positions 158-746 of SEQ ID NO:1 or SEQ ID NO:2).

[0030] In some embodiments of the present invention, when the respiratory syncytial virus antigen is a glycoprotein of respiratory syncytial virus, the promoter is a CAG promoter (the sequence of which is preferably the nucleotide sequence of positions 158-1890 of SEQ ID NO:3).

[0031] In some embodiments of the present invention, the second region further includes a Kozak sequence.

[0032] In some embodiments of the present invention, the nucleotide sequence of the Kozak sequence is GCCACC.

[0033] In some embodiments of the present invention, the second region further includes a post-transcriptional regulatory element.

[0034] In some embodiments of the present invention, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0035] In some embodiments of the present invention, the nucleotide sequence of the posttranscriptional regulatory element of the marmot hepatitis virus is the nucleotide sequence of positions 2550-3147 of SEQ ID NO:1 or SEQ ID NO:2, or the nucleotide sequence of positions 2866-3463 of SEQ ID NO:3.

[0036] In some embodiments of the present invention, the second region further includes a polyadenosine monophosphate (polyA) signal.

[0037] In some embodiments of the present invention, the polyadenylation signal is selected from one or more of bovine growth hormone polyA (BGH polyA), short polyA, SV40 polyA, and human β-globin polyA; more specifically, bovine growth hormone polyA (BGH polyA).

[0038] In some embodiments of the present invention, the nucleotide sequence of the bovine growth hormone polyA is the nucleotide sequence of positions 3178-3385 ​​of SEQ ID NO:1 or SEQ ID NO:2, or the nucleotide sequence of positions 3494-3701 of SEQ ID NO:3.

[0039] In some embodiments of the present invention, the second region further includes a signal peptide.

[0040] In some embodiments of the present invention, when the respiratory syncytial virus antigen is a fusion protein of respiratory syncytial virus or a mutant fusion protein of respiratory syncytial virus, the second region further includes a signal peptide.

[0041] In some embodiments of the present invention, the signal peptide is selected from one or more of the following: SAP signal peptide, IHC signal peptide, ILC signal peptide, AP signal peptide, CSP signal peptide, IL2 signal peptide, TPA signal peptide, L1 signal peptide, H7 signal peptide, SP1 signal peptide, H1 signal peptide, and RSVF signal peptide; and further, RSVF signal peptide.

[0042] In some embodiments of the present invention, the amino acid sequence of the RSVF signal peptide is the amino acid sequence from position 1 to position 25 of the full-length RSVF protein with accession number ACO83301.1 (GenBank).

[0043] In some embodiments of the present invention, the nucleotide sequence of the RSVF signal peptide is the nucleotide sequence of positions 777-851 of SEQ ID NO:1 or SEQ ID NO:2.

[0044] In some embodiments of the present invention, the N-terminus and / or C-terminus of the respiratory syncytial virus (RSV) antigen contains a protein tag.

[0045] In some embodiments of the present invention, the protein tag is selected from at least one of Poly his(His), FLAG, Strep-Tag II, Poly arg, C-myc, HA, V5, VSV-G, Trx, SUMO, GST, MBP, and NusA; more specifically, Poly his(His).

[0046] In some embodiments of the present invention, the nucleotide sequence of the gene encoding the fusion protein (containing a protein tag) of respiratory syncytial virus is the nucleotide sequence of positions 852-2519 of SEQ ID NO:1.

[0047] In some embodiments of the present invention, the nucleotide sequence encoding the fusion protein mutant of respiratory syncytial virus (containing a protein tag) is the nucleotide sequence of positions 852-2519 of SEQ ID NO:2.

[0048] In some embodiments of the present invention, the nucleotide sequence encoding the respiratory syncytial virus glycoprotein (containing a protein tag) is the nucleotide sequence of positions 1921-2835 of SEQ ID NO:3.

[0049] In some embodiments of the present invention, the nucleic acid molecule further comprises:

[0050] (a3) Third region: It contains the inverted terminal repeat (ITR) sequence of the second adeno-associated virus (AAV).

[0051] In some embodiments of the present invention, the first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence and the second adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence are each independently selected from one or more of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVDJ, goat AAV, bovine AAV, and mouse AAV serotype ITR.

[0052] In some embodiments of the present invention, the first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence and the second adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence are AAV2 ITR.

[0053] In some embodiments of the present invention, the nucleotide sequence of the first adeno-associated virus (AAV) inverted terminal repeat (ITR) is the nucleotide sequence of positions 1-130 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0054] In some embodiments of the present invention, the nucleotide sequence of the second adeno-associated virus (AAV) inverted terminal repeat (ITR) is the nucleotide sequence of positions 3393-3522 of SEQ ID NO:1 or SEQ ID NO:2, or the nucleotide sequence of positions 3709-3838 of SEQ ID NO:3.

[0055] In some embodiments of the present invention, when the respiratory syncytial virus antigen is a respiratory syncytial virus fusion protein or a mutant of a respiratory syncytial virus fusion protein, the nucleic acid molecule comprises: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a signal peptide, a gene encoding the respiratory syncytial virus (RSV) antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR); further, in some embodiments of the present invention, the nucleic acid molecule comprises, from 5'-3', the following in sequence: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a signal peptide, a gene encoding the respiratory syncytial virus (RSV) antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR).

[0056] In some embodiments of the invention, there are optional linkers between the first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence and the promoter, between the promoter and the Kozak sequence, between the gene encoding the respiratory syncytial virus (RSV) antigen and the post-transcriptional regulatory element, between the post-transcriptional regulatory element and the polyadenylation signal, and / or between the polyadenylation signal and the second adeno-associated virus (AAV) inverted terminal repeat (ITR).

[0057] In some embodiments of the present invention, the sequence of the nucleic acid molecule is the nucleotide sequence of positions 1-3522 of SEQ ID NO:1 or SEQ ID NO:2.

[0058] In some embodiments of the present invention, when the respiratory syncytial virus antigen is a respiratory syncytial virus glycoprotein, the nucleic acid molecule comprises: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a gene encoding the respiratory syncytial virus (RSV) antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR); further, in some embodiments of the present invention, the nucleic acid molecule comprises, from 5'-3', the following in sequence: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a gene encoding the respiratory syncytial virus (RSV) antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR).

[0059] In some embodiments of the invention, there are optional linkers between the first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence and the promoter, between the promoter and the Kozak sequence, between the gene encoding the respiratory syncytial virus (RSV) antigen and the post-transcriptional regulatory element, between the post-transcriptional regulatory element and the polyadenylation signal, and / or between the polyadenylation signal and the second adeno-associated virus (AAV) inverted terminal repeat (ITR).

[0060] In some embodiments of the present invention, the sequence of the nucleic acid molecule is the nucleotide sequence of positions 1-3838 of SEQ ID NO:3.

[0061] A second aspect of the present invention provides a recombinant adeno-associated virus (rAAV) vector comprising the nucleic acid molecule of the first aspect of the present invention.

[0062] In some embodiments of the present invention, the recombinant adeno-associated virus (rAAV) vector further comprises: a gene encoding a marker selected from at least one of antibiotic resistance proteins, toxin resistance proteins, colored or fluorescent proteins, and proteins that mediate enhanced cell growth and / or gene amplification.

[0063] In some embodiments of the present invention, the marker is an antibiotic resistance protein.

[0064] In some embodiments of the present invention, the antibiotic is selected from at least one of ampicillin, neomycin, G418, puromycin, and cyprodinil; more particularly ampicillin.

[0065] In some embodiments of the present invention, the recombinant adeno-associated virus (rAAV) vector includes a replication origin selected from at least one of f1 phage ori, RK2oriV, pUC ori, and pSC101ori.

[0066] In some embodiments of the present invention, the replication origin is pUC ori.

[0067] In some embodiments of the present invention, the sequence of the recombinant adeno-associated virus (rAAV) vector is shown as any one of SEQ ID NO:1-SEQ ID NO:3.

[0068] A third aspect of the invention provides a host cell comprising a nucleic acid molecule of the first aspect of the invention or a recombinant adeno-associated virus (rAAV) vector of the second aspect of the invention.

[0069] In some embodiments of the present invention, the host cell does not contain reproductive material.

[0070] In some embodiments of the present invention, the host cell is a 293T cell.

[0071] A fourth aspect of the present invention provides a recombinant adeno-associated virus (rAAV) comprising: (b1) the nucleic acid molecule of the first aspect of the present invention.

[0072] In some embodiments of the present invention, the recombinant adeno-associated virus further comprises: (b2) capsid protein.

[0073] In some embodiments of the present invention, the capsid protein is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAVDJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAVV708K, AAV2-HBKO, AAVDJ8, AAVPHP.B, AAVPHP.eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1 / AAV2 chimeric, bovine AAV or mouse AAV capsid, and rAAV2 / HBoV1 serum capsid protein; further, it is AAV5 capsid protein.

[0074] In some embodiments of the present invention, the recombinant adeno-associated virus (rAAV) is a single-stranded AAV (ssAAV).

[0075] A fifth aspect of the present invention provides a method for preparing a recombinant adeno-associated virus according to the fourth aspect of the present invention, which is packaged from a recombinant adeno-associated virus vector according to the second aspect of the present invention.

[0076] In some embodiments of the present invention, the method for preparing the recombinant adeno-associated virus includes the following steps: transfecting production cells with the recombinant adeno-associated virus vector, packaging plasmid pAAV-RC, and helper plasmid pHelper of the second aspect of the present invention.

[0077] In some embodiments of the present invention, the mass ratio of the recombinant adeno-associated virus vector, the packaging plasmid pAAV-RC, and the helper plasmid pHelper is 10:(1.25-80):(1.25-80); further, it is 10:(10-26.15):(10-26.15).

[0078] In some embodiments of the present invention, the method for preparing the recombinant adeno-associated virus further includes a purification step.

[0079] In some embodiments of the present invention, purification is performed using the iodixanol method.

[0080] A sixth aspect of the present invention provides the use of the nucleic acid molecule of the first aspect, the recombinant adeno-associated virus vector of the second aspect, the host cell of the third aspect, and / or the recombinant adeno-associated virus of the fourth aspect in the preparation of a drug.

[0081] The drug is used for at least one of (c1)-(c2):

[0082] (c1) Prevention and / or treatment of respiratory syncytial virus infection;

[0083] (c2) Prevention and / or treatment of diseases caused by respiratory syncytial virus.

[0084] In some embodiments of the present invention, the diseases caused by the respiratory syncytial virus include at least one of pneumonia, bronchiolitis, and otitis media.

[0085] In some embodiments of the present invention, the drug comprises a vaccine.

[0086] A seventh aspect of the present invention provides a medicament comprising: a nucleic acid molecule of the first aspect of the present invention, a recombinant adeno-associated virus vector of the second aspect, a host cell of the third aspect, and / or a recombinant adeno-associated virus of the fourth aspect.

[0087] In some embodiments of the present invention, the drug comprises: recombinant adeno-associated virus of the fourth aspect of the present invention.

[0088] In some embodiments of the present invention, the medicament further comprises a pharmaceutically acceptable carrier.

[0089] In some embodiments of the present invention, the dosage form of the drug is a dosage form suitable for children or a dosage form suitable for adults.

[0090] In some embodiments of the present invention, the dosage form is a gastrointestinal dosage form or a non-gastrointestinal dosage form.

[0091] In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powder, tablet, granule, capsule, sustained-release, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0092] In some embodiments of the present invention, the non-gastrointestinal drug delivery dosage forms include at least one of the following: injectable dosage forms (e.g., injections, including various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections); respiratory dosage forms (e.g., sprays, aerosols, powder inhalers, etc.); skin dosage forms (e.g., topical solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, ophthalmic ointments, mouthwashes, sublingual tablets, adhesive tablets, films, etc.); and cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., for use in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0093] In some embodiments of the present invention, the drug is a nasal drop or an injection.

[0094] In some embodiments of the present invention, the drug is administered to animals.

[0095] In some embodiments of the present invention, the animal is a mammal; further selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and even further selected from humans.

[0096] In some embodiments of the invention, the drug is used in at least one of (c1)-(c2) in the sixth aspect of the invention.

[0097] In some embodiments of the present invention, the medicament further comprises other active ingredients for preventing and treating respiratory syncytial virus infection and / or diseases caused by respiratory syncytial virus.

[0098] In some embodiments of the present invention, the diseases caused by the respiratory syncytial virus include at least one of pneumonia, bronchiolitis, and otitis media.

[0099] In some embodiments of the present invention, the drug comprises a vaccine.

[0100] An eighth aspect of the invention provides a method according to any one of (d1)-(d2):

[0101] (d1) A method for preventing and / or treating respiratory syncytial virus, comprising administering an effective amount of the drug of the seventh aspect of the present invention to a subject;

[0102] (d2) A method for preventing and / or treating diseases caused by respiratory syncytial virus, wherein an effective amount of the drug of the seventh aspect of the present invention is administered to a subject.

[0103] In some embodiments of the present invention, the subject described in (d1)-(d2) is an animal; further, a mammal; even further selected from humans, cats, cattle, sheep, pigs, dogs, chickens, ducks, geese, rabbits, and mice; and even further, humans.

[0104] In some embodiments of the present invention, the diseases caused by respiratory syncytial virus (RSV) described in (d1)-(d2) include at least one of pneumonia, bronchiolitis, and otitis media.

[0105] In some embodiments of the present invention, taking mice as an example, the dosage administered in (d1)-(d2) is 10. 10 -10 12 vg; further for 5×10 10 -2×10 11 vg; further to 1×10 11 -2×10 11 vg.

[0106] The beneficial effects of this invention are:

[0107] This invention discloses for the first time the construction of a drug (vaccine) for the prevention and / or treatment of RSV infection or diseases caused by recombinant adeno-associated virus vector. Based on this, this invention provides a nucleic acid molecule comprising: (a1) a first region containing a first adeno-associated virus (AAV) inverted terminal repeat (ITR); and (a2) a second region containing a gene encoding a respiratory syncytial virus (RSV) antigen. The recombinant adeno-associated virus containing this nucleic acid molecule can produce good humoral and cellular immune effects, and has a certain serum neutralizing effect on both RSV A and B subtypes. In particular, when the antigen is a respiratory syncytial virus fusion protein or a mutant of a respiratory syncytial virus fusion protein, the recombinant adenovirus vector containing this nucleic acid molecule can successfully express the RSV pre-fusion F protein (Pre F). Attached Figure Description

[0108] Figure 1 shows the plasmid map of pAAV-RSV-F.

[0109] Figure 2 shows the plasmid map of pAAV-RSV-Fm.

[0110] Figure 3 shows the plasmid map of pAAV-RSV-G.

[0111] Figures 4A-4B show the results of Western blotting (WB) experiments verifying the antigen expression of pAAV-RSV-F, pAAV-RSV-Fm, and pAAV-RSV-G: In Figure 4A-4B, the results of Western blotting experiments verifying the antigen expression of pAAV-RSV-F and pAAV-RSV-Fm are shown, with 1, 2, and 3 representing pAAV-RSV-F, pAAV-RSV-Fm, and control, respectively; in Figure 4B, the results of Western blotting experiments verifying the antigen expression of pAAV-RSV-G are shown, with 1 and 2 representing pAAV-RSV-G and control, respectively.

[0112] Figure 5 shows the conformation verification results of the F protein expressed by rAAV-RSV-F and rAAV-RSV-Fm (N=5).

[0113] Figure 6 shows the serum-specific IgG expression levels in Balb / c mice after intramuscular immunization with rAAV5-RSV-F (N=5).

[0114] Figure 7 shows the serum-specific IgG expression levels in Balb / c mice after intramuscular immunization with rAAV5-RSV-Fm (N=5).

[0115] Figure 8 shows the serum-specific IgG expression levels in Balb / c mice after intramuscular immunization with rAAV5-RSV-G (N=5).

[0116] Figure 9 shows the serum neutralizing antibody levels in Balb / c mice after intramuscular immunization with rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G (N=5).

[0117] Figure 10 shows the CD8+ levels in Balb / c mice after intramuscular injection of rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G. + Figure showing the content of IFN-γ positive cells in T lymphocytes (N=5).

[0118] Figure 11 shows the CD4 count in Balb / c mice after intramuscular injection of rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G. + Figure showing the content of IFN-γ positive cells in T lymphocytes (N=5).

[0119] Figure 12 shows the serum specific IgG expression level results after Balb / c mice were immunized with rAAV5-RSV-F via nasal drops (N=5).

[0120] Figure 13 shows the serum specific IgG expression level results after Balb / c mice were immunized via nasal drops (N=5).

[0121] Figure 14 shows the serum specific IgG expression level results after Balb / c mice were immunized with rAAV5-RSV-G via nasal drops (N=5).

[0122] Figure 15 shows the serum neutralizing antibody levels in Balb / c mice after nasal mucosal immunization with rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G (N=5).

[0123] Figure 16 shows the CD8+ levels in Balb / c mice after intramuscular injection of rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G. + Figure showing the content of IFN-γ positive cells in T lymphocytes (N=5).

[0124] Figure 17 shows the CD4 count in Balb / c mice after intramuscular injection of rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G. + Figure showing the content of IFN-γ positive cells in T lymphocytes (N=5).

[0125] Figure 18 shows the results of the purity determination of rAAV5-RSV-F, rAAV5-RSV-Fm and rAAV5-RSV-G viruses using the Coomassie brilliant blue staining method. Detailed Implementation

[0126] The present invention will be further described in detail below through specific embodiments.

[0127] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0128] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0129] The present invention will be further described in detail below with reference to specific embodiments. These descriptions are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in commonly used reference books in the field, such as "Molecular Cloning: A Laboratory Manual" (3rd edition, Science Press, 2005), or according to the conditions recommended by the reagent manufacturers.

[0130] Example

[0131] I. Construction of plasmids expressing RSV F, RSV Fm, or RSV G proteins

[0132] Plasmids expressing RSV F, RSV Fm, or RSV G proteins are based on the rAAV vector and are named pAAV-RSV-F, pAAV-RSV-Fm, and pAAV-RSV-G, respectively.

[0133]

[0134]

[0135]

[0136] The plasmids were prepared by Guangzhou Yunzhou Biotechnology Co., Ltd.

[0137] II. Western Blot (WB) Verification of the Correct Expression of Target Antigen by Plasmids pAAV-RSV-F, pAAV-RSV-Fm, and pAAV-RSV-G

[0138] ① 293T cells in logarithmic growth phase and in good condition (cultured in DMEM medium + 10% (v / v) fetal bovine serum) were seeded into 12-well plates at 2.5 × 10⁶ cells / wells. 5 Cells / mL / well were incubated in a 37℃ 5% CO2 incubator for 24 hours before plasmid transfection.

[0139] ② Take 6 1.5mL EP tubes and add 100μL of 1% (v / v) FBS DMEM medium to each tube. Add 2.5μg of pAAV-RSV-F, pAAV-RSV-Fm and pAAV-RSV-G to 3 of the tubes respectively. Add 5μL of PEIpro transfection reagent to the other 3 EP tubes. Mix the EP tubes containing the transfection reagent with the EP tubes containing the plasmid one by one to prepare the transfection complex solution. Let it stand at room temperature for 15min.

[0140] ③ Take out the 12-well plate containing 293T cells, discard the culture medium in the wells, add 800μL of 1% (v / v) FBS DMEM medium to each well, then add the corresponding transfection complex solution, and incubate at 37℃ 5% CO2 for 48h before collecting the samples.

[0141] ④ After receiving the samples, Western blot experiments were performed. The antigen expression of pAAV-RSV-F, pAAV-RSV-Fm and pAAV-RSV-G was verified using RSV-F(A2)Antibody (sino biological, 11049-R302) and Anti-HRSV-A G / Major surface glycoprotein G Antibody (3D3) (Antibody system, RVV0805), respectively.

[0142] The results are shown in Figure 4: the proteins expressed by pAAV-RSV-F and pAAV-RSV-Fm can correctly bind to RSV-F (A2) antibody, and the protein expressed by pAAV-RSV-G can correctly bind to hRSV-G antibody (3D3). That is, the proteins expressed by pAAV-RSV-F, pAAV-RSV-Fm, and pAAV-RSV-G bind to their corresponding antibodies and are visualized, indicating that the expressed proteins are correct and the plasmids are constructed correctly.

[0143] III. Preparation of recombinant adeno-associated virus rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G

[0144] (a) Packaging using the three-plasma packaging method: 2.5 × 10 6 HEK293T cells were seeded into T75 cell culture flasks and cultured for 48 hours. Then, three plasmids were transfected (target gene plasmids (pAAV-RSV-F, pAAV-RSV-Fm, or pAAV-RSV-G, pAAV-Cap5Rep2 (Yaohai Biotechnology, JC202012004), pAAV-Helper (Yaohai Biotechnology, JC202012003) according to the three plasmid ratios in Table 1), and PEI pro transfection reagent was added. After 72 hours of viral transfection, subsequent purification operations were performed.

[0145] (II) Purification was performed using the iodixanol method. The specific steps were as follows: collect cells by pipetting, 200×g, incubate at room temperature for 5 min, and collect the supernatant by centrifugation; resuspend the cell pellet with cell lysis buffer at a concentration of 1×10⁻⁶. 7 Cells / mL resuspended and thoroughly mixed by pipetting; rapidly frozen in liquid nitrogen, then thawed in a 37°C water bath, repeating the freeze-thaw cycle 4 times. After the freeze-thaw cycle, centrifuged at 200×g at room temperature for 5 min and collected the supernatant. Combined the cell culture supernatant with the supernatant collected after repeated freeze-thaw cycles to obtain the upstream virus solution. Added PEG8000 to a final concentration of 8% (w / v), mixed by inversion, and incubated overnight at 4°C. After overnight incubation, centrifuged at 11000×g at 4°C for 30 min. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 10 mL of PBS and thoroughly mixed by shaking in a constant temperature shaking incubator at 37°C, 250 rpm, for 15 min to obtain the crude pure virus solution. Iodixanol density gradient ultracentrifugation: Using a 10 mL syringe, 9 mL of 15% iodixanol solution, 6 mL of 25% iodixanol solution, 5 mL of 40% iodixanol solution, and 5 mL of... were added sequentially to a Beckman ultracentrifuge tube. 60% iodixanol solution and 10 mL of crude purified virus solution were used. The mixture was centrifuged on a Beckman ultracentrifuge using a Type 70Ti rotor at 350,000 × g, 18°C, for 60 min. After centrifugation, 3-4 mL of the liquid from the 40% iodixanol solution separation was drawn using a 5 mL syringe; this was the rAAV virus solution. The virus solution was then concentrated using PBS (containing 0.05% poloxamer 188) and a 50 kDa ultrafiltration tube, followed by sterile filtration using a 0.22 μm syringe filter. The final harvested virus solutions were named rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G.

[0146] (III) Titer determination by ddPCR: Sample pretreatment: Add 5 μL of the sample to be tested to 45 μL of PCR diluent; add 5 μL of the pretreated sample to 45 μL of DNase I digestion solution and incubate at 37℃ for 30 minutes; after incubation, add 50 μL of PCR lysis buffer to each tube and incubate at 95℃ for 10 minutes; use PCR diluent to serially dilute the sample 10-fold to make the final concentration conform to the detection range of the ddPCR instrument (1000 copies / μL-10000 copies / μL); prepare the ddPCR reaction solution using ddPCR Supermix for Probes (Bio-rad) and load it into a 96-well plate for ddPCR, 15 μL / well; add 5 μL of PCR diluent to 15 μL / well of the reaction solution as a blank control (NTC), and add 5 μL of the diluted sample to 15 μL / well of the reaction solution as the sample solution, to obtain a 20 μL system; place it in a droplet generator to automatically generate droplets. After droplet generation, the sample plate was heat-sealed and amplified using a PCR amplification instrument. Once amplification was complete, the PCR plate was transferred to a droplet digital PCR instrument (Bio-rad) for detection. When the target gene plasmid was pAAV-RSV-F, the results are shown in Table 1: the highest viral titer was achieved and the purification yield was stable when the mass ratios of the target gene plasmid (pAAV-RSV-F), pAAV-Cap5Rep2, and pAAV-Helper were 21.67:56.67:21.67 and 21.67:21.67:56.67.

[0147] Table 1. Results of viral load in different proportions of target gene plasmids (pAAV-RSV-F), pAAV-Cap2Rep5, and pAAV-Helper.

[0148] Note: The upstream titer in the table represents the result of the upstream virus solution in step (II), the purified sample titer represents the result of the rAAV virus solution in step (II), the 10 concentrated samples represent the result of the concentrated rAAV virus solution in step (II), and the sterilized samples represent the result of the concentrated and sterilized rAAV virus solution (i.e., rAAV5-RSV-F) in step (II).

[0149] (IV) Coomassie Brilliant Blue staining method for rAAV5-RSV (rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G obtained in step (II), wherein the mass ratio of the target gene plasmid, pAAV-Cap5Rep2, and pAAV-Helper is 21.67:21.67:56.67) Virus purity determination: Sample gel chromatography: 2.5 × 10⁻⁶ 11The vg sample stock solution was mixed with 5× Loading Buffer and placed in a dry thermostat at 98℃ for 5 min to denature. After denaturation, the sample was loaded into the gel wells, with a marker loading volume of 5 μL. The electrophoresis apparatus was set to 120V and run for 80 min. Coomassie Brilliant Blue staining: PAGE staining was performed using an eStain L1 protein staining system. Band observation: The presence of impurities was observed, and the bands were observed to be displayed in a ratio of vp1:vp2:vp3 of 1:1:10. The results are shown in Figure 18: the rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G obtained in step (II) have high purity.

[0150] IV. Verification of the specific protein conformations expressed by rAAV5-RSV-Fm and rAAV5-RSV-F in 293T cells after in vitro infection

[0151] ① 293T cells in logarithmic growth phase and in good condition (cultured in DMEM medium + 10% (v / v) fetal bovine serum) were seeded into 12-well plates at 2.5 × 10⁶ cells / wells. 5 Cells / mL / well were incubated in a 37℃ 5% CO2 incubator for 24 hours before viral infection.

[0152] ② Discard the supernatant, according to 2×10 11 Virus doses of vg / mL / well were added to rAAV5-RSV-F and rAAV5-RSV-Fm (prepared according to the above "III. Preparation of Recombinant Adeno-Associated Virus rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G", wherein the mass ratio of the target gene plasmid, pAAV-Cap5Rep2, and pAAV-Helper was 21.67:21.67:56.67), with cells without viral intervention serving as the blank control group.

[0153] ③ After 48 hours of intervention, cells were collected according to the group, and fluorescent antibodies D25, AM14, and 4D7 were added respectively. The cells were incubated on ice for 20 minutes, washed with 1 mL of PBS, centrifuged at 300g for 5 minutes, and the supernatant was discarded. This process was repeated once. The cells were resuspended in 400 μL of PBS and prepared for instrumental analysis.

[0154] ④ The results are presented in a bar chart with the horizontal axis representing different groups and the vertical axis representing antibody binding rate (%).

[0155] The results are shown in Figure 5: AM14 antibody mainly binds to the Pre-F trimer conformation, while D25 antibody mainly binds to the antigen sites of the Pre-F conformation. In comparison, the 4D7 antibody primarily binds to the post-F trimer conformation; both rAAV5-RSV-F and rAAV5-RSV-Fm can express RSV F protein. Specifically, the binding rates of rAAV5-RSV-F to 4D7, D25, and AM14 antibodies after cell infection were 29.7%, 19%, and 20.1%, respectively, indicating that rAAV5-RSV-F mainly expresses the fused RSV F protein (post-F protein) or an intermediate conformation protein, meaning the F protein expressed by rAAV5-RSV-F is predominantly post-F. The binding rates of rAAV5-RSV-Fm to 4D7, D25, and AM14 antibodies after cell infection were 4.76%, 33.5%, and 43.3%, respectively, indicating that rAAV5-RSV-Fm mainly expresses the pre-F trimer protein, with little or no expression of the post-F protein, meaning the F protein expressed by rAAV5-RSV-Fm is predominantly post-F. With the F trimer structure as the main component, the rAAV vector can successfully express the pre-RSV fusion F protein (Pre F).

[0156] V. Intramuscular immunization of Balb / c mice with rAAV5-RSV vaccines (rAAV5-RSV-F, rAAV5-RSV-Fm, rAAV5-RSV-G)

[0157] ① Grouping: Fifty 6-8 week old Balb / c mice were randomly divided into 10 groups of 5 mice each. The groups were: blank control group, low-dose rAAV5-RSV-F group, medium-dose rAAV5-RSV-F group, high-dose rAAV5-RSV-F group, low-dose rAAV5-RSV-Fm group, medium-dose rAAV5-RSV-Fm group, high-dose rAAV5-RSV-Fm group, low-dose rAAV5-RSV-G group, medium-dose rAAV5-RSV-G group, and high-dose rAAV5-RSV-G group. The dosage group; rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G were prepared in the above-mentioned "III. Preparation of Recombinant Adeno-Associated Virus rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G", wherein the mass ratio of the target gene plasmid, pAAV-Cap5Rep2, and pAAV-Helper was 21.67:21.67:56.67.

[0158] ② Immunization: A single immunization regimen was adopted, with injection administered on day 0 in the right hind limb at a volume of 100 μL. The blank control group received PBS, while the drug group received 5 × 10⁻⁶ PBS. 10 vg, 1×10 11 vg, 2×10 11 VG was administered via intramuscular injection in low-dose, medium-dose, and high-dose groups.

[0159] ③ Analysis of IgG antibody levels and serum antibody neutralization assay for humoral immunity: Blood samples were collected on days 15, 30, 60, 90, and 120; 0.1-0.2 mL of blood was collected from each mouse, incubated at 4°C for 60 minutes, then centrifuged at 3000 rpm for 15 minutes. The supernatant serum was used for IgG antibody detection and antibody neutralization experiments on days 90 and 120, respectively.

[0160] The IgG antibody level was detected using ELISA, as follows: RSV-F Protein (ECD, His Tag) (blank control group, rAAV5-RSV-F and rAAV5-RSV-Fm vaccine group plate-forming protein) and RSV (subtype A, strain Long) Glycoprotein G Protein (ECD, His Tag) (blank control group, rAAV5-RSV-G vaccine group plate-forming protein) were prepared into 0.5 μg / mL solutions using 0.1 M carbonate buffer (pH 9.6). 100 μL of each solution was added to a 96-well ELISA plate, sealed, wrapped with aluminum foil, and incubated overnight at 4°C. The next day, the plate was incubated at 37°C for 1 hour (while simultaneously removing the blocking buffer and PBS to room temperature). The plate was washed three times with PBST (PBS + 0.1% (v / v) Tween 20). Add 300 μL of washing buffer to each well and wash using a plate washer; after washing, add 250 μL / well of 2% skim milk powder; incubate at room temperature for 1 hour, then wash the plate 3 times; add primary antibody: serum dilutions for IgG detection: 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, 1:109350, for a total of 8 dilutions; add secondary antibody: dilute HRP-labeled GOAT with PBS. Anti-mouse IgG (dilution 1:5000): Add 100 μL of the diluted secondary antibody to the corresponding wells and incubate at 37°C for 1 hour. Wash the plate 3 times. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 15 minutes. Add 100 μL of TMB stop solution to each well. After stopping the chromogenic reaction, immediately measure the absorbance using a microplate reader at a wavelength of 450 nm and a background wavelength of 570 nm. The results are shown in Figure 6-8, with the horizontal axis representing the detection time and the vertical axis representing the IgG antibody titer. rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G can produce good humoral immunity through intramuscular injection. Among them, high doses (2×10⁻⁶) of rAAV5-RSV-Fm and rAAV5-RSV-G produce the best humoral immunity. 11The vg group induced a certain level of humoral immune response in mice as early as day 15 post-immunization; the specific antibody titers in mice in the low- and medium-dose rAAV5-RSV-Fm and rAAV5-RSV-G groups, as well as the rAAV5-RSV-F group, were at a low level; as time progressed to day 30, the serum specific IgG levels in mice in the rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G groups significantly increased, with the high-dose rAAV5-RSV-Fm group (2×10⁻⁶) showing the highest level. 11 The antibody titer for vg was the most significant, reaching the highest level among all experimental groups. This time point is consistent with the vector characteristics of rAAV; the peak expression of single-chain rAAV vectors in vivo is 3-4 weeks, and they can be stably expressed in vivo for a long period. Therefore, up to day 60 post-immunization, the results showed that the serum-specific IgG antibody titers of mice in the rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G groups continued to increase, especially in the medium-dose rAAV5-RSV-Fm group (1×10⁻⁶). 11 The highest titer (log10) of specific IgG antibody in the serum of mice immunized with vg could reach 5.63, and it was stably expressed until the detection endpoint Day 120. The overall humoral immunization effect of rAAV5-RSV-F and rAAV5-RSV-Fm was better than that of the rAAV5-RSV-G design.

[0161] The antibody neutralization assay was performed using Hep-2 cells at a concentration of 2.0 × 10⁻⁶ cells / cells. 4 100 μL / well of cell suspension was seeded into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Serum samples were serially diluted 2-fold from 1:4 to 1:1024 (divisions were 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:512, and 1:1024). The diluted serum was then transferred sequentially to the corresponding neutralization plates according to the above dilution gradient (100 μL per well, starting from the 1:1024 dilution). Live RSV virus: HRSV (strain Long, subtype A, ATCC VR-26) and HRSV (strain 9320, subtype B, ATCC VR-955) (provided and tested by the Level 2 Biosafety Laboratory of Guangdong Provincial Center for Disease Control and Prevention) were diluted to 2 × 10⁻⁶. 5TCID50 / mL was added to each well of the corresponding neutralization plate at a rate of 50 μL. After gentle shaking and homogenization, the plates were incubated at 37℃ in a CO2 incubator for 2 hours. Cytopathic effect (CPE) was recorded on day 7, with the endpoint titer being the reciprocal of the highest serum dilution that did not produce CPE. Results are shown in Figure 9 and Tables 2 and 3: rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G all showed serum neutralization activity against both RSV subtypes A and B. In particular, serum neutralizing antibody levels on day 90 showed that the rAAV5-RSV-Fm group consistently induced neutralizing antibody levels, with the high-dose group inducing the highest levels of neutralizing antibodies against RSV-A and RSV-B subtypes, at 1:512 and 1:256, respectively. Serum neutralizing antibody levels on day 120... The results of level detection showed that the serum of mice immunized in the AAV5-RSV-Fm group had good neutralizing activity and a dose-response relationship. The highest neutralizing antibody titer against RSV-B type reached 1:512 on day 120 after immunization in the high-dose rAAV5-RSV-Fm group. Combined with the serum IgG antibody results, it can be seen that a single intramuscular immunization of mice with rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G can induce an effective humoral immune response, especially rAAV5-RSV-Fm, which showed the best effect.

[0162] Table 2. Results of Respiratory Syncytial Virus Serum Antibody Neutralization Assay - Results on Day 90 after Intramuscular Immunization (Selected Mice)

[0163] Table 3. Results of Respiratory Syncytial Virus Serum Antibody Neutralization Assay - Results on Day 120 after Intramuscular Immunization (Selected Mice)

[0164] ④ Flow cytometry analysis of cellular immunity levels: On day 75, anticoagulated blood was collected from mice under different treatments. 50 μL of whole blood was collected from each mouse for flow cytometry experiments. 1 mL of erythrocyte lysis buffer was added to each tube, and the cells were lysed for 5 min. The cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. 1 mL of Cell Staining Buffer was added, and the cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. 100 μL of Cell Staining Buffer was added to resuspend the cells. An appropriate amount of fluorescently labeled flow cytometry surface staining antibody was added to the cell suspension, mixed well, and incubated on ice in the dark for 15-20 min (Note: Incubation time and temperature can be adjusted according to staining indicators). 1 μL of PE / Cyanine7 anti-mouse CD3; 0.5 mL of PerCP anti-mouse CD4; 2 μL of FITC anti-mouse CD8a were added. 1 mL of Cell Staining Buffer was added, and the cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. The cells were washed twice. 150 μL of Cyto-Fast was added to each tube. TM Mix the Fix Perm Solution thoroughly; incubate at room temperature in the dark for 20 minutes; during incubation, add 10×Cyto-Fast solution to the solution. TM Perm Wash Solution diluted to 1×; after incubation, add 1 mL of 1× Cyto-Fast to each tube. TM Centrifuge with PermWash Solution at 300×g for 5 min, discard the supernatant, and wash twice; add 100 μL of 1×Cyto-Fast TM Resuspend cells in Perm Wash Solution, add appropriate amounts of fluorescently labeled flow cytometry intracellular factor staining antibodies (APC anti-mouse TNF-α - 1 μL; PE anti-mouse IFN-γ - 1 μL), mix well, incubate at room temperature in the dark for 20 min, then add 1 mL of 1×Cyto-Fast. TM PermWash Solution, centrifuged at 300×g for 5 min, discarded the supernatant; added 1 mL Cell Staining Buffer to resuspend the cells, centrifuged at 300×g for 5 min, discarded the supernatant; added 800 μL Cell Staining Buffer to resuspend the cells; data were collected. The results are shown in Figures 10-11: rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G all produced cellular immune effects. In particular, rAAV5-RSV-Fm and rAAV5-RSV-G showed better cellular immune effects and significantly increased CD4+. + T lymphocytes and CD8 +The expression of IFN-γ on T lymphocytes was enhanced, and the high-dose group showed significantly better results than the medium-dose and low-dose groups, suggesting that the cellular immune response was activated and could produce an effective immune response.

[0165] VI. Balb / c mice were immunized via nasal drop administration of rAAV5-RSV vaccines (rAAV5-RSV-F, rAAV5-RSV-Fm, rAAV5-RSV-G) via mucosal immunization.

[0166] ① Grouping: Forty 6-8 week old Balb / c mice were randomly divided into 8 groups of 5 mice each. Because the low-dose neutralizing antibody effect of intramuscular injection of rAAV5-RSV-F and rAAV5-RSV-G was not obvious, they were removed in the intranasal immunization. All three vaccines were tested by intranasal immunization using medium and high dose groups. The groups were set as follows: blank control group, AAV empty shell group, rAAV5-RSV-F medium-dose group, rAAV5-RSV-F high-dose group, rAAV5-RSV-Fm medium-dose group, rAAV5-RSV-Fm high-dose group, rAAV5-RSV-G medium-dose group, and rAAV5-RSV-G high-dose group; rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G were prepared in the above-mentioned "III. Preparation of Recombinant Adeno-Associated Virus rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G", wherein the mass ratio of the target gene plasmid, pAAV-Cap5Rep2, and pAAV-Helper was 21.67:21.67:56.67.

[0167] ② Immunization: A single immunization regimen was adopted, administered on day 0 via nasal drops (20 μL). The blank control group received PBS, while the AAV empty-shell group received AAV empty-shell virus solution (the preparation method of AAV empty-shell virus solution is the same as in "Preparation of Recombinant Adeno-Associated Virus rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G", the only difference being that it does not contain the target gene plasmid; the mass ratio of pAAV-Cap5Rep2 to pAAV-Helper is 21.67:56.67). The drug group received 1×10 11 vg, 2×10 11 VG was administered via nasal drops in the medium- and high-dose groups, while the AAV empty shell group received 2 × 10 11 The dose is administered via nasal drops.

[0168] ③ Analysis of IgG antibody levels and serum antibody neutralization assay for humoral immunity: Blood samples were collected on days 15, 30, 60, and 90; 0.1-0.2 mL of blood was collected from each mouse, incubated at 4°C for 60 minutes, then centrifuged at 3000 rpm for 15 minutes. The supernatant serum was used for IgG antibody detection and antibody neutralization assay on day 60, respectively.

[0169] The IgG antibody level was detected by ELISA, as follows: RSV-F Protein (ECD, His Tag) (blank control group, AAV empty shell group, rAAV5-RSV-F and rAAV5-RSV-Fm vaccine group plate coating protein) and RSV (subtype A, strain Long) Glycoprotein G Protein (ECD, His Tag) (blank control group, AAV empty shell group, rAAV5--RSV-G vaccine group plate coating protein) were prepared into 0.5 μg / mL solutions using 0.1 M carbonate buffer (pH 9.6). 100 μL of each solution was added to a 96-well ELISA plate, sealed, wrapped with aluminum foil, and incubated overnight at 4°C. The next day, the plate was incubated at 37°C for 1 hour (while simultaneously removing the blocking buffer and PBS to room temperature). The plate was washed three times with PBST (PBS + 0.1% (v / v) Tween 20), with 300 μL of washing buffer added to each well, using a plate washer.After washing the plate, add 250 μL / well of 2% skim milk powder; incubate at room temperature for 1 hour, then wash the plate 3 times; add primary antibody: serum dilutions for IgG detection at the following ratios: 1:50, 1:150, 1:450, 1:1350, 1:4050, 1:12150, 1:36450, and 1:109350, for a total of 8 dilutions; add secondary antibody: dilute HRP-labeled Goat anti-mouse antibody with PBS. IgG (dilution 1:5000): Add 100 μL of the diluted secondary antibody to the corresponding wells and incubate at 37°C for 1 hour. Wash the plate 3 times. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 15 minutes. Add 100 μL of TMB stop solution to each well. Immediately after stopping the chromogenic reaction, measure the absorbance using a microplate reader at a wavelength of 450 nm and a background wavelength of 570 nm. The results graph shows the detection time on the x-axis and the IgG antibody on the y-axis. The titers are presented in a bar chart, as shown in Figures 12-14. Nasal instillation of rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G resulted in good humoral immunity. On day 30 after nasal instillation, the serum levels of RSV-specific IgG antibodies in mice of the rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G groups were significantly elevated, indicating the onset of a humoral immune response. Later than intramuscular immunization; by days 60 and 90, serum levels in mice in the rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G groups steadily increased, with the highest serum levels in the high-dose rAAV5-RSV-F and rAAV5-RSV-Fm groups; overall, after intranasal immunization, serum-specific IgG levels in mice in the rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G groups showed a generally stable increase, but overall specific IgG levels... The heterologous IgG titer was lower than that of intramuscular immunization, suggesting that nasal instillation of rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G mucosal immunization can achieve a certain level of humoral immune response. The results of specific antibody IgG in the AAV empty shell group were the same as those in the control group, suggesting that the immunogenicity of the rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G groups is related to the expressed antigen. The AAV vector has no RSV-specific immunogenicity, indicating that it has good safety.

[0170] Hep-2 cells were fed at a rate of 2.0 × 10⁻⁶ 4100 μL / well of cell suspension was seeded into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Serum samples were serially diluted 2-fold from 1:4 to 1:1024 (divisions were 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:512, and 1:1024). The diluted serum was then transferred sequentially to the corresponding neutralization plates according to the above dilution gradient (100 μL per well, starting from the 1:1024 dilution). Live RSV virus: HRSV (strain Long, subtype A, ATCC VR-26) and HRSV (strain 9320, subtype B, ATCC VR-955) (provided and tested by the Level 2 Biosafety Laboratory of Guangdong Provincial Center for Disease Control and Prevention) were diluted to 2 × 10⁻⁶. 5 Add 50 μL of TCID50 / mL to each well of the corresponding neutralization plate, shake gently to mix, and then incubate at 37℃ in a CO2 incubator for 2 hours. Record the cytopathic effect (CPE) of each well on day 7 of the experiment, with the reciprocal of the highest dilution of serum that does not produce cytopathic effect as the endpoint titer. The results are shown in Figure 15 and Table 4: rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G all showed some serum neutralization against both RSV subtypes A and B. The overall neutralizing antibody levels in the rAAV5-RSV-Fm and rAAV5-RSV-F groups were higher than those in the rAAV5-RSV-G group, consistent with the trend observed with intramuscular immunization, suggesting that RSV-F protein is a better antigen target in the design of mucosal immunization vaccines. Furthermore, comparing the neutralizing antibody results of rAAV5-RSV-F and rAAV5-RSV-Fm, the overall neutralizing antibody level in the rAAV5-RSV-Fm group was higher than that in the rAAV5-RSV-F group. The medium-dose group showed the highest neutralizing antibody efficacy against RSV-A and RSV-B subtypes, reaching 1:1024. This suggests that the vaccine design can successfully express RSV-Pre The F protein is structurally related and has an antigenic site that neutralizing antibodies can bind to. Combined with the results of serum-specific IgG detection by ELISA, the rAAV5-RSV-Fm vaccine group can induce an effective humoral immune response with neutralizing capacity by a single intranasal immunization of mice, and the ability to induce neutralizing antibodies is higher than that of intramuscular immunization.

[0171] Table 4. Results of Respiratory Syncytial Virus Serum Antibody Neutralization Assay - Results on Day 60 after Intranasal Immunization (Selected Mice)

[0172] ④ Flow cytometry analysis of cellular immunity levels: On day 30, anticoagulated blood was collected from mice under different treatments. 50 μL of whole blood was collected from each mouse for flow cytometry experiments. 1 mL of erythrocyte lysis buffer was added to each tube, and the cells were lysed for 5 min. The cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. 1 mL of Cell Staining Buffer was added, and the cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. 100 μL of Cell Staining Buffer was added to resuspend the cells. An appropriate amount of fluorescently labeled flow cytometry surface staining antibody was added to the cell suspension, mixed well, and incubated on ice in the dark for 15-20 min (Note: Incubation time and temperature can be adjusted according to staining indicators). 1 μL of PE / Cyanine7 anti-mouse CD3; 0.5 mL of PerCP anti-mouse CD4; 2 μL of FITC anti-mouse CD8a were added. 1 mL of Cell Staining Buffer was added, and the cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. The cells were washed twice. 150 μL of Cyto-Fast was added to each tube. TM Mix the Fix Perm Solution thoroughly; incubate at room temperature in the dark for 20 minutes; during incubation, add 10×Cyto-Fast solution to the solution. TM Perm Wash Solution diluted to 1×; after incubation, add 1 mL of 1× Cyto-Fast to each tube. TM Centrifuge with PermWash Solution at 300×g for 5 min, discard the supernatant, and wash twice; add 100 μL of 1×Cyto-Fast TM Resuspend cells in Perm Wash Solution, add appropriate amounts of fluorescently labeled flow cytometry intracellular factor staining antibodies (APC anti-mouse TNF-α - 1 μL; PE anti-mouse IFN-γ - 1 μL), mix well, incubate at room temperature in the dark for 20 min, then add 1 mL of 1×Cyto-Fast. TM PermWash Solution, centrifuged at 300×g for 5 min, discarded the supernatant; added 1 mL Cell Staining Buffer to resuspend the cells, centrifuged at 300×g for 5 min, discarded the supernatant; added 800 μL Cell Staining Buffer to resuspend the cells; data were collected. The results are shown in Figures 16-17: rAAV5-RSV-F, rAAV5-RSV-Fm, and rAAV5-RSV-G all produced cellular immune effects. In particular, rAAV5-RSV-Fm and rAAV5-RSV-G showed better cellular immune effects and significantly increased CD4+. + T lymphocytes and CD8 +The expression of IFN-γ on T lymphocytes suggests activation of cellular immune response, which can generate an effective immune response.

[0173] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A nucleic acid molecule comprising: (a1) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR); and (a2) a second region comprising a gene encoding a respiratory syncytial virus antigen; the respiratory syncytial virus antigen is selected from one or more of a fusion protein of respiratory syncytial virus, a fusion protein mutant of respiratory syncytial virus, a glycoprotein of respiratory syncytial virus; the fusion protein mutant of respiratory syncytial virus has mutations N42I, and S190P compared to the fusion protein of respiratory syncytial virus.

2. The nucleic acid molecule of claim 1, wherein: the respiratory syncytial virus antigen is selected from any one of a fusion protein of respiratory syncytial virus, a fusion protein mutant of respiratory syncytial virus, a glycoprotein of respiratory syncytial virus; further selected from any one of a fusion protein of respiratory syncytial virus, a fusion protein mutant of respiratory syncytial virus; further a fusion protein mutant of respiratory syncytial virus; preferably, the second region further comprises a promoter; preferably, the promoter is one or more of a constitutive promoter, an inducible promoter; further a constitutive promoter; preferably, the constitutive promoter is selected from one or more of a CMV promoter, an EF1A promoter, an EFS promoter, a CAG promoter, a CBh promoter, an SFFV promoter, an MSCV promoter, an SV40 promoter, an mPGK promoter, an hPGK promoter, and a UBC promoter; preferably, the inducible promoter is selected from one or more of a tetracycline-regulated promoter, an alcohol-regulated promoter, a steroid-regulated promoter, a metal-regulated promoter, a pathogenesis-regulated promoter, a temperature / heat-inducible promoter, and a light-regulated promoter, an IPTG-inducible promoter; preferably, the second region further comprises a Kozak sequence; preferably, the second region further comprises a post-transcriptional regulatory element; preferably, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element; preferably, the second region further comprises a polyadenylation signal; preferably, the polyadenylation signal is selected from one or more of a bovine growth hormone poly A, a short polyA, an SV40 polyA, and a human beta globin poly A; preferably, the second region further comprises a signal peptide; preferably, the signal peptide is selected from one or more of a SAP signal peptide, an IHC signal peptide, an ILC signal peptide, an AP signal peptide, a CSP signal peptide, an IL2 signal peptide, a TPA signal peptide, an L1 signal peptide, an H7 signal peptide, an SP1 signal peptide, an H1 signal peptide, an RSV F signal peptide.

3. The nucleic acid molecule of any one of claims 1-2, wherein: the nucleic acid molecule further comprises: (a3) a third region comprising a second adeno-associated virus (AAV) inverted terminal repeat (ITR). Preferably, each of the first adeno-associated virus (AAV) inverted terminal repeat (ITR), the second adeno-associated virus (AAV) inverted terminal repeat (ITR) is independently selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, mouse AAV serotype ITRs; Preferably, when the respiratory syncytial virus antigen is a fusion protein of respiratory syncytial virus, or a fusion protein mutant of respiratory syncytial virus, the nucleic acid molecule comprises: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a signal peptide, a gene encoding the respiratory syncytial virus antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR); Further preferably, the nucleic acid molecule comprises, in the order from 5’ to 3’: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a signal peptide, a gene encoding the respiratory syncytial virus antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR); Preferably, there is an optional linker between the first adeno-associated virus (AAV) inverted terminal repeat (ITR) and the promoter, between the promoter and the Kozak sequence, between the gene encoding the respiratory syncytial virus antigen and the post-transcriptional regulatory element, between the post-transcriptional regulatory element and the polyadenylation signal, and / or between the polyadenylation signal and the second adeno-associated virus (AAV) inverted terminal repeat (ITR); Preferably, the nucleic acid molecule has the sequence of SEQ ID NO: 1, or nucleotides 1-3522 of SEQ ID NO: 2; Preferably, when the respiratory syncytial virus antigen is a glycoprotein of respiratory syncytial virus, the nucleic acid molecule comprises: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a gene encoding the respiratory syncytial virus antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR); Further preferably, the nucleic acid molecule comprises, in the order from 5’ to 3’: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a promoter, a Kozak sequence, a gene encoding the respiratory syncytial virus antigen, a post-transcriptional regulatory element, a polyadenylation signal, and a second adeno-associated virus (AAV) inverted terminal repeat (ITR); Preferably, there is an optional linker between the first adeno-associated virus (AAV) inverted terminal repeat (ITR) and the promoter, between the promoter and the Kozak sequence, between the gene encoding the respiratory syncytial virus antigen and the post-transcriptional regulatory element, between the post-transcriptional regulatory element and the polyadenylation signal, and / or between the polyadenylation signal and the second adeno-associated virus (AAV) inverted terminal repeat (ITR). Preferably, the sequence of the nucleic acid molecule is the nucleotide sequence of SEQ ID NO: 3 from position 1 to position 3838.

4. A recombinant adeno-associated virus vector comprising the nucleic acid molecule of any one of claims 1-3.

5. The recombinant adeno-associated virus vector of claim 4, wherein: the recombinant adeno-associated virus vector further comprises a gene encoding a marker selected from at least one of an antibiotic resistance protein, a toxin resistance protein, a colored or fluorescent protein, and a protein that mediates enhanced cell growth and / or gene amplification; Preferably, the marker is an antibiotic resistance protein. Preferably, the antibiotic is selected from at least one of ampicillin, neomycin, G418, puromycin, and blasticidin. Preferably, the recombinant adeno-associated virus vector comprises an origin of replication selected from at least one of f1 phage ori, RK2 oriV, pUC ori, and pSC101 ori. Preferably, the sequence of the recombinant adeno-associated virus vector is set forth in any one of SEQ ID NO: 1-SEQ ID NO:

3.

6. A host cell comprising the nucleic acid molecule of any one of claims 1-3 or the recombinant adeno-associated virus vector of any one of claims 4-5.

7. A recombinant adeno-associated virus comprising: (b1) the nucleic acid molecule of any one of claims 1-3.

8. The recombinant adeno-associated virus of claim 7, wherein: the recombinant adeno-associated virus further comprises: (b2) a capsid protein; Preferably, the capsid protein is selected from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2 R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, AAV2-HBKO, AAV DJ8, AAVPHP.B, AAVPHP.eB, AAV BR1, AAVHSC15, AAVHSC17, a goat AAV, an AAV1 / AAV2 chimeric, a bovine AAV, or a mouse AAV capsid, or a rAAV2 / HBoV1 serotype capsid protein. Preferably, the recombinant adeno-associated virus is a single-stranded AAV.

9. A method for preparing the recombinant adeno-associated virus according to any one of claims 7-8, wherein the recombinant adeno-associated virus is packaged by the recombinant adeno-associated virus vector according to any one of claims 4-5.

10. The method for preparing according to claim 9, wherein: the method for preparing the recombinant adeno-associated virus comprises the following steps: transfecting the recombinant adeno-associated virus vector according to any one of claims 4-5, the packaging plasmid pAAV-RC, and the helper plasmid pHelper into production cells; preferably, the mass ratio of the recombinant adeno-associated virus vector, the packaging plasmid pAAV-RC, and the helper plasmid pHelper is 10:(1.25-80):(1.25-80); preferably, the method for preparing the recombinant adeno-associated virus further comprises a purification step.

11. Use of the nucleic acid molecule according to any one of claims 1-3, the recombinant adeno-associated virus vector according to any one of claims 4-5, the host cell according to claim 6, and / or the recombinant adeno-associated virus according to any one of claims 7-8 in the preparation of a medicament; the medicament is used for at least one of (c1)-(c2): (c1) preventing and / or treating a respiratory syncytial virus infection; (c2) preventing and / or treating a disease caused by a respiratory syncytial virus.

12. The use according to claim 11, wherein: the disease caused by a respiratory syncytial virus comprises at least one of pneumonia, bronchiolitis, and otitis media; preferably, the medicament comprises a vaccine.

13. A medicament comprising: the nucleic acid molecule according to any one of claims 1-3, the recombinant adeno-associated virus vector according to any one of claims 4-5, the host cell according to claim 6, and / or the recombinant adeno-associated virus according to any one of claims 7-8.

14. The medicament according to claim 13, wherein: the medicament further comprises a pharmaceutically acceptable carrier; preferably, the dosage form of the medicament is a dosage form suitable for children or a dosage form suitable for adults; preferably, the dosage form is a dosage form for gastrointestinal administration or a dosage form for non-gastrointestinal administration; preferably, the dosage form for gastrointestinal administration comprises at least one of a powder, a tablet, a granule, a capsule, a sustained-release preparation, a solution, a dry suspension, an effervescent tablet, an emulsion, a suspension, a syrup, a drop, a chewable tablet; preferably, the dosage form for non-gastrointestinal administration comprises at least one of an injection administration dosage form, a respiratory administration dosage form, a skin administration dosage form, a mucosa administration dosage form, a cavity administration dosage form; preferably, the medicament is a nasal drop or an injection; preferably, the subject of administration of the medicament is an animal; preferably, the animal is a mammal; further selected from a human, a cat, a cow, a sheep, a pig, a dog, a chicken, a duck, a goose, a rabbit, a mouse.

15. The medicament according to any one of claims 13-14, wherein: the medicament is used for at least one of (c1)-(c2): (c1) preventing and / or treating a respiratory syncytial virus infection; (c2) preventing and / or treating a disease caused by a respiratory syncytial virus. Preferably, the medicament further comprises other active ingredients for the prevention and / or treatment of respiratory syncytial virus infection and / or disease caused by respiratory syncytial virus; Preferably, the disease caused by respiratory syncytial virus comprises at least one of pneumonia, bronchiolitis and otitis media. Preferably, the medicament comprises a vaccine.

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