An immune composition product for the prevention or treatment of respiratory syncytial virus-related diseases and its preparation method.

Abstract

This invention relates to the field of biomedical technology, specifically to an immune composition product for the prevention or treatment of respiratory syncytial virus (RSV)-related diseases and its preparation method. Specifically, this invention provides a RSV nanoparticle vaccine comprising an antigen component and a particulate protein component, wherein the antigen component and the particulate protein component form an immunogenic complex. The antigen component comprises the pre-fusion F protein of RSV, and the particulate protein component comprises nanoparticle proteins. This nanoparticle vaccine can better maintain the pre-fusion conformation of the RSV F protein, induce higher titers of neutralizing antibodies, and exhibits good stability and a simple manufacturing process, making it a very promising RSV vaccine candidate.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to immune composition products for the prevention or treatment of respiratory syncytial virus (RSV)-related diseases and their preparation methods. Background Technology

[0002] Respiratory syncytial virus (RSV) is an enveloped virus belonging to the genus Orthopneumovirus in the family Pneumoviridae, order Single-stranded transvirales. RSV is the leading cause of acute lower respiratory tract infections in children under 5 years of age and a significant cause of hospitalization and death from respiratory diseases in the elderly. Developing effective interventions to prevent and treat RSV infection is crucial.

[0003] Respiratory syncytial virus (RSV) is a typical non-segmented, single-stranded, negative-sense RNA virus. Its genome encodes 11 viral proteins: nucleocapsid protein (N), phosphoprotein (P), polymerase subunit protein (L), matrix protein (M), anti-termination factor (M2-1), RNA regulatory factor (M2-2), two non-structural proteins (NS1 and NS2), and three transmembrane proteins: adhesion protein (G), fusion protein (F), and small hydrophobic protein (SH). The G protein mediates RSV virion attachment to the host cell membrane. After RSV attachment, the F protein promotes the fusion of the viral envelope with the host cell membrane, subsequently releasing a helical ribonucleoprotein complex (RNP) into the host cytoplasm. The F protein is the primary binding site for neutralizing antibodies, thus becoming a major target protein in vaccine development. It belongs to class I fusion glycoproteins and is cleaved by proteases during maturation, generating two polypeptide chains, F1 and F2, linked by disulfide bonds. Like other viral fusion glycoproteins, the RSV F protein undergoes irreversible structural rearrangement during fusion, refolding from an unstable pre-fusion conformation to a stable post-fusion conformation. The post-fusion state of the RSV F protein has been shown to induce only a slight increase in neutralizing antibodies, while the pre-fusion state (preF) can induce a strong neutralizing antibody response.

[0004] Scientists spent decades obtaining stable pre-fusion F protein mutants. Peter Kwong's research group pioneered the introduction of a monomeric disulfide bond mutation (“DS”) between residues 155 and 290, and a cavity-filling mutation (“Cav1”) in the F protein to maintain the pre-fusion conformation of the RSV F protein, resulting in a stable F trimer protein named “DS-Cav1”. Based on DS-Cav1, a second-generation RSV pre-fusion F protein mutant (Sc9-10DS-Cav1 A149C Y458C, abbreviated as SC9-10 in this application) deleted the fusion peptide between the F subunits (F1 and F2) and linked them with a flexible chain. Additionally, a monomeric disulfide bond was introduced between residues 149 and 459 to further stabilize the pre-fusion conformation of the F trimer protein. Compared to the first-generation DS-Cav1, SC9-10 exhibits enhanced immunogenicity, stability, and yield characteristics.

[0005] Based on the design of DS-Cav1 and its derivatives, RSV vaccines have been successfully developed and approved for marketing. These include GlaxoSmithKline's subunit vaccine Arexvy, Pfizer's bivalent subunit vaccine Abrysvo, and Moderna's mRNA vaccine mResvia. Arexvy and mResvia are only approved for use in adults 60 years of age and older. Arexvy is not recommended for infants and young children, possibly because it contains the AS01E adjuvant; it is also not recommended for pregnant women due to the potential risk of preterm birth. Abrysvo has been approved for use in adults 60 years of age and older, as well as pregnant women at 32 to 36 weeks of gestation, due to its better safety profile (adjuvant-free formulation) and subsequent efficacy data. None of the above vaccines have been approved for marketing in China.

[0006] In recent years, recombinant self-assembled nanoparticle (NP) technology has developed rapidly. Repeated presentation of antigens on NP vectors can significantly improve the immunogenicity of antigens and induce the production of potent neutralizing antibodies. Several NP platforms, such as ferritin, AP205, Qβ, mi3, and I53-50, have been used to present viral surface glycoproteins derived from influenza virus, Epstein-Barr virus, and SARS-CoV-2. For RSV, stable pre-fusion F protein mutants have been presented using NPs. For example, CN112638411B disclosed the display of RSV-F pre-fusion F protein trimers (SC9-10) on HBsAg VLP, but only verified the VLP-induced IgG level and did not verify whether SC9-10 displayed by HBsAg could maintain a stable pre-fusion conformation. WO2024155561A2 disclosed the display of DS-Cav1, SC-TM and SC9-10 using ferritin and E2p respectively. It was found that the ferritin fusion constructs of DS-Cav1 and SC-TM failed to form nanoparticles or trimer structures. The SC9-10 trimer spikes seemed to be a "lollipop" shaped and hollow open conformation, rather than a "thumb" shaped pre-fusion closed trimer spike conformation. The conclusion suggested that the existing three representative RSV pre-fusion F protein designs DS-Cav1, SC9-10 and SC-TM are not suitable for nanoparticle display. Therefore, it is evident that using NP to present pre-fusion F protein mutants may not be able to maintain a stable pre-fusion conformation, and NP design still needs to be optimized to obtain nanoparticle vaccines that can maintain the pre-fusion conformation of F protein.

[0007] Leveraging the advantages of self-assembled nanoparticle technology, nanoparticle vaccines exhibiting a stable pre-fusion conformation of the F protein have enormous development potential and application demand. Currently, there are no RSV nanoparticle vaccines marketed in China, and very few are under development. Therefore, developing RSV nanoparticle vaccines exhibiting a stable pre-fusion conformation of the F protein is of great significance. Summary of the Invention

[0008] The purpose of this invention is to provide an immune composition product for the prevention or treatment of respiratory syncytial virus (RSV)-related diseases and its preparation method, thereby addressing the current problems of insufficient RSV vaccine technology and supply. To achieve the above objective, the technical solution adopted by this invention includes the following aspects:

[0009] In a first aspect, the present invention provides an RSV immunogenic complex comprising RSV pre-fusion F protein.

[0010] The RSV pre-fusion F protein described in this invention, referred to as "preF" in this invention, means that compared with the natural RSV F protein, it has increased retention of the pre-fusion conformation.

[0011] The pre-fusion conformation of the RSVF protein described in this invention refers to the RSVF protein containing residues 62-69 and 196-209 at its distal membrane tip, and also including epitopes of monoclonal antibodies 5C4, D25, and AM22. The recombinant RSVF protein in the pre-fusion conformation can specifically bind to antibodies that bind to the pre-fusion conformation of the RSVF protein, rather than the post-fusion conformation, such as 5C4, D25, or AM22 antibodies. Example specific antibodies for the post-fusion conformation include 4D7.

[0012] The present invention provides an RSV immunogenic complex comprising an antigenic component and a particulate protein component, wherein the antigenic component comprises preF.

[0013] In some embodiments, the present invention provides an immunogenic complex comprising:

[0014] (1) Antigen component, which contains RSV preF;

[0015] (2) Particulate protein component, which includes nanoparticle protein.

[0016] In some embodiments, the present invention provides an immunogenic complex comprising:

[0017] (1) Antigen component, which contains the RSV pre-fusion F protein preF;

[0018] (2) Particulate protein component, which includes nanoparticle protein mi3.

[0019] The antigen component and the particulate protein component are covalently bound by peptide binding pairs. The antigen component contains binding peptide 1, and the particulate protein component contains binding peptide 2. The antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2.

[0020] In some embodiments, the present invention provides an immunogenic complex comprising:

[0021] (1) Antigen component, which contains RSV preF and binding peptide 1;

[0022] (2) Particulate protein component, which includes nanoparticle protein and binding peptide 2.

[0023] Wherein, binding peptide 1 and binding peptide 2 are peptide binding pairs, and the antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2, for example, SpyTag / SpyCatcher.

[0024] In some embodiments, the present invention provides an immunogenic complex comprising:

[0025] (1) Antigen component, which includes RSV preF, linker peptide 1 and binding peptide 1;

[0026] (2) Particulate protein component, which includes nanoparticle protein, linker peptide 2 and binding peptide 2;

[0027] The antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2.

[0028] In some embodiments, the present invention provides an immunogenic complex comprising:

[0029] (1) Antigen component, which consists of RSV preF, linker peptide 1 and binding peptide 1;

[0030] (2) The particulate protein component, which consists of nanoparticle protein, linker peptide 2 and binding peptide 2;

[0031] The antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2.

[0032] In some embodiments, in any of the immunogenic complexes provided by the present invention, the antigen component is formed by fusing RSVpreF at its C-terminus with binding peptide 1 via linker peptide 1.

[0033] In some embodiments, in any of the immunogenic complexes provided by the present invention, the particulate protein component is formed by fusing nanoparticle protein at the N-terminus with linking peptide 2 and binding peptide 2.

[0034] In a preferred embodiment, in any of the immunogenic complexes provided by the present invention, the antigen component from the N-terminus to the C-terminus is, in sequence: RSV preF, linker peptide 1, and binding peptide 1; the particulate protein component from the N-terminus to the C-terminus is, in sequence: binding peptide 2, linker peptide 2, and nanoparticle protein; the antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2 to form an immunogenic complex.

[0035] In some embodiments, in any of the immunogenic complexes provided by the present invention, the antigen component and / or the particulate protein component contains a purification tag, such as a histidine tag.

[0036] In some embodiments, the RSV preF of the present invention is known in the prior art, such as the RSV preF disclosed in WO2009079796, WO2014160463, and WO2017172890.

[0037] In some embodiments, the RSV preF of the present invention is selected from DS-Cav1 and Sc9-10 DS-Cav1 A149CY458C (hereinafter referred to as "SC9-10").

[0038] The amino acid sequence of RSV preF of the present invention has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% identity with SEQ ID NO: 1.

[0039] In some embodiments, the amino acid sequence of the RSV preF of the present invention is shown in SEQ ID NO: 2 or SEQ ID NO: 3; preferably, the amino acid sequence of the RSV preF of the present invention is shown in SEQ ID NO: 3.

[0040] In some embodiments, the C-terminus of the RSV preF of the present invention is connected to a trimerization domain.

[0041] In some embodiments, the trimerizing domain of the present invention is selected from T4 Fibritin foldon. Preferably, the amino acid sequence of foldon is as shown in YIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 8).

[0042] In some embodiments, the C-terminus of the RSV preF protein of the present invention is linked to a trimerizing domain via a peptide linker. The peptide linker is a commonly used linker peptide; example peptide linkers include the GS linker. Preferably, the peptide linker is GSG.

[0043] In other embodiments, the present invention provides an immunogenic complex comprising:

[0044] (1) Antigen component, which contains RSV preF and linker peptide 1;

[0045] (2) Particulate protein component, which contains nanoparticle protein subunits.

[0046] When the particulate protein component contains a subunit of the nanoparticle protein, the antigen component also contains another subunit of the nanoparticle protein.

[0047] In some embodiments, RSV preF is linked to one subunit of the nanoparticle protein to form a fusion protein, which is then covalently bound to another subunit of the nanoparticle protein.

[0048] In some embodiments, in any of the immunogenic complexes provided by the present invention, the nanoparticle protein is selected from: mi3, ferritin, I53-50, etc.

[0049] In some embodiments, in any of the immunogenic complexes provided by the present invention, the nanoparticle protein I53-50 particles are composed of two subunits, I53-50A and I53-50B.

[0050] In some embodiments, the nanoparticle protein in any of the immunogenic complexes provided by the present invention is mi3, referred to as "NPM" in the present invention.

[0051] In some embodiments, in any of the immunogenic complexes provided by the present invention, the binding peptide 1 contains an amino acid sequence as shown in SEQ ID NO: 9.

[0052] In some embodiments, in any of the immunogenic complexes provided by the present invention, the binding peptide 2 contains an amino acid sequence as shown in SEQ ID NO: 10.

[0053] In some embodiments, in any of the immunogenic complexes provided by the present invention, the linker peptide 1 and linker peptide 2 are commonly used peptide linkers, and flexible linkers, rigid linkers, and semi-flexible / semi-rigid linkers can be used.

[0054] In some embodiments, in any of the immunogenic complexes provided by the present invention, the linker peptide 1 comprises (GGGGS). n Or (EAAAK) n The amino acid sequence, where n can be an integer greater than 0 and less than or equal to 5. In some embodiments, in any immunogenic complex provided by the present invention, the linker peptide 1 is selected from (GGGGS)3, GGGSGGSGSGGSGGGSGGGS, (EAAAK)3, or GGSGGSGSEKAAKAEEAAR. In some embodiments, the linker peptide 1 is selected from SAIG.

[0055] In some embodiments, in any of the immunogenic complexes provided by the present invention, the linker peptide 2 comprises (GGS). n (SGGSGG) n Or (GSGGSGGSG) n The amino acid sequence, where n can be an integer greater than 0 and less than or equal to 10. In some embodiments, in any immunogenic complex provided by the present invention, the linker peptide 2 is preferably GGSGGSGGS, GGSGGSGGSGGS, SGGSGG, or GSGGSGGSG.

[0056] Preferably, the RSV fusion-pre-fusion F protein of the present invention is expressed using the signal peptide shown in SEQ ID NO: 4 or SEQ ID NO: 5; the preferred signal peptide sequence is SEQ ID NO: 4.

[0057] In some embodiments, in any of the immunogenic complexes provided by the present invention, the C-terminus of the pre-fusion F protein of RSV is linked to foldon, and then linked to binding peptide 1 (named "4T" or Tag in this application) via linker peptide 1. Simultaneously, a histidine purification tag (such as 6His) can be added to the C-terminus of the fusion protein. The coding gene encoding the above fusion protein is inserted into a eukaryotic cell expression vector (e.g., pcDNA3.4) and expressed in CHO cells to obtain a fusion protein with the antigen component preF-4T. The antigen component is subjected to nickel column affinity chromatography and molecular sieve chromatography to obtain a high-purity protein, specifically in the forms shown in DS-Cav1-Tag (SEQ ID NO:15) and SC9-10-Tag (SEQ ID NO:16).

[0058] In some embodiments, the C-terminus of the pre-fusion F protein of the present invention is linked to a trimerization domain via a peptide linker, and the C-terminus of the trimerization domain is linked to binding peptide 1 via a peptide linker. The peptide linker is a commonly used peptide linker; an example peptide linker includes a GS linker. Preferably, the peptide linker is GSGGSG.

[0059] In some embodiments, in any of the immunogenic complexes provided by the present invention, the particulate protein component is a fusion protein formed by linker peptide 2 and binding peptide 2 at the N-terminus of the nanoparticle protein; preferably, the nanoparticle protein is NPM. Specifically, in some alternative embodiments, binding peptide 2 (named "4C") is linked to the encoding gene of the nanoparticle protein via linker peptide 2, inserted into a prokaryotic expression vector (such as pET-28a(+), pET-30a(+)), and expressed in E. coli cells to obtain a fusion protein of binding peptide 2 and nanoparticle protein. The fusion protein can be purified by chromatography, such as by anion exchange chromatography or hydrophobic chromatography, to obtain the product. The formed particulate protein component is named 4C-NPM.

[0060] Specifically, in some alternative embodiments, under suitable reaction conditions, any of the above-mentioned antigen components are covalently bound to the particulate protein component. This coupling is achieved through the covalent bonding of binding peptide 1 of the antigen component and binding peptide 2 of the particulate protein component, thereby forming the immunogenic complex preF-NPM. The specific immunogenic complexes are named DS-Cav1-NPM and SC9-10-NPM.

[0061] In other embodiments, the present invention provides an immunogenic complex comprising:

[0062] (1) Antigen components, which include RSV pre-fusion F protein and linker peptide 1;

[0063] (2) A particulate protein component comprising nanoparticle protein subunits; preferably, the nanoparticle protein subunits are I53-50A or I53-50B subunits.

[0064] In some embodiments, in any of the immunogenic complexes provided by the present invention, the nanoparticle protein I53-50 comprises I53-50A or I53-50B subunits; preferably, I53-50A comprises the amino acid sequence shown in SEQ ID NO: 6, and I53-50B comprises the amino acid sequence shown in SEQ ID NO: 7.

[0065] Specifically, in any of the immunogenic complexes provided by this invention, the pre-fusion F protein of the RSV is linked to one subunit of the nanoparticle protein to form a fusion protein, and the fusion protein then binds to another subunit of the nanoparticle protein. Preferably, the subunit of the nanoparticle protein is I53-50A or I53-50B. Further, in some alternative embodiments, the preF in the antigen component binds to the I53-50A subunit of the nanoparticle protein at its C-terminus via linker peptide 1 to form a preF-I53-50A fusion protein; then, the fusion protein binds to the I53-50B subunit of the nanoparticle protein to form preF-I53-50.

[0066] As described above, when the nanoparticle protein I53-50 is selected, I53-50 contains two subunits, I53-50A and I53-50B. The aforementioned preF, which may or may not contain a specific signal peptide, is linked to I53-50A via linker peptide 1. A purification tag (such as 6His) can be added to the C-terminus. The gene encoding the above fusion protein is inserted into a eukaryotic cell expression vector (such as pcDNA3.4), and expressed and purified in CHO cells. The resulting fusion protein is named preF-I53-50A. At the same time, a purification tag (such as 6His) can be added to the C-terminus of I53-50B, and the gene encoding the above protein is inserted into a prokaryotic cell expression vector (such as pET-30a(+)). The protein is expressed and purified in E. coli cells and named I53-50B. Then, under suitable reaction conditions, preF-I53-50A and I53-50B are covalently bound to form the immunogenic complex preF-I53-50, which are specifically named DS-Cav1-I53-50 and SC9-10-I53-50.

[0067] Preferably, any immunogenic complex provided by the present invention contains preF-I53-50A (the expression protein uses the signal peptide shown in SEQ ID NO:4, and the fusion protein containing preF as shown in SEQ ID NO:2 or 3 is obtained by linking it with I53-50A as shown in SEQ ID NO:6 via linker peptide 1), such as DS-Cav1-I53-50A (SEQ ID NO:13) or SC9-10-I53-50A (SEQ ID NO:14).

[0068] In some embodiments, the present invention provides an immunogenic complex having any one or more of the following features (1)-(5):

[0069] (1) The antigen component further includes a trimerization domain, which is fused to the C-terminus of preF;

[0070] (2) The binding peptide 1 is fused to the C-terminus of the trimerization domain;

[0071] (3) The amino acid sequence of the antigen component is shown in SEQ ID NO: 16 or SEQ ID NO: 15;

[0072] (4) The preF is expressed using the signal peptide shown in SEQ ID NO: 4 or SEQ ID NO: 5;

[0073] (5) The antigen component and / or the particulate protein component contains a purification tag, which is a histidine tag.

[0074] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following items (1)-(6):

[0075] (1) The amino acid sequence of the preF is shown in SEQ ID NO: 2 or SEQ ID NO: 3;

[0076] (2) The C end of the preF is connected to the foldon;

[0077] (3) The amino acid sequence of the binding peptide 1 is shown in SEQ ID NO:9;

[0078] (4) The amino acid sequence of the binding peptide 2 is shown in SEQ ID NO:10;

[0079] (5) The nanoparticle protein is selected from NPM, and the amino acid sequence of the NPM is shown in SEQ ID NO:11;

[0080] (6) The nanoparticle protein subunits are selected from I53-50A or I53-50B, the amino acid sequence of I53-50A is shown in SEQ ID NO: 6, and the amino acid sequence of I53-50B is shown in SEQ ID NO: 7.

[0081] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following items (1)-(4):

[0082] (1) The amino acid sequence of preF is shown in SEQ ID NO: 2 or 3;

[0083] (2) The amino acid sequence of linker peptide 1 is shown in the figure.

[0084] (3) The nanoparticle protein subunit is fused to the C-terminus of preF via linker peptide 1. The nanoparticle protein subunit is selected from I53-50A, and the amino acid sequence of I53-50A is shown in SEQ ID NO: 6.

[0085] (4) Another nanoparticle protein subunit is selected from I53-50B, the amino acid sequence of which is shown in SEQ ID NO: 7.

[0086] In some embodiments, the present invention provides an immunogenic complex comprising any one or more of the following items (1)-(8):

[0087] (1) The amino acid sequence of preF is shown in SEQ ID NO: 2 or SEQ ID NO: 3;

[0088] (2) The amino acid sequence of linker peptide 1 is shown in GSG;

[0089] (3) foldon is fused to the C-terminus of preF via linker peptide 1, and the amino acid sequence of foldon is shown in SEQ ID NO: 8;

[0090] (4) The amino acid sequence of binding peptide 1 is shown in SEQ ID NO: 9;

[0091] (5) Binding peptide 1 is fused to the C-terminus of foldon via GSGGSG;

[0092] (6) The nanoparticle protein is NPM, and its amino acid sequence is shown in SEQ ID NO:11;

[0093] (7) The amino acid sequence of linker peptide 2 is shown in GGSGGSGGSGGS;

[0094] (8) The amino acid sequence of binding peptide 2 is shown in SEQ ID NO:10.

[0095] In some embodiments, the present invention provides an immunogenic complex, preF expressed as a signal peptide with an amino acid sequence such as SEQ ID NO: 4 or SEQ ID NO: 5; the antigen component and / or the particulate protein component contains a purification tag, such as a histidine tag.

[0096] In some embodiments, the present invention provides an immunogenic complex composed of an antigenic component and a particulate protein component, wherein the amino acid sequence of the antigenic component is shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16, and the amino acid sequence of the particulate protein component is shown in SEQ ID NO:7 or SEQ ID NO:12.

[0097] In some embodiments, the present invention provides an immunogenic complex having a stable preF conformation or maintaining a stable preF conformation.

[0098] In a preferred embodiment, the immunogenic complex of the present invention induces higher titers of IgG and / or neutralizing antibodies compared to the naked antigen, wherein the naked antigen is preF that has not been shown by nanoparticle proteins. Furthermore, the present invention also provides a method for preparing any of the above-described immunogenic complexes, comprising the following steps:

[0099] (1) The antigen component and the particulate protein component encoding genes were respectively ligated into expression vectors to construct recombinant expression plasmids and expression host cells, express the target protein and purify it;

[0100] (2) The antigen component obtained in step (1) is co-incubated with the particulate protein component to obtain an immunogenic complex.

[0101] In some embodiments, the method for preparing the immunogenic complex of the present invention includes:

[0102] (1) a) preparing an expression vector encoding the antigen component of the present invention, introducing the expression vector into a host cell, culturing the host cell under conditions suitable for expressing the antigen component, and purifying the antigen component; and b) co-culturing the antigen component obtained in (1) a) with the particulate protein component of the present invention to obtain the immunogenic complex; optionally, the method further includes: preparing an expression vector encoding the particulate protein component, introducing the expression vector into a host cell, culturing the host cell under conditions suitable for expressing the particulate protein component, and purifying the particulate protein component;

[0103] Or (2) a) prepare an expression vector encoding the particulate protein component, introduce the expression vector into a host cell; culture the host cell under conditions suitable for expressing the particulate protein component; and purify the particulate protein component; and b) co-incubate the particulate protein component obtained in (2) a) with the antigen component of the present invention to obtain the immunogenic complex; optionally, the method further includes preparing an expression vector encoding the antigen component, introducing the expression vector into a host cell; culturing the host cell under conditions suitable for expressing the antigen component; and purifying the antigen component.

[0104] This invention provides a method for preparing an immunogenic complex for the prevention or treatment of RSV-related diseases:

[0105] (1) The encoding genes of the preF antigen component and the particulate protein component were respectively ligated into expression vectors to construct recombinant plasmids;

[0106] (2) Construct host cells capable of expressing the RSV antigen components and particulate protein components;

[0107] (3) The recombinant strain was used to express the fusion protein, and the fusion protein was purified;

[0108] (4) The above antigen components and particulate protein components are co-incubated to undergo a conjugation reaction and obtain an immunogenic complex.

[0109] Preferably, the immunogenic complex obtained in step (4) above is purified to obtain the vaccine stock solution.

[0110] Preferably, in the method for preparing an immunogenic complex for RSV-related diseases, the plasmid expressing the RSV antigen component in step (1) can be pcDNA3.4, and the plasmid expressing the particulate protein component can be pET-28a(+) or pET-30a(+).

[0111] In step (2) of the method for preparing an immunogenic complex for the prevention or treatment of RSV-related diseases according to the present invention, the host cell expressing the water RSV antigen is CHO, and the host cell expressing the particulate protein component carrier is E. coli.

[0112] In the immunogenic complex for the prevention or treatment of RSV-related diseases described in this invention, the binding ratio of preF-4T to 4C-NPM is 6:1, and the binding conditions are pH 7.4, 0.1M Tris-HCl, 25% (w / v) Sucrose, incubation at 4°C for 24 hours; the binding ratio of preF-I53-50A to I53-50B is 1:3, and the binding conditions are pH 7.4, 20mM Tris-HCl, 150mM NaCl, incubation at room temperature for 2 hours. Endotoxin levels are all less than 100 EU / ml, meeting the requirements for large-scale production.

[0113] Secondly, the present invention provides the encoding nucleic acids for the aforementioned RSV pre-fusion F protein and antigenic components. The specific nucleic acid sequences are readily obtainable by those skilled in the art using conventional methods such as codon tables. Preferably, the encoding nucleic acid sequences for preF and antigenic components are optimized according to the preferences of CHO host cells.

[0114] This invention also provides the nucleic acids encoding the aforementioned particulate protein components and nanoparticle proteins. The specific nucleotide sequences are readily obtainable by those skilled in the art using conventional methods such as codon tables. Preferably, the nucleic acid sequences encoding the particulate protein components and nanoparticle proteins are optimized according to the preferences of E. coli host cells.

[0115] Preferably, the nucleic acid sequence of preF-I53-50A provided by the present invention is shown in SEQ ID NO: 17 (DS-Cav1-I53-50A) or SEQ ID NO: 18 (SC9-10-I53-50A).

[0116] Preferably, the nucleic acid sequence of the preF-Tag provided by the present invention is shown in SEQ ID NO: 19 (DS-Cav1-Tag) or SEQ ID NO: 20 (SC9-10-Tag).

[0117] The present invention also provides vectors comprising the nucleotide sequences of the present invention, including cloning or expression vectors, and host cells transformed with said vectors.

[0118] In some embodiments, the vector used in this invention is selected from pcDNA3.4, pET-28a(+), and pET-30a(+).

[0119] In some embodiments, the host cell expressing the antigen component vector is CHO, and the host cell expressing the particulate protein vector is E. coli. Preferably, preF-Tag and preF-I53-50A are expressed in CHO host cells, and NPM and I53-50B are expressed in E. coli host cells.

[0120] The present invention also provides a kit comprising the immunogenic composition of the present invention, or a nucleic acid sequence, or a vector, or a host cell.

[0121] The present invention also provides any of the following products related to immunogenic complexes:

[0122] (1) A nucleic acid sequence encoding the antigenic component and / or particulate protein component described in this invention;

[0123] (2) A vector containing the nucleic acid sequence described in (1);

[0124] (3) A host cell containing the nucleic acid sequence of (1) or the vector of (2);

[0125] (4) A kit comprising the nucleic acid sequence of (1), or the vector of (2), or the host cell of (3).

[0126] The nucleic acid sequences of the antigen components include SEQ ID NO: 20 and SEQ ID NO: 19, the vector of the nucleic acid sequences is a plasmid, and the host cells of the vector are CHO and E. coli.

[0127] Thirdly, the present invention provides an immune composition comprising the immunogenic complex of the present invention and a pharmaceutically acceptable carrier.

[0128] The present invention also provides an immune composition comprising any of the above-described immunogenic complexes and a pharmaceutically acceptable carrier; preferably, the pharmaceutically acceptable carrier is selected from stabilizers, surfactants and buffers, wherein the stabilizer is sucrose, the surfactant is polysorbate 20, and the buffer is disodium hydrogen phosphate dihydrate and / or disodium hydrogen phosphate dihydrate.

[0129] In some embodiments, the pharmaceutically acceptable carrier of the present invention comprises stabilizers, surfactants, and buffers.

[0130] Preferably, the pharmaceutically acceptable carrier of the present invention is sucrose, polysorbate 20, disodium hydrogen phosphate dihydrate, and sodium dihydrogen phosphate dihydrate.

[0131] In some embodiments, the immunogenic composition of the present invention contains an immunogenic complex in an amount of 20-150 μg / dose, preferably 30-120 μg / dose, and more preferably 30 μg / dose, 40 μg / dose, 50 μg / dose, 60 μg / dose, 70 μg / dose, 80 μg / dose, 90 μg / dose, 100 μg / dose, 110 μg / dose, or 120 μg / dose.

[0132] In some embodiments, the immunogenic composition of the present invention contains an immunogenic complex at a dose of 30 μg / dose, 60 μg / dose, or 120 μg / dose.

[0133] Preferably, the present invention provides an immune composition containing an immunogenic complex of SC9-10-NPM.

[0134] In some embodiments, the immune composition provided by the present invention is an injection solution or a lyophilized preparation, preferably a lyophilized preparation.

[0135] In some embodiments, the immune composition provided by the present invention is a lyophilized formulation comprising an RSV preF-NPM or preF-I53-50 immunogenic complex, a stabilizer, a surfactant, and a buffer; preferably, the stabilizer is sucrose, the surfactant is polysorbate 20, and the buffer is disodium hydrogen phosphate dihydrate and disodium dihydrogen phosphate dihydrate.

[0136] In some embodiments, the immune composition provided by the present invention is a lyophilized formulation comprising an RSV preF-NPM or preF-I53-50 immunogenic complex, sucrose, polysorbate 20, disodium hydrogen phosphate dihydrate, and sodium dihydrogen phosphate dihydrate. Each unit dose of the lyophilized formulation comprises: 30 μg-120 μg of the immunogenic complex; 42.50-170 mg of sucrose; 0.075-0.30 mg of polysorbate 20; 0.75-3.0 mg of disodium hydrogen phosphate dihydrate; and 0.125-0.50 mg of sodium dihydrogen phosphate dihydrate.

[0137] In some embodiments, the immune composition provided by the present invention is a lyophilized preparation comprising 60 μg of RSV preF-NPM or preF-I53-50 immunogenic complex, 85.00 mg of sucrose, 0.15 mg of polysorbate 20, 1.50 mg of disodium hydrogen phosphate dihydrate and 0.25 mg of disodium hydrogen phosphate dihydrate.

[0138] Preferably, the immune composition provided by the present invention is a lyophilized preparation, the components of which are: SC9-10-NPM 60μg, sucrose 85.00 mg, polysorbate 20 0.15 mg, disodium hydrogen phosphate dihydrate 1.50 mg and sodium dihydrogen phosphate dihydrate 0.25 mg.

[0139] In some embodiments, the immune composition provided by the present invention is an injection solution comprising an RSV preF-NPM or preF-I53-50 immunogenic complex, a stabilizer, a surfactant, a buffer, and a pH adjuster; preferably, the stabilizer is sucrose, the surfactant is polysorbate 20, the buffer is disodium hydrogen phosphate dihydrate or sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0140] In some embodiments, the immunogenic composition provided by the present invention is an injection solution comprising an RSV preF-NPM or preF-I53-50 immunogenic complex, sucrose, polysorbate 20, disodium hydrogen phosphate dihydrate, and sodium dihydrogen phosphate dihydrate. Each unit dose of the injection solution comprises: 30 μg-120 μg of RSV preF-NPM or preF-I53-50 immunogenic complex; 42.50-170 mg of sucrose; 0.075-0.30 mg of polysorbate 20; 0.75-3.0 mg of disodium hydrogen phosphate dihydrate; and 0.125-0.50 mg of sodium dihydrogen phosphate dihydrate.

[0141] In some embodiments, the immune composition provided by the present invention is an injection solution containing 60 μg of RSV preF-NPM or preF-I53-50 immunogenic complex, 85.00 mg of sucrose, 0.15 mg of polysorbate 20, 1.50 mg of disodium hydrogen phosphate dihydrate and 0.25 mg of disodium hydrogen phosphate dihydrate.

[0142] Preferably, the immune composition provided by the present invention is an injection solution, the components of which are: SC9-10-NPM 60μg, sucrose 85.00 mg, polysorbate 20 0.15 mg, disodium hydrogen phosphate dihydrate 1.50 mg and sodium dihydrogen phosphate dihydrate 0.25 mg.

[0143] Fourthly, the present invention provides a vaccine comprising the immune composition and adjuvant described herein.

[0144] Preferably, the immunogenic complex in the vaccine of the present invention is SC9-10-NPM.

[0145] The present invention further provides a respiratory syncytial virus (RSV) vaccine comprising any of the above-described immune compositions and an adjuvant, wherein the adjuvant is selected from at least one of the following: aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvants, water-oil adjuvants, cytokines, CpG DNA, genetically engineered attenuated toxins, immunostimulatory complexes, and liposomes.

[0146] The RSV vaccine described in this invention uses a squalene-based adjuvant containing squalene. In mouse experiments, a good immunizing effect was achieved when the immunogenic complex was administered at a dose of 0.2 μg / dose in combination with the adjuvant.

[0147] In some embodiments, the adjuvant used in this invention is an aluminum adjuvant or MF59 or an MF59 bioanalyst.

[0148] In some embodiments, the adjuvant used in this invention is MF59 or an MF59 bioanalyst.

[0149] The respiratory syncytial virus vaccine of the present invention contains 30-120 μg of the immunogenic complex per unit dose of vaccine for human use, preferably 30 µg, 60 µg or 120 µg.

[0150] The squalene adjuvant of the present invention contains: squalene, Span 85, Tween 80 and 10 mM citrate buffer.

[0151] The squalene adjuvant of the present invention contains: (w / w) squalene 0.5%-5%, Span 85 0.05%-1%, Tween 80 0.05%-1%, and 10mM citrate buffer.

[0152] The squalene adjuvant of the present invention contains (w / w) 1.5%-5% squalene, 0.05%-1% Span 85, 0.05%-1% Tween 80, and 10mM citrate buffer.

[0153] The squalene adjuvant of the present invention preferably contains: (w / w) 2%-4.5% squalene, 0.2%-0.5% Span 85, 0.2%-0.5% Tween 80, and 10mM citrate buffer. More preferably, the amounts of squalene are 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, and 4.4% (w / w); more preferably, Span 85 is 0.3%-0.4% (w / w); and more preferably, Tween 80 is 0.3%-0.4% (w / w).

[0154] The squalene-based water-oil adjuvant used in specific embodiments of the present invention may be composed of (w / w) 3.9% squalene, 0.47% Span 85, 0.47% Tween 80, and 10mM citrate buffer.

[0155] The squalene-based water-oil adjuvant used in specific embodiments of the present invention may be composed of (w / w) 4.3% squalene, 0.5% Span 85, 0.5% Tween 80, and 10mM citrate buffer.

[0156] The squalene-based water-oil adjuvant used in specific embodiments of the present invention may be composed of (w / w) 4.03% squalene, 0.5% Span 85, 0.5% Tween 80, 0.016% citric acid, and 0.264% sodium citrate.

[0157] The squalene-based water-oil adjuvant used in specific embodiments of the present invention may be composed of (w / w) 3.0225% squalene, 0.375% Span 85, 0.375% Tween 80, 0.012% citric acid, and 0.198% sodium citrate.

[0158] The squalene-based water-oil adjuvant used in specific embodiments of the present invention may be composed of (w / w) 2.015% squalene, 0.25% Span 85, 0.25% Tween 80, 0.08% citric acid, and 0.132% sodium citrate.

[0159] The squalene-based water-oil adjuvant used in specific embodiments of the present invention may be composed of (w / w) squalene 0.403%, Span 85 0.05%, Tween 80 0.05%, citric acid 0.0016%, and sodium citrate 0.0264%.

[0160] The above (w / w) refers to the percentage by mass, that is, the percentage by which each component of the adjuvant is present in the total mass of the vaccine (antigen + adjuvant).

[0161] The present invention discloses a respiratory syncytial virus vaccine, wherein the vaccine comprises an immunizing composition of 30-120 μg / 0.5 ml and an adjuvant, wherein the adjuvant is an MF59 biosimilar and comprises, by weight percentage, 1.5%-5% squalene, 0.05%-1% Span 85, 0.05%-1% Tween 80 and 10 mM citrate buffer.

[0162] Furthermore, the preferred composition of the squalene adjuvant (adjuvant 1) used in the specific embodiments of the present invention is: 10.50 mg (4.2%) of squalene, 1.25 mg (0.5%) of Span 85, 1.25 mg (0.5%) of Tween 80, 0.04 mg (0.264%) of citric acid, and 0.66 mg (0.016%) of sodium citrate (w / w). This adjuvant can be used in conjunction with the SC9-10-NPM injection formulation.

[0163] The present invention discloses a respiratory syncytial virus vaccine comprising an immunizing composition of 60 μg / 0.5 ml and an adjuvant, wherein the adjuvant comprises 10.50 mg squalene, 1.25 mg Span 85, 1.25 mg Tween 80, 0.04 mg citric acid, and 0.66 mg sodium citrate.

[0164] In this invention, the control group used AS01E adjuvant. AS01E consists of: 25 μg of saponin QS-21, 25 μg of 3-O-deacyl-4'-monophosphatidyl ester A (MPL), 0.5 mg of dioleoylphosphatidylcholine (DOPC), and 0.125 mg of cholesterol per 0.5 mL. The AS01E adjuvant used in this invention is the commercially available Arexvy vaccine from GSK. ® The adjuvant product sold in combination with RSV PreF3 antigen.

[0165] The present invention further provides a complete kit, characterized in that it comprises a respiratory syncytial virus vaccine as described in the present invention, as well as instruments and containers required for administering the vaccine.

[0166] This invention provides a respiratory syncytial virus (RSV) vaccine comprising an SC9-10-NPM immune composition (i.e., an immune composition containing SC9-10-NPM, which can be formulated into a lyophilized or injectable preparation) and an adjuvant (in liquid form). The SC9-10-NPM immune composition and adjuvant are packaged separately in vials. The adjuvant contains 10.50 mg of squalene, 1.25 mg of Span 85, 1.25 mg of Tween 80, 0.04 mg of sodium citrate, and 0.66 mg of citric acid. When this adjuvant is used in conjunction with the SC9-10-NPM lyophilized preparation or the injectable preparation, the concentration of each component in the adjuvant vial is half that of the former (diluted by half). The SC9-10-NPM immune complex is available in three strengths: 30 μg / dose, 60 μg / dose, or 120 μg / dose.

[0167] For SC9-10-NPM immunization compositions in lyophilized formulation: Before clinical vaccination, all liquid should be drawn from the adjuvant bottle into the bottle containing the SC9-10-NPM lyophilized formulation, mixed well and used.

[0168] In a preferred embodiment, the RSV vaccine provided by the present invention comprises an SC9-10-NPM immunization composition.

[0169] This invention provides the use of a respiratory syncytial virus immunogenic complex, an immune composition, or a vaccine in the preparation of a medicament for the prevention or treatment of respiratory syncytial virus-related diseases.

[0170] This invention provides a method for generating an immune response in a subject, comprising administering to the subject an effective amount of the immunogenic complex, immunogenic composition, or vaccine described in this invention. Preferably, the method generates protective immunity against respiratory syncytial virus.

[0171] This invention provides a method for preventing respiratory syncytial virus (RSV) infection, comprising administering to a subject an effective amount of the immunogenic complex, immunogenic composition, or vaccine described in this invention. Preferably, the method generates protective immunity against RSV.

[0172] This invention provides a method for immunizing a subject against respiratory syncytial virus (RSV), comprising administering to the subject an effective amount of the immunogenic complex, immunogenic composition, or vaccine of the present invention. Preferably, the method generates protective immunity against RSV.

[0173] The immunogenic complexes, immune compositions, or vaccines described in this invention are used to generate neutralizing antibodies against respiratory syncytial virus.

[0174] All reagents used in this invention are commercially available.

[0175] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0176] (1) The present invention uses different nanoparticle proteins NPM and I53-50 to demonstrate the pre-fusion F protein of RSV. Both can obtain RSV nanoparticles with uniform particle size, uniform distribution without aggregation, stable product performance, qualified endotoxin, and suitable for non-clinical development and antibody immunogenicity testing.

[0177] (2) Compared with the naked antigen, both DS-Cav1 and SC9-10 displayed by NPM nanoparticles exhibited more stable pre-fusion conformations. Among them, SC9-10-NPM showed the highest affinity for the pre-fusion conformation-specific antibody D25 of RSV F protein and could also induce high levels of IgG and neutralizing antibodies. Furthermore, under the same dosage conditions, the neutralizing antibody responses induced by SC9-10-NPM in combination with adjuvant 1 against both RSV strains were significantly higher than those induced by the commercially available vaccine Arevxy. The neutralizing antibody titers induced by SC9-10-NPM / Alhydrogel were similar to those of the control vaccine. Notably, even at a dose of only 1 / 5 of the control vaccine dose (i.e., 0.2 μg SC9-10-NPM vs 1 μg Arevxy), the neutralizing antibody responses induced by SC9-10-NPM / adjuvant 1 were still significantly higher than those induced by the control vaccine. These results confirm that SC9-10-NPM is a promising RSV vaccine candidate.

[0178] (3) For safety reasons, the vaccine of the present invention avoids the use of the potent adjuvant AS01E used in Arexvy, but the vaccine of the present invention can maintain similar or higher efficacy. In the cotton rat model, the SC9-10-NPM of the present invention can reduce the viral load in the experimental group regardless of whether the adjuvant is added.

[0179] (4) All four types of nanoparticles of the present invention have good thermal stability and have been stored at 4℃, 25℃ and 37℃ for up to four weeks, or at -80℃ for up to four months. Among them, preF-NPM has more outstanding overall stability.

[0180] (5) In this invention, the particulate protein component is prepared by E. coli fermentation and chromatography purification, and the preF antigen is prepared by CHO cell reactor culture and chromatography purification. Both are suitable for large-scale industrial production and have advantages such as high expression levels, stable process and yield, and simple operation. The yield of one batch of recombinant particulate protein component can be used to bind multiple batches of preF antigen, thereby improving production efficiency. Compared with ordinary recombinant protein vaccines, the nanoparticle vaccine of this invention has the advantage of higher immune protection at the same or lower doses, which can save the cost of large-scale production.

[0181] (6) The preparation method of recombinant particulate protein components provided by the present invention does not require special equipment, is easy to scale up, is suitable for industrial production, has a short production time, and the process is simple and stable, which can reduce the cost of large-scale industrial production; the protein products prepared by the recombinant particulate protein component method provided by the present invention effectively reduce the side effects caused by the residues of impurities, host proteins, exogenous DNA, antibiotics, bacterial endotoxins and other substances in the particles, and improve safety. Attached Figure Description

[0182] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or examples will be briefly introduced below.

[0183] Figure 1 SDS-PAGE identification results of RSV preF-Tag and RSV preF-NPM. Lane 1 is DS-Cav1-Tag, lane 2 is DS-Cav1-NPM, lane 3 is SC9-10-Tag, lane 4 is SC9-10-NPM, and lane 5 is unbound NPM (Naked NPM).

[0184] Figure 2 The results of SDS-PAGE identification of RSV preF-I53-50A and RSV preF-I53-50 are as follows: Lane 1 is DS-Cav1-I53-50A, Lane 2 is DS-Cav1-I53-50, Lane 3 is SC9-10-I53-50A, Lane 4 is SC9-10-I53-50, and Lane 5 is I53-50B.

[0185] Figure 3 The results of non-reducing SDS-PAGE identification of RSV preF-Tag and RSV preF-NPM are as follows: Lane 1 is DS-Cav1-Tag, Lane 2 is DS-Cav1-NPM, Lane 3 is SC9-10-Tag, Lane 4 is SC9-10-NPM, and Lane 5 is Naked NPM that has not bound to preF.

[0186] Figure 4 The results of non-reducing SDS-PAGE identification of RSV preF-I53-50A and RSV preF-I53-50 are as follows: Lane 1 is DS-Cav1-I53-50A, Lane 2 is DS-Cav1-I53-50, Lane 3 is SC9-10-I53-50A, Lane 4 is SC9-10-I53-50, and Lane 5 is I53-50B.

[0187] Figure 5 DLS detection results of RSV preF-NPM.

[0188] Figure 6 DLS detection results of RSV preF-I53-50.

[0189] Figure 7 Electron microscopy results of DS-Cav1-NPM.

[0190] Figure 8 Electron microscopy results of SC9-10-NPM.

[0191] Figure 9 Electron microscopy results of DS-Cav1-I53-50.

[0192] Figure 10 Electron microscopy results of SC9-10-I53-50.

[0193] Figure 11 : Enzyme-linked immunosorbent assay (ELISA) curves of RSV preF-Tag, preF-NPM, preF-I53-50 binding to pre-fusion specific antibody D25.

[0194] Figure 12 : Enzyme-linked immunosorbent assay (ELISA) curves of RSV preF-Tag, preF-NPM, preF-I53-50 and the fusion-specific antibody 4D7.

[0195] Figure 13 Stability test results of four types of nanoparticles: DS-Cav1-NPM, DS-Cav1-I53-50, SC9-10-NPM, and SC9-10-I53-50.

[0196] Figure 14 The results of ELISA detection of total IgG titer against antigen preF in mice after immunization with four nanoparticle vaccines: DS-Cav1-I53-50, DS-Cav1-NPM, SC9-10-I53-50, and SC9-10-NPM.

[0197] Figure 15 The serum of mice immunized with four nanoparticle vaccines—DS-Cav1-I53-50, DS-Cav1-NPM, SC9-10-I53-50, and SC9-10-NPM—was analyzed using an ELISA that competes with the D25 antibody to detect the neutralizing antibody titer.

[0198] Figure 16 The titers of neutralizing antibodies against RSV A2 strain were detected in the serum of mice immunized with four nanoparticle vaccines: DS-Cav1-I53-50, DS-Cav1-NPM, SC9-10-I53-50, and SC9-10-NPM, using live virus neutralization analysis.

[0199] Figure 17The results of live virus neutralization analysis of serum from mice immunized with SC-9-10-NPM vaccines with different adjuvants and without adjuvants were used to detect the neutralizing antibody titers against RSV strains. The strain in Figure A is RSV A2 strain, and the strain in Figure B is B18537 strain.

[0200] Figure 18 The neutralizing antibody titer in serum collected from cotton rats on day 42 after immunization was detected by a micro-neutralization assay. The strain in Figure A is RSV A2, and the strain in Figure B is B18537.

[0201] Figure 19 Viral load data from the nasal turbinate bones, tracheal tissue, and lung tissue of cotton rats. Figure A shows the nasal turbinate bones, Figure B shows the tracheal tissue, and Figure C shows the lung tissue. Detailed Implementation

[0202] The principles and features of the present invention are described below with reference to examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. Before further describing specific embodiments of the invention, it should be understood that the scope of protection of the invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the invention is for describing specific embodiments and not for limiting the scope of protection of the invention. Test methods in the following embodiments without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be used. Unless otherwise defined, all technical and scientific terms used in the invention have the same meaning as commonly understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, based on the knowledge of the prior art by those skilled in the art and the description of the invention, any prior art methods, equipment, and materials similar to or equivalent to those described, used, and materials in the embodiments of the invention can be used to implement the invention. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional reagent companies.

[0203] Example 1: Recombinant expression and purification of RSV preF-Tag

[0204] 1. Construction of a recombinant RSV preF-Tag cell line:

[0205] The amino acid sequences of RSV pre-F antigens DS-Cav1 and SC9-10 were obtained according to patent CN109069611B. Foldon and Tag (4T) sequences were introduced at the C-terminus. A fusion gene was obtained using conventional molecular cloning methods. The sequence of the fusion gene was codon-optimized for CHO cells. The encoding nucleic acid sequence of the recombinant protein DS-Cav1-Tag is SEQ ID NO: 19, and the encoding nucleic acid sequence of SC9-10-Tag is SEQ ID NO: 20. The signal peptide used was MELLILKANAITTILTAVTFCFASG (SEQ ID NO: 4) or MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 5). Following conventional molecular cloning techniques, the fusion gene was cloned into the vector pcDNA3.4 to construct a stably transfected Chinese hamster ovary (CHO) cell pool. Positive cell pools were screened, and cell expansion culture was completed, with a cell viability exceeding 95%.

[0206] 2. Expression and purification of recombinant proteins:

[0207] The selected CHO cell lines secreted and expressed the fusion proteins DS-Cav1-Tag and SC9-10-Tag. After expression for a certain period, the cell supernatant was harvested. High-purity fusion proteins were purified from the clarified supernatant using a series of orthogonal chromatography methods with the AKTA system (Cytiva). The supernatant was then replaced with buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) using a tangential flow filtration (TFF) system (Cobetter) equipped with a Pellicon 2 Biomax 10 kDa MWCO membrane (Merck-Millipore). The target proteins were collected for SDS-PAGE electrophoresis. Total protein was quantified using the Pierce BCA (Thermo Fisher Scientific) protein assay kit via the BCA assay method, following the kit's instructions.

[0208] 3. Experimental Results:

[0209] (1) Figure 1 To reconstruct the identification results of SDS-PAGE, lane 1 is the band of DS-Cav1-Tag, and lane 3 is the band of SC9-10-Tag.

[0210] (2) Figure 3 The results are from non-reducing SDS-PAGE, where lane 1 is the band of DS-Cav1-Tag and lane 3 is the band of SC9-10-Tag.

[0211] Example 2: Preparation and purification of protein nanoparticles (NPMs)

[0212] 1. Construction of a recombinant 4C-NPM cell line:

[0213] According to conventional molecular cloning methods, the nanoparticle protein NPM is linked to binding peptide 2 at the N-terminus via linker peptide 2, thereby forming a binding peptide 2-NPM fusion protein, namely 4C-NPM (as shown in SEQ ID NO: 12).

[0214] The encoding gene of 4C-NPM was expressed in *E. coli*. After harvesting the cells, the target protein was released by high-pressure homogenization and the liquid was clarified, primarily to remove cell debris and impurity proteins. The clarification was mainly achieved through heat treatment. A two-step heating method was used, applying both a first and second heating step to the supernatant after *E. coli* disruption (i.e., "two-step heating"). The impurity removal effect of the two-step heating step and the purity of the recombinant particle protein components were calculated.

[0215] 2. Specific purification procedures:

[0216] 60g of E. coli wet cells collected by centrifugation were resuspended in 240ml of buffer (20mM Tris-HCl, 2mM MPMSF, pH 9.0), and homogenized using a high-pressure homogenizer at 1000 bar. After centrifugation, 280ml of supernatant was collected, and 40ml of this supernatant was subjected to a two-step heating process. SDS-PAGE analysis was performed on the homogenized supernatant, the supernatant from the first heating and centrifugation step, and the resuspended precipitate from the second heating and centrifugation step.

[0217] As shown in Table 1, in the first heating step, the pH was adjusted to 9.0, and the solution was heated in a water bath at 80°C for 1 hour. After returning to room temperature, approximately 35 ml of supernatant was collected by centrifugation. In the second heating step, 35 ml of a buffer solution containing 100 mM Tris-HCl, 5 mM EDTA, 4% Triton, and pH 7.4 was added, followed by 7 ml of a 1 M Tris-HCl solution at pH 7.4. The solution was then mixed. The solution was heated in a 60°C water bath for 10 min, and the precipitate was immediately collected by centrifugation. The precipitate was then reconstituted using a buffer solution containing 20 mM Tris-HCl, 5 mM EDTA, and pH 9.0.

[0218] Table 1. Two-step heating extraction method for recombinant particulate protein components.

[0219] Adding different concentrations of urea and sodium chloride after the two-step heating process and before chromatographic purification can significantly reduce impurities other than the target recombinant particulate protein. The preferred process conditions for pretreatment of recombinant particulate protein component samples before Fractogel DEAE M chromatography are soaking in 8M urea and 50-200mM sodium chloride.

[0220] The recombinant particle protein fraction sample solution was purified using ion exchange and hydrophobic chromatography. The first step of the chromatographic purification used Fractogel DEAE M chromatography; specific steps and parameters are shown in Table 2. The Fractogel DEAE M elution sample was first diluted with buffer, and then 50% (w / v) sucrose stabilizer was added; specific parameters are shown in Table 3. Then, the sample was purified using Octyl Bestarose 4FF hydrophobic chromatography (the second step of chromatographic purification); specific steps and parameters are shown in Table 4.

[0221] Step 1 chromatography: Chromatography packing material - Fractogel DEAE M, retention time - 12.5 min.

[0222] Table 2 First-step chromatography method

[0223]

[0224] Table 3. Sample dilution method before the second step of chromatography.

[0225]

[0226] Second step chromatography: chromatography packing material - Octyl Bestarose 4FF, retention time - 12.5 min.

[0227] Table 4. Second-step chromatography method

[0228]

[0229] Results and Analysis: Purity testing revealed that after further purification using the above combination of chromatographic media, the purity of the product could reach over 99.0%.

[0230] Example 3: Preparation and characterization of RSV preF-NPM

[0231] I. Combining RSV preF-Tag and NPM:

[0232] 1. Preparation of RSV preF-NPM binding products:

[0233] The preF-Tag (DS-Cav1-Tag and SC9-10-Tag) were mixed with the nanoparticle protein NPM prepared in Example 2 at a BCA protein concentration ratio of 6:1 and incubated at 4°C for 24 hours.

[0234] 2. Purification of RSV preF-NPM binding products:

[0235] Uncoupled preF-Tags were separated by gel filtration chromatography (SEC) using a HiLoad 16 / 600 Superdex 200 pg column on an AKTA (Cytiva) system. The pre-equilibration buffer was 20 mM Tris-HCl, 25% sucrose (w / v), pH 7.4. Following separation, the coupled nanoparticles (DS-Cav1-NPM, SC9-10-NPM) were analyzed by SDS-PAGE.

[0236] II. Particle Characterization:

[0237] 1. Experimental materials: RSV preF-NPM particles prepared in this embodiment.

[0238] 2. Detection method:

[0239] (1) Dynamic light scattering (DLS)

[0240] DLS testing was performed using a Zetasizer Lab instrument manufactured by Malvern, equipped with a 633 nm helium-neon laser. Particle size measurements were conducted at a 90° angle, and data were acquired and analyzed using Malvern's ZS XPLORER software. Approximately 1 mL of a 0.25 mg / mL sample was used for each measurement, placed in a disposable polystyrene cuvette (model DTS0012) provided by Malvern, and performed at a controlled temperature of 25 °C. The viscosity and refractive index parameters used in the measurements were adjusted for the 25 °C condition. Each sample was tested three times.

[0241] (2) Negative staining electron microscope (TEM)

[0242] 3–4 µL of preF-NPM sample at a concentration of 100 ng / µL was added to a glow discharge-treated carbon film copper grid for adsorption for 1 minute. The grid was then rinsed with Milli-Q ultrapure water and blotted dry with filter paper. Negative staining was performed using 0.75% uranium formate for 1 minute. Electron micrographs of preF-NPM were taken using a Thermo Fisher Scientific FEI Tecnai 12 transmission electron microscope equipped with an Orius SC200 CCD camera and operating at 120 kV.

[0243] III. Experimental Results:

[0244] (1) Figure 1 To reconstruct the results of SDS-PAGE, lane 2 is the band of DS-Cav1-NPM, lane 4 is the band of SC9-10-NPM, and lane 5 is the band of 4C-NPM.

[0245] (2) Figure 3 The results are from non-reducing SDS-PAGE, where lane 2 is the band of DS-Cav1-NPM, lane 4 is the band of SC9-10-NPM, and lane 5 is the band of 4C-NPM.

[0246] (3) Figure 5 The results are shown in the DLS analysis of preF-NPM. The particle diameter of DS-Cav1-NPM is 45.27 nm, the particle diameter of SC9-10-NPM is 45.64 nm, and the particle diameter of uncoupled NPM (Naked NPM) is 26.91 nm.

[0247] (4) Figures 7-8 The electron microscopy negative staining results of DS-Cav1-NPM and SC9-10-NPM show that all samples formed relatively uniform nanoparticles.

[0248] The above results indicate that the RSV preF and NPM provided by this invention assemble normally and have a reasonable molecular weight range.

[0249] Example 4: Expression, purification, combination, and particle characterization of preF-I53-50A and I53-50B.

[0250] The combination of preF and I53-50 is achieved by expressing and purifying the preF-I53-50A fusion protein and I53-50B separately, and then combining the two to obtain preF-I53-50.

[0251] I. Expression and purification of I53-50B

[0252] 1. Induced expression: The gene encoding I53-50B was expressed in Escherichia coli.

[0253] 2. Centrifugation for bacterial collection: After the bacterial culture is completed, centrifuge to collect the bacterial cells, discard the culture medium, resuspend the bacterial cells in 10 mL of 300 mM NaCl, 50 mM Tris 7.4, 1 mM DTT, and 0.75% CHAPS solution, and vortex thoroughly in a shaker.

[0254] 3. Ultrasonic disruption: Place the centrifuge tube containing the resuspended bacterial solution on ice for ultrasonic disruption.

[0255] 4. Centrifuge to collect the target protein: 15000 rpm, 4 ℃, 30 min, collect the cell lysate supernatant. The sequence of the target protein I53-50B is shown in SEQ ID NO: 7.

[0256] 5. Histrap purification of the target protein: Wash buffer consisted of 300 mM NaCl, 50 mM Tris pH 7.4, 1 mM DTT, 0.75% CHAPS, and 30 mM Imidazole; Elution buffer consisted of 300 mM NaCl, 50 mM Tris pH 7.4, 1 mM DTT, 0.75% CHAPS, and 300 mM Imidazole. Purification was performed using a Histrap Excel-5 mL or Histrap Bogelong-10 mL column. The column was equilibrated with 5 CV of wash buffer. The target protein was filtered through a 0.22 μm filter and diluted to 45 mL with wash buffer. Injection was performed using S1. Impurities were washed away with 10 CV of wash buffer, and the target protein was eluted with 5 CV of Elution buffer.

[0257] 6. Replacement buffer: Replace the target protein solution eluted by Histrap with a 300 mM NaCl, 50 mM Tris (pH 7.4), and 0.75% CHAPS solution using a concentration tube. Then measure the protein concentration and store at an appropriate temperature for subsequent binding reactions.

[0258] II. Expression, purification, and binding reaction of RSV preF-I53-50A target protein

[0259] 1. The encoding genes of preF-I53-50A, SEQ ID NO: 17 (DS-Cav1-I53-50A) and SEQ ID NO: 18 (SC9-10-I53-50A), were inserted into eukaryotic cells expressing pcDNA3.4 to construct a stable transfected Chinese hamster ovary (CHO) cell pool. Positive cell pools were screened out, and cell expansion culture was completed. The cell viability was higher than 95%.

[0260] 2. Centrifuge to collect cell expression supernatant, then filter through a filter membrane.

[0261] 3. High-purity fusion protein was purified using a series of orthogonal chromatography methods based on the AKTA system (Cytiva).

[0262] 4. The molecular sieve was used for further purification. The elution buffer was 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. The purified preF-I53-50A was identified by SDS-PAGE, and the protein concentration of preF-I53-50A was determined. The protein was then stored at -80℃. The amino acid sequence of the target protein DS-Cav1-I53-50A is shown in SEQ ID NO: 13, and the amino acid sequence of SC9-10-I53-50A is shown in SEQ ID NO: 14.

[0263] 5. preF-I53-50A and I53-50B were mixed at a mass ratio of 1:3 and a binding experiment was conducted. The binding conditions were pH 7.4, 20mM Tris-HCl, 150mM NaCl, 25℃, and the reaction was carried out at room temperature for 2 hours to obtain the bound products DS-Cav1-I53-50 and SC9-10-I53-50.

[0264] 6. The product after molecular sieve separation and purification is identified by SDS-PAGE and the particle size is detected by DLS.

[0265] III. Particle Characterization:

[0266] 1. Experimental materials: preF-I53-50 particles prepared in this example

[0267] 2. Detection method: Same as in Example 3.

[0268] IV. Results:

[0269] (1) Figure 2To reconstruct the SDS-PAGE results, lane 1 is the band of DS-Cav1-I53-50A, lane 2 is the band of DS-Cav1-I53-50, lane 3 is the band of SC9-10-I53-50A, lane 4 is the band of SC9-10-I53-50, and lane 5 is the band of I53-50B.

[0270] (2) Figure 4 The results are from non-reducing SDS-PAGE, where lane 1 is the band for DS-Cav1-I53-50A, lane 2 is the band for DS-Cav1-I53-50, lane 3 is the band for SC9-10-I53-50A, lane 4 is the band for SC9-10-I53-50, and lane 5 is the band for I53-50B. (3) Figure 6 The results are as follows: DLS detection results for preF-I53-50. The particle diameter of DS-Cav1-I53-50 is 38.69 nm, the particle diameter of SC9-10-I53-50 is 51.15 nm, and the particle diameter of I53-50 (NakedI53-50) not coupled with preF is 33.73 nm.

[0271] (4) Figures 9-10 The electron microscopy negative staining results of DS-Cav1-I53-50 and SC9-10-I53-50 show that all samples formed relatively uniform nanoparticles.

[0272] The above results indicate that the RSV preF and I53-50 provided by this invention are assembled normally, and the molecular weight range is reasonable.

[0273] Example 5: Detection of the immunological properties of the pre-fusion conformation of preF-NPM and preF-I53-50

[0274] Prior art knows that D25 and 4D7 are specific antibodies that specifically bind to the prefusion and postfusion conformations of RSV F (see Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody, McLellan JS et al., Science 340:1113-7, 2013; Stability Characterization of a Vaccine Antigen Based on the Respiratory Syncytial Virus Fusion Glycoprotein, Flynn JA et al., PLoS One 11:e0164789, 2016). Antibodies D25 and 4D7 were expressed and purified using conventional molecular cloning methods. The binding affinity of the preF and preF nanoparticles of this invention to the two antibodies was detected using enzyme-linked immunosorbent assay (ELISA).

[0275] Experimental results showed that both DS-Cav1 and SC9-10 had a strong affinity for the pre-fusion specific antibody D25. Figure 11 This indicates that the mutations introduced in DS-Cav1 and SC9-10 successfully stabilized their structural conformations in the pre-fusion state. SC9-10 has a stronger affinity for D25 than DS-Cav1, but a weaker interaction with the post-fusion specific antibody 4D7. Figure 12 Following protein display on nanoparticles, both DS-Cav1 and SC9-10 exhibited effective binding to D25, indicating that the pre-fusion conformation of DS-Cav1 and SC9-10 on the nanoparticles was fully preserved. Notably, the binding affinity of the D25 antibody to DS-Cav1-NPM or SC9-10-NPM was higher than that to DS-Cav1 / SC9-10 or DS-Cav1 / SC9-10-I53-50, suggesting that the NPM platform can further enhance the stability of the pre-F antigen. Overall, multivalent display of RSV pre-F on nanoparticles effectively improves its antigenicity. Among them, SC9-10-NPM showed the highest affinity for D25 and is a very promising RSV vaccine candidate.

[0276] Example 6: Stability of preF-NPM and preF-I53-50 proteins

[0277] I. Experimental Methods:

[0278] 1. Experimental Instruments: Uncle All-in-One Protein Stability Analyzer

[0279] 2. Operation method: The stability of the DS-Cav1-NPM, DS-Cav1-I53-50, SC9-10-NPM, and SC9-10-I53-50 nanoparticle vaccine candidates obtained in the above examples was monitored for a long time under different temperature conditions.

[0280] II. Experimental Results

[0281] DS-Cav1 / Sc9-10 combined NPM and I53-50 nanoparticles were stored at 4°C, 25°C, and 37°C for up to four weeks, or at -80°C for up to four months. SDS-PAGE analysis showed that all four types of nanoparticles exhibited good stability, showing no signs of degradation after four weeks at 4°C or six months at -80°C. However, from the third day of storage at 25°C or 37°C, the DS-Cav1 and SC9-10 combined I53-50 nanoparticles began to show irreversible degradation. Therefore, preF-NPM showed better overall stability than preF-I53-50. Figure 13 ).

[0282] Example 7: Mouse Immunization Experiment

[0283] I. Experimental Materials

[0284] 1. Laboratory animals: 6- to 8-week-old female BALB / c mice purchased from Charles River Company were housed in a specific pathogen-free (SPF) environment.

[0285] 2. Test sample and reference sample

[0286] ① Test vaccine stock solution: The test vaccine protein stock solution was prepared by Guangzhou Pano Biotechnology Co., Ltd.: including DS-Cav1-NPM and SC9-10-NPM prepared in Example 3, and DS-Cav1-I53-50 and SC9-10-I53-50 prepared in Example 4.

[0287] ② The adjuvant of the tested vaccine was adjuvant 1 (MF59-bio): squalene 10.50 mg (4.2%), Span 85 1.25 mg (0.5%), Tween 80 1.25 mg (0.5%), citric acid 0.04 mg (0.264%), sodium citrate 0.66 mg (0.016%) (w / w).

[0288] The preparation steps are as follows: Span 85 is dispersed in the squalene oil phase, and Tween 80 is dispersed in the aqueous phase. The mixtures are then high-speed mixed using a smart laboratory homogenizer (FLUKO brand) to form a crude emulsion. The crude emulsion is then processed using an ATS-AH high-pressure pilot-scale homogenizer (ATS Engineering Limited) to obtain a fine emulsion. Finally, after sterilization and filtration, adjuvant 1 with a particle size of approximately 145-165 nm is obtained.

[0289] ③ Vaccine control: GSK's marketed vaccine Arexvy®, with AS01E as the adjuvant (per 0.5 ml: 25 μg QS-21, 25 μg 3D-MPL, 0.5 mg DOPC, 0.125 mg cholesterol).

[0290] II. Experimental Methods

[0291] 1. Immunogenicity test

[0292] Immunization was administered via intramuscular injection (im) using a primary immunization-boost immunization regimen (primary immunization on day 0, booster immunization on day 14). The antigen molecules were thoroughly mixed with PBS, Alhydrogel®, or adjuvant 1 before injection. Blood samples were collected on day 28. Whole blood was incubated overnight at 4°C to induce coagulation, followed by centrifugation at 16,000×g for 10 minutes at room temperature to separate the serum. The serum was transferred to new Eppendorf tubes and frozen for subsequent analysis. Table 5 shows the immunization protocols for four nanoparticle antigen immunogenicity assays, and Table 6 shows the immunization protocols for live virus neutralization assays using SC9-10-NPM with different adjuvants.

[0293] Table 5. Immunization regimens for comparative immunogenicity studies of different nanoparticle vaccines

[0294]

[0295] Table 6. Immunization regimens for immunogenicity, dosage, and adjuvant studies of SC9-10-NPM in mice.

[0296]

[0297] 2. IgG endpoint titer ELISA detection

[0298] The endpoint titer of total anti-preF IgG in the serum of immunized animals was determined using an indirect ELISA method. 96-well NuncMaxiSorp microplates (Thermo Fisher Scientific) were coated with preF antigen (100 ng / 50 μL / well) and incubated overnight at 4°C. The plates were then washed twice with PBS containing 0.05% Tween 20 (PBST), and blocked with 200 μL / well of PBS-diluted casein blocking buffer (Thermo Fisher Scientific) at room temperature for 1 hour, followed by two more washes with PBST. Proportionally diluted serum samples were added to each well and incubated at room temperature for 1 hour. The plates were then washed four times with PBST, and HRP-labeled goat anti-mouse IgG antibody (Abcam) diluted 1:5000 was added and incubated at room temperature for 1 hour. The plates were then washed six times with PBST and developed with tetramethylbenzidine (TMB, Thermo Fisher Scientific, USA) at room temperature for 10 minutes. The reaction was terminated by adding 1 N HCl, and the absorbance was recorded. Data were read at wavelengths of 450 nm–620 nm. The endpoint titer was defined as the dilution factor corresponding to the intersection of the X-axis of the dilution curve and the absorbance value exceeding the optical density (OD) of negative mouse serum plus two standard deviations or 0.15 (whichever is higher).

[0299] 3. Competitive ELISA detection

[0300] The level of specific antibodies against preF in serum was detected using a competitive ELISA. The simplified procedure is as follows: A 96-well ELISA plate was coated with D25 antibody (200 ng / 50 μL / well) and incubated overnight at 4°C. Immunized mouse serum was serially diluted 3-fold and mixed with 0.1 μg / mL pre-F antigen at a 1:1 volume ratio. 100 μL of this mixture was then added to the D25-coated plate and incubated at room temperature for 1 hour. HRP-labeled Anti-6 × His tag® antibody (Abcam) was then added, and the plate was incubated at room temperature for another 1 hour. Finally, TMB substrate was added for color development. The titer of neutralizing antibodies in serum was determined using the same method.

[0301] 4. Live virus neutralization test

[0302] The levels of neutralizing antibodies against RSV A2 and B18537 strains in mouse serum were detected using Hep-2 cells. The simplified procedure was as follows: 10,000 Hep-2 cells per well were seeded in a 96-well plate and incubated overnight at 37°C with 5% CO2. On the day of the experiment, 12.5 μL of immunized mouse serum was added to 87.5 μL of DMEM, serially diluted 4-fold, and then mixed with 100 TCID45. 50Mix 75 μL of RSV A2 or B18537 strain and incubate at 37°C for 2 hours. During virus-serum incubation, wash the 96-well cell culture plate three times with PBS, then transfer the incubation mixture to the cell plate and incubate again at 37°C for 2 hours. Continue culturing for 5 to 7 days after inoculation, and observe the cytopathic effect (CPE) using an inverted microscope. Serum neutralizing antibody titer (VNT) 50 It is defined as the reciprocal of the serum dilution factor that can provide 50% inhibition of viral growth.

[0303] 5. Statistical Analysis

[0304] The results were analyzed using Graphpad Prism 9.1.2 software. Differences were analyzed using the unpaired t-test or one-way ANOVA. Two groups were defined as statistically significant when P < 0.05.

[0305] III. Results Analysis:

[0306] 1. Figure 14 The results of ELISA assays for IgG endpoint titers showed that all four nanoparticle formulations induced strong humoral immune responses. Notably, mice immunized with DS-Cav1 / SC9-10-NPM nanoparticles exhibited a higher binding antibody response compared to mice immunized with DS-Cav1 / SC9-10-I53-50 nanoparticles.

[0307] 2. Figure 15 The results of the competitive ELISA assay showed that mice immunized with DS-Cav1 / Sc-9-10-NPM nanoparticles produced significantly higher titers of D25 competitive neutralizing antibodies than those DS-Cav1 / SC-9-10-I53-50.

[0308] 3. Figure 16 For live virus neutralization assay results, the results were similar to those of competitive ELISA. Animals immunized with DS-Cav1 / Sc9-10 conjugated with NPM showed higher nAb titers, and the nAb response induced by SC9-10 nanoparticles was generally higher than that DS-Cav1 nanoparticles.

[0309] 4. Figure 17Figure AB presents the results of live virus neutralization assays with SC-9-10-NPM formulated with different adjuvants. The results showed that the nAb response induced by adjuvanted SC9-10-NPM was significantly higher than that of the control group without adjuvant, with adjuvant 1 (MF59-bio) inducing the highest nAb titer. At the same doses (1 μg and 5 μg), the nAb responses induced by SC9-10-NPM / MF59-bio against both viral strains were significantly higher than those induced by the commercially available vaccine Arevxy (except for the 5 μg group against strain B). The nAb titers induced by SC9-10-NPM / Alhydrogel were similar to those of the control vaccine, while the immunogenicity of unadjuvanted SC9-10-NPM was lower than that of the control vaccine. Notably, even at only 1 / 5 the dose of the control vaccine (i.e., 0.2 μg SC9-10-NPM vs 1 μg Arevxy), the nAb response induced by SC9-10-NPM / MF59-bio was still significantly higher than that of the control vaccine. These results confirm that SC9-10-NPM is a promising RSV vaccine candidate.

[0310] Example 8: Cotton Mouse Invasive Experiment

[0311] I. Experimental Methods

[0312] SPF-grade male cotton rats were administered candidate vaccines or control formulations via intramuscular injection on days 0 and 21, including 60 μg of SC-9-10-NPM (with or without adjuvant) and 60 μg of the control vaccine Arexvy. A PBS control group and a healthy animal control group were also established. Serum samples were collected on day 42 to detect neutralizing antibody titers against RSV A2 and B18537 strains. On day 42, the cotton rats were administered 1×10⁻⁶ ozonally. 6 The rats were challenged with PFU containing RSV A2 strain. Animals were sacrificed on day 46, and the lung tissue was divided into two parts: the right lung was rapidly frozen in HBSS solution at a volume of 10 times its weight; the left lung was perfused with 0.8 mL of 4% paraformaldehyde and fixed in 4% paraformaldehyde. In addition, nasal turbinate bone and tracheal tissue were collected, rapidly frozen in HBSS solution at a volume of 10 times its weight, and stored at –80°C for subsequent analysis. Table 7 shows the immunization and challenge protocol for the rats.

[0313] Table 7 Immunization and Challenge Protocols for Cotton Rats

[0314]

[0315] II. Determination of serum neutralizing antibody titers using a micro-neutralization assay

[0316] Serum samples were heat-inactivated at 56°C for 30 minutes. First, the serum was diluted to a starting concentration of 1:20 with DMEM, followed by a series of 3-fold serial dilutions, for a total of 8 dilution gradients. Simultaneously, a separate 96-well microplate was prepared for virus back titration validation, with 100 μL of serially diluted 2-fold virus solution added to each well. Finally, 50 μL of diluted serum and 50 μL of diluted virus (200 TCID50) were added to each well of the assay plate. 50 The cells were incubated at 37°C in a 5% CO2 incubator for 2 hours. After incubation, Hep-2 cells were seeded into detection and re-titering plates at a density of 25,000 cells per well. A cell control group (uninfected with the virus) and a virus control group (infected with the virus but without other treatment) were included in the experiment. All cells were cultured at 37°C in a 5% CO2 incubator for 5 days. Subsequently, the cells were fixed with 80% acetone at 4°C for 15 minutes and then air-dried. Primary and secondary antibodies were added to each well sequentially, and the cells were incubated at 37°C for 1 hour. Premixed TMB chromogenic solution was then added, and the reaction was terminated with 1% hydrochloric acid. The absorbance was read at 450 nm using a microplate reader. The serum neutralizing antibody titer (NT) was... 50 The calculations and data analysis were performed using GraphPad Prism software, and the formulas used are as follows:

[0317] %Antibody activity = 100 − [OD(sample − cell control) / OD(virus control − cell control) × 100]

[0318] Curve fitting was analyzed using a logarithmic (inhibitor)-response model with varying slopes.

[0319] III. Detection of RSV titer in tissue

[0320] Mouse lung, nasal turbinate, and tracheal tissues were homogenized for virus detection. RSV titers in the tissues were detected using a plaque assay. HEp-2 cells were seeded in 12-well cell culture plates and cultured overnight at 37°C with 5% CO2 to form a monolayer. The tissue homogenate was centrifuged, and the supernatant was collected and serially diluted 8-fold (50 μL supernatant + 350 μL DMEM). The diluted mixture was added to the cell culture plates and incubated at 37°C for 4 hours to promote virus adsorption. After incubation, the cells were fixed with paraformaldehyde, followed by the addition of primary and secondary antibodies and appropriate incubation. A mixture of chromogenic solutions A and B was prepared and added to each well, incubated for 15 minutes in the dark. After chromogenic development, ddH2O was added to terminate the reaction, and the wells were rinsed with water and then dried at 37°C. Finally, the plate surface was scanned and the number of plaques was recorded. The viral titer in the sample was calculated and expressed as a logarithmic value of plaque-forming units (PFU) per gram of tissue homogenate.

[0321] IV. Experimental Results

[0322] 1. Figure 18 AB represents the detection of neutralizing antibody titers in serum collected from cotton rats on day 42 post-immunization using a micro-neutralization assay. The results showed that, compared with the control group, Sc-9-10-NPM with adjuvant 1 induced higher neutralizing antibody titers against both viral strains.

[0323] 2. Figure 19 AC data represent viral load in the lung, nasal turbinate, and tracheal tissues of mice. Results showed that, compared to the PBS control group, viral load in the nasal turbinate, trachea, and lungs was significantly reduced in all vaccine groups. SC9-10-NPM reduced viral load in the groups, whether or not adjuvant was added.

[0324] In summary, the above embodiments and accompanying drawings are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An immunogenic complex for the prevention or treatment of respiratory syncytial virus-related diseases, characterized in that, Include: (1) Antigen component, which contains the RSV pre-fusion F protein preF; (2) Particulate protein component, which includes nanoparticle protein mi3.

2. The immunogenic complex according to claim 1, characterized in that, The amino acid sequence of the preF has at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% identity with SEQ ID NO:

1.

3. The immunogenic complex according to claim 1, characterized in that, The amino acid sequence of the preF is shown in SEQ ID NO:3 or SEQ ID NO:

2.

4. The immunogenic complex according to claim 1, characterized in that, The antigen component and the particulate protein component are covalently bound by a peptide binding pair, wherein the antigen component contains binding peptide 1 and the particulate protein component contains binding peptide 2, and the antigen component and the particulate protein component are covalently bound by binding peptide 1 and binding peptide 2.

5. The immunogenic complex according to claim 4, characterized in that, Binding peptide 1 contains the amino acid sequence shown in SEQ ID NO: 9, and binding peptide 2 contains the amino acid sequence shown in SEQ ID NO:

10.

6. The immunogenic complex according to claim 4, characterized in that, It has one or more of the following characteristics (1)-(5): (1) The antigen component further includes a trimerizing domain; (2) The binding peptide 1 is fused to the C-terminus of the trimerization domain; (3) The amino acid sequence of the antigen component is shown in SEQ ID NO: 16 or SEQ ID NO: 15; (4) The preF is expressed using the signal peptide shown in SEQ ID NO: 4 or SEQ ID NO: 5; (5) The antigen component and / or the particulate protein component contains a purification label.

7. The immunogenic complex according to claim 6, characterized in that, The antigen component contains a trimerizing domain fused to the C-terminus of preF, and the purification tag is a histidine tag.

8. The method for preparing the immunogenic complex according to any one of claims 1-7, characterized in that, include: (1) a) Prepare an expression vector encoding the antigen component, introduce the expression vector into a host cell, culture the host cell under conditions suitable for expressing the antigen component, and purify the antigen component; and b) co-culture the antigen component obtained in (1) a) with a particulate protein component according to any one of claims 1-7 to obtain the immunogenic complex. or (2) a) Prepare an expression vector encoding the particulate protein component, introduce the expression vector into a host cell, culture the host cell under conditions suitable for expressing the particulate protein component, and purify the particulate protein component; and b) co-incubate the particulate protein component obtained in (2) a) with the antigen component of any one of claims 1-7 to obtain the immunogenic complex.

9. The method for preparing the immunogenic complex according to claim 8, characterized in that, The expression vector is a plasmid.

10. The method for preparing the immunogenic complex according to claim 8, characterized in that, The method (1) of claim 8 further includes preparing an expression vector encoding the particulate protein component, introducing the expression vector into a host cell, culturing the host cell under conditions suitable for expressing the particulate protein component, and purifying the particulate protein component.

11. The method for preparing the immunogenic complex according to claim 8, characterized in that, The method (2) of claim 8 further includes preparing an expression vector encoding the antigen component, introducing the expression vector into a host cell, culturing the host cell under conditions suitable for expressing the antigen component, and purifying the antigen component.

12. An immune composition, characterized in that, The invention comprises the immunogenic complex according to any one of claims 1-7, and further comprises a pharmaceutically acceptable carrier.

13. The immune composition according to claim 12, characterized in that, The pharmaceutically acceptable carriers include stabilizers, surfactants, and buffers.

14. The immune composition according to claim 13, characterized in that, The stabilizer is sucrose, the surfactant is polysorbate 20, and the buffer is disodium hydrogen phosphate dihydrate and / or sodium dihydrogen phosphate dihydrate.

15. The immune composition according to claim 12, characterized in that, The immune composition is a lyophilized preparation, and each unit dose of the lyophilized preparation contains: 30 μg-120 μg of immunogenic complex, 42.50-170 mg of sucrose, 0.075-0.30 mg of polysorbate 20, 0.75-3.0 mg of disodium hydrogen phosphate dihydrate, and 0.125-0.50 mg of disodium hydrogen phosphate dihydrate.

16. The immune composition according to claim 15, characterized in that, Each unit dose of the lyophilized formulation contains: 60 μg of immunogenic complex, 85.00 mg of sucrose, 0.15 mg of polysorbate 20, 1.50 mg of disodium hydrogen phosphate dihydrate, and 0.25 mg of disodium hydrogen phosphate dihydrate.

17. A respiratory syncytial virus vaccine, characterized in that, It comprises the immune composition and adjuvant as described in claim 12, 15 or 16.

18. The vaccine according to claim 17, characterized in that, The adjuvant is selected from aluminum adjuvants, MF59, or MF59 biosimilars.

19. The vaccine according to claim 17, characterized in that, The vaccine comprises an immunizing composition of 30-120 μg / 0.5 ml and an adjuvant, wherein the adjuvant is an MF59 biosimilar comprising squalene, Span 85, Tween 80 and 10 mM citrate buffer.

20. The vaccine according to claim 17, characterized in that, The vaccine comprises an immunizing composition of 60 μg / 0.5 ml and an adjuvant, wherein the adjuvant comprises 10.50 mg squalene, 1.25 mg Span 85, 1.25 mg Tween 80, 0.04 mg citric acid, and 0.66 mg sodium citrate.

21. The vaccine according to claim 19, characterized in that, The vaccine comprises an immunizing composition of 60 μg / 0.5 ml and an adjuvant, wherein the adjuvant comprises 10.50 mg squalene, 1.25 mg Span 85, 1.25 mg Tween 80, 0.04 mg citric acid, and 0.66 mg sodium citrate.

22. The immunogenic complex according to any one of claims 1-7, or the immune composition according to claim 12, 15 or 16, or the vaccine according to claim 17 or 19, for the prevention, resistance or treatment of diseases caused by respiratory syncytial virus.

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