Rabies virus g protein mutant, method for preparing same, and use thereof

Abstract

Provided are a rabies virus G protein mutant, a preparation method and application thereof, and relate to the technical field of biological medicine. The rabies virus G protein mutant is obtained by point mutation on the extracellular domain of the G protein. The mutant realizes soluble expression of the extracellular domain of the G protein, and can induce production of high-titer neutralizing antibodies. Compared with commercial human rabies vaccine and canine rabies vaccine, the mutant has more excellent cellular immune and antibody immune responses, and is expected to reduce the number of inoculations, shorten the immunization program and reduce the immunization dose.

Description

Rabies virus G protein mutant and its preparation method and application Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a rabies virus G protein mutant and a preparation method and application thereof. Background Art

[0002] Rabies is a zoonotic disease caused by the rabies virus (RABV), an acute infectious disease that primarily invades the central nervous system and has a mortality rate of almost 100%. Rabies virus belongs to the genus Lyssavirus, family Rhabdoviridae. The virus particle is bullet-shaped, 100-300 nm long and approximately 75 nm in diameter. Its genome encodes five structural proteins: the RNA-dependent RNA polymerase (L protein), the nucleoprotein (N protein), the phosphoprotein (P protein), the matrix protein (M protein), and the surface glycoprotein (G protein). The G protein is embedded in the outer lipid membrane of the rabies virus particle. The G protein trimer forms the spike, which is the site of viral binding to host receptors and contains the primary recognition epitope for neutralizing antibodies. The G protein can also be glycosylated, and glycosylation plays a crucial role in its stability, antigenicity, and biological activity during its expression and secretion. Therefore, the G protein is an important target protein in the development of rabies vaccines, antiviral drugs, and antibody diagnostic reagents.

[0003] The gene coding region of the G protein is 1575 nucleotides long and encodes 524 amino acids, of which amino acids 1-19 are signal peptides. After the signal peptide is removed, a mature protein of 505 amino acids is formed, which includes three domains: the extracellular domain, the transmembrane domain, and the intracellular domain. Among them, the extracellular domain is the G protein segment exposed on the surface of the virus particle. It is the functional segment that performs receptor recognition and membrane fusion. It is also the truly effective antigen segment in vaccine preparation and the target of neutralizing antibodies. Studies have shown that most of the eukaryotic expression studies of rabies virus G protein are full-length proteins. Due to the high hydrophobicity of the transmembrane region, the G protein obtained is very easy to aggregate in the solution to form highly heterogeneous protein polymers, making it difficult to effectively purify. Literature has reported that only the extracellular domain of the G protein of the wild-type CVS-11 strain will form irregular aggregates. The extracellular segment of the rabies virus G protein (RABV-G-ecto) was recombinantly modified, and the region where amino acids 73 to 79 in the first fusion loop of the protein and amino acids 117 to 125 in the second fusion loop were replaced with a connecting peptide composed of 5 amino acids (GGSGG), respectively. The result was a soluble recombinant RABV-G-ecto protein with uniform properties in solution (see "Structural Analysis of Rabies Virus Glycoprotein Reveals pH-Dependent Conformational Changes and Interactions with a Neutralizing Antibody", Yang et al., Cell Host & Microbe 27, 441-453, 2020.3.11; CN109627294B). Another paper reported that while purified RABV G protein expressed solely from the extracellular domain of the rabies virus G gene (Genbank accession number: KT221113.1) produced high titers of anti-G protein antibodies, it failed to induce neutralizing antibodies in mice, indicating that the transmembrane region of the rabies virus G protein is crucial for the conformation and biological activity of the extracellular domain (see "Construction and Characterization of a Stable Secretory Cell Line Expressing the RABV G Extracellular Domain," Li Qingqing, Southern Medical University, CNKI, March 1, 2018). This suggests that obtaining a soluble G protein extracellular domain with the correct spatial folding and the ability to induce neutralizing antibodies is challenging, and that using the G protein extracellular domain in the preparation of rabies vaccines still presents many technical challenges that need to be overcome.

[0004] At the same time, studies have shown that the conformation of G protein changes during receptor binding and membrane fusion, switching between pre- and post-fusion forms with host cells. Under acidic conditions, G protein is in a post-fusion state, while under neutral and alkaline conditions, it is in a pre-fusion state. It is generally believed that the pre-fusion state of G protein can more effectively induce the production of neutralizing antibodies. Furthermore, G protein can also change from a trimer structure to a monomeric structure. Therefore, this characteristic of G protein is a difficulty and a key point in the design and later processing of protein vaccines.

[0005] At present, rabies vaccines are mainly purified vaccines cultured from primary hamster kidney cells, chicken embryo cells, human diploid cells and Vero cells. Among them, human diploid cell vaccines have a low incidence of adverse reactions, mild symptoms and good immune effects after vaccination, but the cell proliferation is slow, the virus production is low, and the vaccine cost is high, so it cannot be widely used. In addition to traditional rabies vaccines, new vaccines are also being rapidly developed, such as recombinant protein-based vaccines or mRNA vaccines. In addition, since the rabies vaccines currently used for humans are based on a 4-injection or 5-injection immunization program, which is long and has a low compliance rate among vaccine recipients, it also imposes a heavy economic burden on poor people. In April 2022, India launched the world's first new rabies vaccine, which is also the only recombinant protein vaccine. Its immunization program is three injections after exposure, which further shortens the immunization program and reduces the immunization dose.

[0006] There is currently no recombinant rabies protein vaccine on the market in China, so it is of great significance to develop a rabies vaccine with more effective, safer and long-lasting immunity.

[0007] Summary of the Invention

[0008] In response to the above technical problems, the present invention transforms the extracellular domain of rabies virus G protein as the target protein to obtain a G protein antigen with stable conformation and strong immunogenicity.

[0009] A. Viral membrane fusion protein

[0010] The rabies virus G protein is a type of viral membrane fusion protein. A key step in enveloped virus infection is the fusion between the viral membrane and the host cell membrane, which is mediated by viral membrane fusion proteins. Among them, viral membrane fusion proteins are divided into class I (such as the membrane fusion proteins of human immunodeficiency virus and respiratory syncytial virus), class II (such as the membrane fusion proteins of dengue virus) and class III (such as the membrane fusion proteins of rhabdoviruses and herpes viruses). Viral membrane fusion proteins are conformationally metastable proteins that, after being triggered by certain factors that come into contact with the host cell, transform from a higher-energy pre-fusion conformation to a lower-energy post-fusion conformation through an unstable intermediate state. The intermediate state exposes a hydrophobic fusion peptide or fusion loop that is inserted into the host cell membrane, and the transition to the low-energy post-fusion conformation pulls the two membranes together, promoting the fusion of the viral envelope and the host cell membrane. Because epitopes that stimulate neutralizing antibody production are often contained in the prefusion conformation and, in some cases, are absent in the postfusion form, immunization with membrane fusion proteins in the prefusion conformation is clinically desirable because it has the potential to induce more potent neutralizing antibodies than immunization with proteins in the postfusion conformation.

[0011] Rabies virus G protein belongs to Class III viral membrane fusion proteins. Specifically, the structure of rabies virus G protein (RABV-G for short) exists in two structural states, the pre-fusion conformation of the curved hairpin structure on the surface of the virus and the post-fusion conformation of the extended conformation that mediates membrane fusion. Since the natural structure of RABV-G is a pre-fusion conformation under alkaline conditions and a post-fusion conformation under acidic conditions, its structure is unstable. At the same time, the pre-fusion conformation will gradually transform to the post-fusion conformation as the storage time changes, so that its antigenic sites are reduced, thereby leading to a decrease in immunogenicity. It can be seen that as the main antigen of rabies virus, the conformation of G protein can affect its immunogenicity. Therefore, stabilizing the pre-fusion conformation of G protein can effectively improve its immunogenicity and enhance the immune protection effect.

[0012] B. Wild-type RABV-G

[0013] The present invention provides a wild-type RABV-G, the full-length amino acid sequence of which is derived from the G protein of the Pasteur Virus strain (ie, the PV strain) (Genbank accession number is AAA47218.1).

[0014] The present invention provides a wild-type RABV-G extracellular domain, the amino acid sequence of which is derived from the 20th to 459th amino acids of the G protein of the PV strain (Genbank accession number is AAA47218.1).

[0015] In some embodiments, the wild-type RABV-G of the present invention is derived from a strain other than PV strain.

[0016] Alternatively, the wild-type RABV-G of the present invention is derived from the G protein of the following strains: Pitman Moore (GenBank accession number CAI43218.1), HEP-Flury (GenBank accession number BAC53868.1), MOR1-DG (GenBank accession number AAK92057.1), MOR3-HM (GenBank accession number AAK92058.1), NeiMeng1025C (GenBank accession number ABY19509.2), FRA1-FX (GenBank accession number AAK92050.1), CNX8601 (GenBank accession number AAG347 22.1), CHI1-BK (GenBank accession number AAK92060.1), MAU1-CL (GenBank accession number AAK92072.1), Yunnan_Zt07 (GenBank accession number ABX79939.1), RVD (GenBank accession number AAO72525.1), nukovo-32 (GenBank accession number OP642459), CVS-11 (GenBank sequence number ADJ29911.1), or CTN (GenBank accession number AY009100) strains.

[0017] In some embodiments, the present invention provides a wild-type RABV-G extracellular domain, whose amino acid sequence has a sequence corresponding to or aligned with amino acids 20-459 of the G protein of the above-mentioned PV strain.

[0018] In some embodiments, the amino acid sequence of the wild-type RABV-G extracellular domain of the present invention comprises the amino acid sequence shown in SEQ ID NO: 1.

[0019] Preferably, the amino acid sequence of the wild-type RABV-G extracellular domain of the present invention is shown in SEQ ID NO: 1.

[0020] Here, "corresponding" or "aligned" has the meaning commonly understood by those skilled in the art. Specifically, "corresponding" or "aligned" means that after homology or sequence identity comparison between a reference sequence and a given sequence, identical residues are found between the two sequences, and the positions of these identical residues in the given sequence are said to correspond or align with those in the reference sequence. For example, one skilled in the art can easily align the G proteins of other strains with the G protein of the PV strain using common programs such as Align to determine the positions of amino acids 20-459 of the PV strain G protein in the G protein of the other strain.

[0021] In some embodiments, the present invention provides a wild-type RABV-G extracellular domain, whose amino acid sequence is at least 90% identical to SEQ ID NO: 1.

[0022] Preferably, the present invention provides a wild-type RABV-G extracellular domain, whose amino acid sequence is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1.

[0023] C. RABV-G mutants

[0024] The present invention provides a RABV-G mutant, which comprises a RABV-G extracellular domain mutant.

[0025] In some embodiments, the present invention provides RABV-G mutants with increased stability of the prefusion conformation.

[0026] Preferably, the RABV-G mutant of the present invention is specifically a RABV-G extracellular domain mutant, and the RABV-G extracellular domain mutant has improved stability of the pre-fusion conformation.

[0027] As a non-limiting example, the stability of the prefusion conformation of a RABV-G mutant can be determined by incubating the RABV-G mutant with an antibody specific for the prefusion conformation at neutral and acidic pH and measuring antibody binding by ELISA or FACS.

[0028] The present invention provides a RABV-G extracellular domain mutant, wherein the RABV-G extracellular domain mutant has one, two or more amino acid mutations or substitutions at positions 261, 266, 270, 271, 384 and 424 of the wild-type RABV-G extracellular domain, or has one, two or more amino acid mutations or substitutions at positions corresponding to or aligned with the above amino acid positions, wherein the mutations or substitutions can improve the stability of the pre-fusion conformation of the G protein.

[0029] In some embodiments, the present invention provides a RABV-G extracellular domain mutant, wherein the RABV-G extracellular domain mutant has two amino acid mutations or substitutions at positions 261, 266, 270, 271, 384, and 424 of the wild-type RABV-G extracellular domain, or has two amino acid mutations or substitutions at positions corresponding to or aligned with the above-mentioned amino acid positions, wherein the mutations or substitutions can improve the stability of the prefusion conformation of the G protein.

[0030] Preferably, the mutation or substitution of the two amino acids is selected from the following combinations: H261+H270; D266+H270.

[0031] In some embodiments, the present invention provides a RABV-G extracellular domain mutant, wherein the RABV-G extracellular domain mutant has one, two or more amino acid mutations or substitutions at positions 261, 266, 270, 271, 384 and 424 of SEQ ID NO: 1, wherein the mutations or substitutions can improve the stability of the prefusion conformation of the G protein.

[0032] In some embodiments, the present invention provides a RABV-G extracellular domain mutant, wherein the RABV-G extracellular domain mutant has two amino acid mutations or substitutions at positions 261, 266, 270, 271, 384, and 424 of SEQ ID NO: 1, and the mutations or substitutions can improve the stability of the prefusion conformation of the G protein.

[0033] Preferably, the two mutations or substitutions in the above SEQ ID NO: 1 are selected from the following combinations: H261+H270; D266+H270.

[0034] Furthermore, the residue at the aforementioned site to be mutated or substituted is mutated to proline or substituted by proline.

[0035] In some embodiments, the residue at the aforementioned site to be mutated or substituted is mutated to leucine or substituted with leucine.

[0036] The present invention provides a RABV-G extracellular domain mutant, wherein the RABV-G extracellular domain mutant has one, two or more amino acid mutations or substitutions selected from H261L, D266P, H270P, L271P, H384P and H424L in SEQ ID NO: 1, and the mutations or substitutions can improve the stability of the pre-fusion conformation of the G protein.

[0037] In some embodiments, the RABV-G extracellular domain mutant has two amino acid mutations or substitutions selected from H261L, D266P, H270P, L271P, H384P and H424L in SEQ ID NO: 1, and the mutations or substitutions can improve the stability of the prefusion conformation of the G protein.

[0038] Preferably, the two mutations or substitutions in the above SEQ ID NO: 1 are selected from the following combinations: H261L+H270P; D266P+H270P.

[0039] The present invention provides a RABV-G extracellular domain mutant, wherein the RABV-G extracellular domain mutant has an amino acid sequence as shown in any one of SEQ ID NOs: 2-9, as shown in Table 1.

[0040] In some embodiments, the amino acid sequence of the RABV-G extracellular domain mutant of the present invention is shown in any one of SEQ ID NOs: 2-9.

[0041] Preferably, the amino acid sequence of the RABV-G extracellular domain mutant of the present invention is shown in any one of SEQ ID NOs: 2, 4, 5, 6, 7, 8, and 9.

[0042] Preferably, the amino acid sequence of the RABV-G extracellular domain mutant of the present invention is shown in any one of SEQ ID NOs: 4, 5, 8, and 9.

[0043] The RABV-G mutant of the present invention further includes glycosylation modification, and the RABV-G mutant can be N-glycosylated at the Asn37, Asn247 or Asn319 site of the extracellular domain.

[0044] In some embodiments, the RABV-G mutant of the present invention is modified by N-glycosylation at Asn319 of the extracellular domain, or further modified by N-glycosylation at Asn37 or Asn247.

[0045] In some embodiments, the RABV-G mutants described herein are capable of inducing the production of neutralizing antibodies.

[0046] The neutralizing antibodies described in the present invention refer to antibodies that can eliminate the virus's ability to infect after binding to the virus. After the surface antigens of the enveloped virus bind to the neutralizing antibodies, the complement can be activated, leading to the dissolution of the virus.

[0047] Preferably, the RABV-G mutant of the present invention can induce the production of IgG.

[0048] Preferably, the RABV-G mutant of the present invention can induce the production of neutralizing antibodies IgG.

[0049] Preferably, the neutralizing antibody titer induced by the RABV-G mutant of the present invention is higher than that of the commercially available rabies vaccine.

[0050] In some embodiments, the RABV-G mutants described herein can achieve soluble expression and have correct spatial folding.

[0051] In some embodiments, a signal peptide is introduced into one end of the RABV-G mutant of the present invention for extracellular secretory expression of the protein.

[0052] Preferably, a signal peptide is introduced at the N-terminus of the RABV-G mutant of the present invention, and the amino acid sequence of the signal peptide is shown as MEFGLSWVFLVAIIKGVQC (SEQ ID NO: 18).

[0053] In some embodiments, a 6×His tag is introduced into one end of the RABV-G mutant of the present invention for purification of recombinantly expressed proteins.

[0054] Preferably, a 6×His tag is introduced into the C-terminus of the above-mentioned RABV-G mutant.

[0055] Optionally, the RABV-G mutant of the present invention is connected to a 6×His tag via a linker.

[0056] Optionally, the linker is selected from (GGGGS) n 、(GSGGSG) n 、(GGS) n wherein n is an integer greater than 0 and less than or equal to 5. In some embodiments, the linker is preferably GSGGSG.

[0057] Table 1: Amino acid sequences of wild-type and mutant RABV-G extracellular domains of the present invention

[0058] D. RABV-G trimers and multimers

[0059] The present invention provides a RABV-G trimer, wherein the trimer is composed of the RABV-G mutant of the present invention.

[0060] In some embodiments, the RABV-G trimer described herein is composed of the RABV-G extracellular domain mutant described herein.

[0061] Preferably, the RABV-G trimer of the present invention is formed by self-assembly of the RABV-G mutants described in the present invention.

[0062] The present invention provides a method for preparing the RABV-G trimer as follows: a RABV-G mutant is recombinantly expressed using a prokaryotic expression system or a eukaryotic expression system, and the recombinant RABV-G mutant self-assembles to obtain a RABV-G trimer.

[0063] In some embodiments, the present invention provides a method for preparing the RABV-G trimer as follows: recombinantly expressing a RABV-G ectodomain mutant using a prokaryotic expression system or a eukaryotic expression system, and the recombinant RABV-G ectodomain mutant self-assembles to obtain a RABV-G trimer.

[0064] The present invention provides a RABV-G multimer, which is composed of the RABV-G trimer described in the present invention.

[0065] In some embodiments, the RABV-G multimers of the present invention include RABV-G hexamers, nonamers, and dodecamers, among others.

[0066] The present invention provides a method for preparing a RABV-G trimer or multimer, the method comprising the following steps:

[0067] (1) recombinantly expressing the RABV-G mutant of the present invention in CHO cells using conventional molecular cloning techniques;

[0068] (2) After expressing in the expression medium for a certain period of time, the cell supernatant is harvested, centrifuged, and the supernatant is filtered through a membrane;

[0069] (3) purifying the filtered supernatant by passing it through a nickel column;

[0070] (4) collecting a sample containing the target protein, concentrating it by ultrafiltration, and then performing a first molecular sieve purification; optionally, purifying the product of the first molecular sieve purification by an anion exchange column, and then performing a second molecular sieve purification;

[0071] (5) The purified target protein is diluted with buffer to obtain soluble trimers or polymers with uniform particle size.

[0072] In some embodiments, the buffer used in the first molecular sieve purification in the above step (4) is TBS, and the buffer used in the second molecular sieve purification contains a certain concentration of Tween.

[0073] In some embodiments, the buffer in step (5) above is TBS or a buffer containing Tween.

[0074] Furthermore, the content of Tween in steps (4)-(5) is 0.02%-1%.

[0075] Preferably, the content of Tween in steps (4)-(5) is 0.02%, 0.05% or 0.1%, and Tween is selected from Tween-20 or Tween-80, preferably Tween-80.

[0076] E. Immunogenic complex

[0077] The present invention provides an immunogenic complex, which comprises a protein formed by a RABV-G antigen component and a granule protein component through a covalent binding reaction.

[0078] The RABV-G antigen component of the present invention is selected from the RABV-G mutant, RABV-G trimer or RABV-G multimer of the present invention.

[0079] In some embodiments, the immunogenic complex of the present invention is a virus-like particle, which is a structure assembled from viral proteins and does not contain any nucleic acid.

[0080] The present invention provides an immunogenic complex comprising:

[0081] (1) an antigen component comprising the RABV-G antigen component of the present invention;

[0082] (2) A granular protein component comprising nanoparticle protein.

[0083] The present invention provides an immunogenic complex comprising:

[0084] (1) an antigen component comprising the RABV-G antigen component and binding peptide 1 described in the present invention;

[0085] (2) A particle protein component comprising nanoparticle protein and binding peptide 2.

[0086] The present invention provides an immunogenic complex comprising:

[0087] (1) an antigen component comprising the RABV-G antigen component of the present invention, a connecting peptide 1 and a binding peptide 1;

[0088] (2) A particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2.

[0089] In some embodiments, in any of the immunogenic complexes provided herein, the antigenic component is formed by fusing a RABV-G antigenic component at the C-terminus with a binding peptide 1 via a linker peptide 1. The RABV-G mutant and the binding peptide 1 form a fusion protein that assembles into a RABV-G trimer or multimer. Alternatively, the RABV-G mutant is fused at the C-terminus with a binding peptide 1 via a linker peptide 1 and then assembles into a RABV-G trimer or multimer.

[0090] In some embodiments, in any immunogenic complex provided by the present invention, the particle protein component is formed by fusing the N-terminus of the nanoparticle protein to the binding peptide 2 via the connecting peptide 2.

[0091] In some embodiments, the antigen component and the granule protein component are covalently bound to each other via binding peptide 1 and binding peptide 2 to form an immunogenic complex.

[0092] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the binding peptide 1 comprises an amino acid sequence as shown in AHIVMVDAYKPTK (SEQ ID NO: 19), hereinafter referred to as "4T".

[0093] In some embodiments, in any one of the immunogenic complexes provided herein, the binding peptide 2 comprises an amino acid sequence as shown in DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 20), hereinafter referred to as "4C".

[0094] In some embodiments, both the antigen component and the granule protein component comprise a histidine tag.

[0095] In some embodiments, in any immunogenic complex provided by the present invention, the particle protein can be selected from nanoparticle protein, and further can be selected from virus-like particle protein; the antigen component and the particle protein component can both self-assemble to form a particle structure by binding.

[0096] In some embodiments, the self-assembling nanoparticles used in the present invention include: NPM particles, ferritin particles, virus-like particles formed by viral structural proteins, I53-50 particles, etc. Among them, the viral structural proteins include bacteriophage capsid protein AP205, etc.

[0097] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 1 comprises (GGGGS) n 、(EAAAK) n 、(GSGGSG) n 、(GGS) n wherein n is 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 connecting peptide 1 is preferably GSGGSG.

[0098] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 2 comprises (GGS) n 、(GGGGS) n 、(EAAAK) n 、(GSGGSG) n wherein n can be an integer greater than 0 and less than or equal to 10. In some embodiments, in any one of the immunogenic complexes provided by the present invention, the connecting peptide 2 is preferably GGSGGSGGS or GGSGGSGGSGGS.

[0099] In some embodiments, in any of the immunogenic complexes described above, the particle protein component is a fusion protein formed by linking peptide 2 at the N-terminus of the nanoparticle protein with binding peptide 2. Preferably, the nanoparticle protein is NPM, AP205 capsid protein 3 (AP205), or Ferritin. Specifically, in some alternative approaches, binding peptide 2 (i.e., "4C") is linked to the gene encoding the nanoparticle protein via linker peptide 2, inserted into a prokaryotic expression vector (e.g., pET-28a(+) or pET-30a(+)), and expressed in E. coli cells to obtain a fusion protein of binding peptide 2 and the nanoparticle protein. The fusion protein can be purified by chromatography, such as anion exchange chromatography or hydrophobic chromatography, to obtain a product. The nanoparticle protein is preferably NPM, AP205, or Ferritin.

[0100] Preferably, in the immunogenic complex provided by the present invention, the amino acid sequence of the antigen component is shown in any one of SEQ ID NOs: 2-9, and the amino acid sequence of the granule protein component is shown in SEQ ID NO: 22.

[0101] Specifically, in some optional schemes, under appropriate reaction conditions, such as at room temperature, any of the above-mentioned antigen components is subjected to a covalent binding reaction with the granule protein component, and the binding peptide 1 of the antigen component and the binding peptide 2 of the granule protein component are coupled by covalent bonds, thereby forming the immunogenic complex.

[0102] In some embodiments, the present invention provides a method for covalently binding an antigen component to a granular protein component. The antigen component and granular protein component are mixed at a protein concentration ratio of 6:1 as determined by the BCA assay. A 50% sucrose stock solution is added to a final sucrose concentration of approximately 25%. A 1M Tris-HCl (pH 7.4) stock solution is added at 10% of the total reaction volume to stabilize the pH. The binding reaction is carried out at 22°C for 24 hours. The bound product is purified using molecular sieves.

[0103] In some embodiments, the present invention provides an immunogenic complex comprising:

[0104] (1) an antigen component comprising the RABV-G antigen component of the present invention, a connecting peptide 1 and a binding peptide 1;

[0105] (2) a particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2;

[0106] The connecting peptide 1 is any connecting peptide commonly used in the art (such as a flexible connecting peptide, a rigid connecting peptide), including but not limited to (GGS) n 、(GSGGSG)n 、(GGGGS) n or (EAAAK) n The connecting peptide 1 is preferably GSGGSG.

[0107] The connecting peptide 2 is any connecting peptide commonly used in the art (such as a flexible connecting peptide, a rigid connecting peptide), including but not limited to (GGS) n 、(GGGGS) n 、(EAAAK) n 、(GSGGSG) n wherein n can be an integer greater than 0 and less than or equal to 10, preferably GGSGGSGGS or GGSGGSGGSGGS; the nanoparticle protein is NPM, AP205 or ferritin.

[0108] In some embodiments, in any one of the immunogenic complexes provided herein, the nanoparticle protein NPM comprises the amino acid sequence shown in SEQ ID NO:21.

[0109] Preferably, in any immunogenic complex provided by the present invention, the particle protein component comprises NPM-4C, as shown in SEQ ID NO: 22, and NPM-4C comprises a fusion protein obtained by connecting binding peptide 2 to the nanoparticle protein NPM as shown in SEQ ID NO: 21 via connecting peptide 2.

[0110] Furthermore, the present invention provides a method for preparing an immunogenic complex:

[0111] (1) The RABV-G antigen component and granule protein component encoding genes were respectively connected into expression vectors to construct recombinant expression plasmids;

[0112] (2) constructing a recombinant host cell capable of expressing the RABV-G antigen component and the granule protein component in the host cell;

[0113] (3) using the recombinant host cell to express the fusion protein and purify the recombinant fusion protein;

[0114] (4) The above antigen component and the granule protein component are subjected to a covalent binding reaction to obtain an immunogenic complex.

[0115] Preferably, the immunogenic complex obtained in the above step (4) is purified to obtain a vaccine stock solution.

[0116] Preferably, in the method for preparing the immunogenic complex, in step (1), the plasmid for expressing the RABV-G antigen component may be pcDNA3.4, and the vector for expressing the nanoparticle may be pET-28a(+) or pET-30a(+).

[0117] In some embodiments, the immunogenic complexes provided herein can induce the production of neutralizing antibodies.

[0118] In some embodiments, the immunogenic complexes provided herein are used to prepare neutralizing antibodies.

[0119] The above-mentioned neutralizing antibodies refer to antibodies that can eliminate the virus's ability to infect after binding to the virus. After the enveloped virus surface antigen binds to the neutralizing antibody, it can activate complement and lead to the dissolution of the virus. IgG is preferred.

[0120] In step (2) of the method for preparing the immunogenic complex of the present invention, the host cell expressing the RABV-G antigen component is CHO, and the host cell expressing the granule protein carrier is E. coli.

[0121] In step (3) of the method for preparing the immunogenic complex of the present invention, the method for purifying the granule protein can be referred to patent CN114395015B.

[0122] F. Nucleic Acids

[0123] The present invention provides the encoding nucleotides of the above-mentioned RABV-G mutants and RABV-G antigen components. The specific nucleotide sequences can be easily obtained by those skilled in the art through conventional means such as codon tables.

[0124] Preferably, the encoding nucleotide sequence of the RABV-G mutant is obtained by codon optimization.

[0125] Preferably, the above-mentioned coding nucleotide sequence is optimized for codons preferred by CHO cell lines.

[0126] The present invention also provides the coding nucleotides for the above-mentioned granule protein components and nanoparticle proteins. The specific nucleotide sequences can be easily obtained by those skilled in the art through conventional means such as codon tables.

[0127] Preferably, the nucleotide sequences encoding the RABV-G mutants provided by the present invention are shown in SEQ ID NOs: 10-17, see Table 2 for details.

[0128] The present invention also provides vectors comprising the nucleotide sequence of the present invention, including cloning or expression vectors, and host cells transformed with the vectors.

[0129] In some embodiments, the vector used in the present invention is selected from pcDNA3.4, pET-28a(+), and pET-30a(+).

[0130] In some embodiments, the host cell expressing the antigen component vector is CHO, and the host cell expressing the particle protein vector is E. coli.

[0131] Table 2: Nucleotide sequences of RABV-G extracellular domain mutants of the present invention

[0132] G. Immunization Compositions

[0133] The present invention provides an immune composition comprising the RABV-G mutant, RABV-G trimer, RABV-G multimer or immunogenic complex of the present invention.

[0134] Optionally, the immunogenic composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0135] Preferably, the pharmaceutically acceptable carrier includes a stabilizer, an excipient, a surfactant, a buffer, and a pH adjuster. The stabilizer is sucrose and arginine, the excipient is mannitol, the surfactant is Tween-80, the buffer is disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate, and the pH adjuster is hydrochloric acid.

[0136] In some embodiments, the immune composition provided by the present invention is an injection or a lyophilized preparation, preferably a lyophilized preparation.

[0137] In some embodiments, the immunogenic composition of the present invention comprises one or more RABV-G mutants, RABV-G trimers, RABV-G multimers, or immunogenic complexes as described above.

[0138] In some embodiments, the immunological compositions of the present invention can induce the production of neutralizing antibodies.

[0139] In some embodiments, the immunological compositions of the present invention are used to prepare neutralizing antibodies.

[0140] The above-mentioned neutralizing antibodies refer to antibodies that can eliminate the virus's ability to infect after binding to the virus. After the enveloped virus surface antigen binds to the neutralizing antibody, it can activate complement and lead to the dissolution of the virus. IgG is preferred.

[0141] H. Vaccine

[0142] The present invention provides a vaccine comprising the immune composition of the present invention and an adjuvant.

[0143] In some embodiments, the vaccines provided by the present invention are vaccines for human use.

[0144] In some embodiments, the vaccines provided by the present invention are veterinary vaccines.

[0145] The adjuvants used in the present invention can enhance the immune response through a variety of mechanisms, including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. Compared to the use of the immunogen alone, the adjuvant increases the titer of the induced antibodies and / or the binding affinity of the induced antibodies. Preferred adjuvants enhance the intrinsic response to the immunogen without causing conformational changes in the immunogen.

[0146] The adjuvant used in the present invention is selected from: aqueous adjuvants; mineral adjuvants, including aluminum salts such as Al(OH)3, as well as aluminum phosphate, calcium phosphate, etc.; oil emulsion adjuvants, including oil-in-water emulsions, water-in-oil emulsions, Freund's adjuvant, MF59, white oil Span adjuvant, and SAF series; microbial adjuvants, including Corynebacterium brevis, BCG, muramyl diphthalide and its synthetic lipophilic derivatives, bacterial lipopolysaccharides, bacterial toxins, and fat-soluble wax D; liposomal adjuvants, including non-phospholipid liposomes such as Novasomes; cytokine adjuvants, including GM-CSF, IL-2, and IL-12; nucleic acid adjuvants, including CpGDNA; Chinese herbal adjuvants, including propolis, saponins, immunostimulatory complexes ISCOMS, polysaccharides, glycosides, and compound Chinese medicines; chemical adjuvants, including levamisole, cimetidine, erythromycin, etc.

[0147] In some embodiments, the vaccine of the present invention contains an adjuvant selected from Toll-like receptor agonists, including poly(I:C), GLA-LSQ, flagellin, and CpG1018.

[0148] In some embodiments, the vaccine of the present invention contains an adjuvant selected from a composite adjuvant, including AS adjuvant system, such as AS01, AS03, AS04; Covaxin; AS37; PIKACA; BC adjuvant system, such as BCG-CpG-DNA.

[0149] In some embodiments, the polysaccharide adjuvant of the present invention is selected from chitosan or inulin; the immunostimulatory complex adjuvant is selected from Matrix-M; the lipid adjuvant is selected from cationic lipid adjuvants, such as CAF01, anionic lipid adjuvants, such as LPS, monophosphoryl lipid A, liposome nanoparticles (LNP); the saponin adjuvant is selected from QuilA or QS21.

[0150] In some embodiments, the adjuvant of the present invention is selected from nanoadjuvants, such as silver nanoparticles, chiral nanoadjuvants.

[0151] In some embodiments, the adjuvant of the invention is selected from ISA201, ISA206, IMS1313, ISA15A.

[0152] In some embodiments, the adjuvant of the present invention is selected from a synthetic derivative of trehalose, a genetically engineered attenuated toxin, or a biodegradable polymeric microsphere. The genetically engineered attenuated toxin adjuvant is selected from cholera toxin, Escherichia coli heat-labile toxin, or tetanus toxoid.

[0153] The adjuvant of the present invention contains 3%-5% squalene, 0.4%-1% Span 85, 0.4%-1% Tween 80, 10mM citrate or 0.1-0.5% sodium citrate, and 0.01-0.05% citric acid (w / v). The squalene content is preferably 3.5%-4.5%, and more preferably 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.1%, 4.2%, 4.3%, 4.4%, or 4.5% (w / v). The squalene adjuvant may be the commercial adjuvant MF59 or SWE, wherein MF59 is an oil-in-water emulsion composed of 4.3% squalene, 0.5% Tween-80 and 0.5% Span85, and SWE is an oil-in-water emulsion with similar composition to MF59.

[0154] The squalene water-oil adjuvant used in the specific embodiment of the present invention has the following components by weight: squalene 4.2%, Span 85 0.5%, Tween-80 0.5%, citric acid 0.264%, and sodium citrate 0.016%.

[0155] Preferably, the adjuvant used in the present invention is selected from SWE or MF59.

[0156] Preferably, the adjuvant used in the present invention is selected from AS01, AS03 or AS04.

[0157] In some embodiments, the vaccines provided by the present invention can induce the production of neutralizing antibodies.

[0158] In some embodiments, the vaccines provided by the present invention are used to prepare neutralizing antibodies.

[0159] The above-mentioned neutralizing antibodies refer to antibodies that can eliminate the virus's ability to infect after binding to the virus. After the enveloped virus surface antigen binds to the neutralizing antibody, it can activate complement and lead to the dissolution of the virus. IgG is preferred.

[0160] The present invention provides a method for preparing a vaccine for preventing or treating rabies, comprising covalently binding the purified antigen component with the granule protein component via binding peptide 1 and binding peptide 2, and mixing with an applicable adjuvant to prepare an immune composition product. The dosage of the immunogenic complex is 0.25-100 μg / dose, preferably 0.5-50 μg / dose, and more preferably 0.5 μg / dose, 1 μg / dose, 2 μg / dose, 3 μg / dose, 4 μg / dose, 5 μg / dose, 10 μg / dose, 15 μg / dose, 20 μg / dose, 25 μg / dose, 30 μg / dose, 35 μg / dose, 40 μg / dose, 45 μg / dose, and 50 μg / dose.

[0161] The present invention provides a method for preparing a vaccine for preventing or treating rabies, wherein the water-oil adjuvant is squalene, and the saponin adjuvant comprises QS-21. The vaccine stock solution is diluted with a buffer solution (such as TBS) according to the dosage and then mixed with the adjuvant in a 1:1 volume ratio.

[0162] The present invention further provides a complete kit, characterized in that it contains rabies vaccine, and instruments and containers required for vaccinating the vaccine, specifically including needle and syringe instruments, powder carrying containers, and solvent carrying containers.

[0163] I. Medical Use

[0164] The present invention provides medical uses of the above-mentioned RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, and vaccines, including the prevention or treatment of related diseases caused by rabies virus infection.

[0165] Preferably, the RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, and vaccines of the present invention are used for pre-exposure prophylaxis and post-exposure prophylaxis of rabies virus infection.

[0166] The present invention provides pharmaceutical preparations of the above-mentioned RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, and vaccines, which are used to prevent or treat related diseases caused by rabies virus infection.

[0167] The present invention provides vaccination schedules for the aforementioned RABV-G mutants, RABV-G trimers, RABV-G multimers, immunogenic complexes, immune compositions, and vaccines. Pre-exposure prophylaxis typically includes two to three visits to a doctor, two to three intramuscular doses, and boosters scheduled based on exposure risk. Post-exposure prophylaxis typically includes three to five intramuscular doses or four intradermal doses.

[0168] The RABV-G mutant, RABV-G trimer, RABV-G multimer, immunogenic complex, immune composition or vaccine provided by the present invention will generally be administered directly to a subject. Direct delivery can be accomplished by parenteral injection, for example, subcutaneous, intraperitoneal, intravenous, intramuscular, intradermal injection or injection into the interstitial space of a tissue. When administered prophylactically, for example, to residents of a rabies-endemic area or to travelers to a rabies-endemic area, a dosing schedule can consist of three doses, two doses or one dose. Alternatively or in addition, a dose can be administered prophylactically. When administered therapeutically, for example, after rabies exposure, a dosing schedule can consist of five doses, four doses, three doses, two doses or one dose. In a preferred embodiment, one or two doses are administered therapeutically.

[0169] J.Beneficial effects

[0170] Compared with the prior art, the present invention has the following beneficial effects:

[0171] (1) The RABV-G extracellular domain mutant of the present invention achieves soluble expression of the RABV-G extracellular domain and can self-assemble into trimers or polymers, which largely solves the technical difficulties in this field.

[0172] (2) The RABV-G extracellular domain mutant vaccine of the present invention has better cellular immunity and antibody immune responses than commercial human rabies vaccines and canine rabies vaccines.

[0173] (3) The RABV-G extracellular domain mutant vaccine of the present invention can induce the production of high-titer neutralizing antibodies, which is expected to reduce the number of vaccinations, shorten the immunization procedure and reduce the immunization dose.

[0174] (4) The RABV-G extracellular domain mutant vaccine of the present invention is a recombinant protein vaccine. The entire production process and the final product do not involve complete viruses, which is highly safe. It can also reduce the complexity that may be brought to the production process from the source, improve production efficiency, and save the cost of large-scale production.

[0175] (5) The present invention also discovered a method for preparing RABV-G extracellular domain mutant protein particles of different particle sizes. The method is simple to operate, and the prepared protein particles are uniform and have uniform particle sizes, which lays the foundation for the preparation of virus-like particles of different types and particle sizes. BRIEF DESCRIPTION OF THE DRAWINGS

[0176] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific implementation manner or examples will be briefly introduced below.

[0177] Figure 1: SDS-PAGE identification results of RABV-G, where A is RABV-G6, B is RABV-G25, C is RABV-G26, D is RABV-G28, E is RABV-G30, F is RABV-G32, G is RABV-G33, and H is RABV-G36;

[0178] Figure 2: AB are the electron microscopy negative staining results of G6, C is the particle size and uniformity test results, and D is the HPLC test results;

[0179] Figure 3: AUC test results of G6 samples;

[0180] Figure 4: AB is the electron microscopy negative staining result of RABV-G25;

[0181] Figure 5: AB is the electron microscopy negative staining result of RABV-G26;

[0182] Figure 6: AB is the electron microscopy negative staining result of RABV-G28;

[0183] Figure 7: AB is the electron microscopy negative staining result of RABV-G30;

[0184] Figure 8: AB is the electron microscopy negative staining result of RABV-G32;

[0185] Figure 9: AB is the electron microscopy negative staining result of RABV-G33;

[0186] Figure 10: AB is the electron microscopy negative staining result of RABV-G36;

[0187] Figure 11: AB are the electron microscopic negative staining results of G6 after adding 0.02% Tween-80;

[0188] Figure 12: AB are the electron microscopic negative staining results of G6 after adding 0.05% Tween-80;

[0189] Figure 13: AB are the electron microscopic negative staining results of G6 after adding 0.1% Tween-80;

[0190] Figure 14: SDS-PAGE identification results of G6-NPM virus-like particles. A is a virus-like particle with the addition of 0.2% Tween-80, B is a virus-like particle with the addition of 0.05% Tween-80, C is a virus-like particle with the addition of 0.02% Tween-80, and D is a virus-like particle without the addition of Tween-80.

[0191] Figure 15: Neutralizing antibody detection results of immune sera from mice vaccinated with G1 and G6;

[0192] Figure 16: Neutralizing antibody test results of immune sera from mice vaccinated with two doses of G6 and four doses of control vaccine;

[0193] Figure 17: Neutralizing antibody detection results of immune sera from mice vaccinated with G protein mutants. DETAILED DESCRIPTION

[0194] The principles and features of the present invention are described below with reference to examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments and are not intended to limit the scope of protection of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. When numerical ranges are given in the examples, it should be understood that unless otherwise specified in the present 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 present 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 examples, any methods, equipment, and materials of the prior art that are similar or equivalent to the methods, equipment, and materials described in the examples of the present invention may also be used to implement the present invention, based on the prior art knowledge of those skilled in the art and the description of the present invention. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0195] Example 1: Expression and purification of RABV-G extracellular domain mutants

[0196] 1. Expression and purification of RABV-G extracellular domain mutants

[0197] 1. Experimental Materials:

[0198] HisTrap excel was purchased from Cytiva, molecular sieve (Superose 6 10 / 300GL) was purchased from Cytiva, and anion exchange column (Hitrap Q FF) was purchased from Cytiva.

[0199] 2. Experimental methods:

[0200] (1) The gene encoding the signal peptide shown in SEQ ID NO: 18 was introduced into the N-terminus of the gene encoding the RABV-G extracellular domain mutants (SEQ ID NO: 2-9) shown in Table 1. Optionally, the gene encoding the 4T sequence (SEQ ID NO: 19) and the 6×His tag was introduced into the C-terminus. The fusion gene was obtained by conventional molecular cloning methods. The sequence of the fusion gene was codon-optimized for CHO cells and cloned into the SPB-007 vector. A stable transfected cell pool was constructed, and the positive cell pool was screened. The cells were expanded and cultured, and the cell survival rate was greater than 95%;

[0201] (2) After 12 days of expression in the expression medium, the cell supernatant was harvested and centrifuged at 6000 g for 20 min. The supernatant was filtered through a 0.22 μm pore size PES membrane.

[0202] (3) The filtered supernatant was purified by Histrap Excel-5ml nickel column, and the flowthrough was collected; then eluted with 20mM imidazole and 10 volumes of TBS buffer, and the sample was collected; then eluted with 500mM imidazole and 10 volumes of TBS buffer, and the eluted sample was collected. All samples were identified by SDS-PAGE and the target protein was collected;

[0203] (4) Collect samples containing the target protein, concentrate them through a 10 kDa ultrafiltration concentrator, and further purify them through Superose 6 increase 10 / 300 GL molecular sieves in TBS buffer. Collect samples and perform SDS-PAGE electrophoresis. The results are shown in Figure 1A. Determine the protein concentration by BCA method. Keep the sample at an appropriate temperature for subsequent testing. Sample G6 is abbreviated as G6.

[0204] The SDS-PAGE identification results of other variants are shown in Figures 1B-H, where B is RABV-G25, C is RABV-G26, D is RABV-G28, E is RABV-G30, F is RABV-G32, G is RABV-G33, and H is RABV-G36.

[0205] (5) Optionally, G6 is further purified. This includes replacing the G6 protein purified by molecular sieve into 50mM Tris, 50mM NaCl buffer, purifying it through an anion exchange column hitrap Q FF, with solution A being 50mM Tris, 50mM NaCl buffer and solution B being 50mM Tris, 1M NaCl buffer, eluting it through a linear gradient, collecting the sample and further purifying it through Superose6 increase 10 / 300GL molecular sieve, with TBS as the buffer. The target protein is collected for SDS-PAGE electrophoresis detection, the protein concentration is determined by the BCA method, and the protein is stored at an appropriate temperature for subsequent detection.

[0206] 2. Characterization of G6 Protein

[0207] (1) The purified G6 protein was diluted to 0.01 mg / mL with TBS and negatively stained. It was observed that the sample formed relatively uniform nanoparticles, as shown in Figures 2A-B. The particle size and uniformity of the particles were also detected, as shown in Figure 2C. The particle size was uniform, approximately 20.36 nm. The purity of the G6 protein was detected by HPLC, as shown in Figure 2D. The main peak was asymmetric, which may be caused by glycosylation. The purity was approximately 89%.

[0208] (2) The molecular weight and uniformity of the G6 protein sample were determined by AUC. The results are shown in Figure 3. The peak on the left with a molecular weight of approximately 60.8 kDa is a monomer. The self-assembled multimers are mainly composed of 9-mers with a molecular weight of approximately 582 kDa, and a small number of hexamers and 12-mers with molecular weights of approximately 443 kDa and 754 kDa, respectively. This shows that G6 can self-assemble into multimers.

[0209] (3) Samples of other RABV-G extracellular domain mutants purified by nickel and molecular sieve were diluted to 0.01 mg / mL and negatively stained. It was observed that all samples formed relatively uniform nanoparticles. Among them, Figure 4A-B shows the electron microscopic negative staining results of RABV-G25, Figure 5A-B shows the electron microscopic negative staining results of RABV-G26, Figure 6A-B shows the electron microscopic negative staining results of RABV-G28, Figure 7A-B shows the electron microscopic negative staining results of RABV-G30, Figure 8A-B shows the electron microscopic negative staining results of RABV-G32, Figure 9A-B shows the electron microscopic negative staining results of RABV-G33, and Figure 10A-B shows the electron microscopic negative staining results of RABV-G36.

[0210] Example 2: Binding of RABV-G ectodomain mutants to NPM and particle characterization

[0211] 1. Preparation of RABV-G ectodomain mutants of different particle sizes

[0212] Purified G6 protein samples were negatively stained with 0.02%, 0.05%, and 0.1% Tween-80, respectively. Dispersion of the sample resulted in small, uniformly sized protein particles. Figures 11A-B show electron microscopic negative staining of G6 after addition of 0.02% Tween-80, Figures 12A-B show electron microscopic negative staining of G6 after addition of 0.05% Tween-80, and Figures 13A-B show electron microscopic negative staining of G6 after addition of 0.1% Tween-80.

[0213] II. Binding experiments of RABV-G ectodomain mutants and NPM

[0214] G6 diluted with TBS and G6 dispersed with various concentrations of Tween, obtained in Example 1, were mixed with NPM-4C (SEQ ID NO: 22), the particle protein component, at a BCA protein ratio of 6:1. A 50% sucrose stock solution was added to a final concentration of approximately 25% sucrose. A 1M Tris-HCl (pH 7.4) stock solution was added to stabilize the pH at 10% of the total reaction volume. The binding reaction was carried out at 22°C for 24 hours. The bound product was purified by molecular sieve separation (purification buffer: 20 mM Tris-HCl, 150 mM NaCl, 25% Sucrose, pH 7.4), and the G6-NPM fraction was collected. The purified G6-NPM virus-like particles were identified by SDS-PAGE, as shown in Figure 14. A shows VLPs formed by G6 dispersed with 0.2% Tween-80, B shows VLPs formed by G6 dispersed with 0.05% Tween-80, C shows VLPs formed by G6 dispersed with 0.02% Tween-80, and D shows VLPs formed by G6 without Tween-80. This indicates that G6 dispersed with different concentrations of Tween-80 can form VLPs of varying molecular weights upon binding to NPM.

[0215] Example 3: Animal Immunization Experiment 1

[0216] (1) Experimental materials

[0217] Mice: 5-6 weeks old female BALB / c mice (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.)

[0218] Adjuvant: SWE;

[0219] Pseudovirus: Zhongyan Guobang;

[0220] Cells: 293T cells;

[0221] Control vaccines: freeze-dried human rabies vaccine (Vero cells, Liaoning Chengda), freeze-dried human rabies vaccine (human diploid cells, Chengdu Kanghua);

[0222] Other reagents and consumables are commercial conventional reagents and consumables.

[0223] (2) Experimental plan

[0224] As shown in Table 3, animals received one dose of the vaccine intramuscularly on days 0 and 7, respectively, at a dose of 5 μg / dose / animal, mixed with 25 μg of SWE adjuvant. Blood was collected on days 7 and 14 for serum analysis and neutralizing antibody testing.

[0225] Table 3: Animal Immunization Scheme 1

[0226] (3) Neutralizing antibody detection:

[0227] 1. Sample preparation: Inactivate mouse serum in a dry-well incubator at 56°C for 30 min.

[0228] Virus preparation: Take out the pseudovirus from -80℃, thaw at 4℃, and dilute the virus to 5×10 4 TCID50 / mL;

[0229] 2. The first well of serum samples was diluted with a dilution factor of 1:100 (i.e., 1.5 μL sample + 148.5 μL culture medium for dilution, the final dilution factor in the well was 1:250), and each serum sample was diluted with two replicates, and then a 3-fold serial dilution was performed, for a total of 7 concentrations (the last 6 gradients were used for reading);

[0230] 3. Add 50 μL of diluted virus to each well to a concentration of 5×10 4 The virus with TCID50 / mL and blank wells were sealed with sterile liquid, 200 μL / well, and incubated in a cell culture incubator at 37°C and 5% CO2 for 1 h;

[0231] 4. After the sample and virus have been incubated for 1 hour, add 100 μL of a 0.5×10 6 / mL of 293T cells, and then cultured in a 37°C, 5% CO2 cell culture incubator for 24 h;

[0232] 7. Detection: Remove the 96-well white plate and Bio-Lite detection reagent, equilibrate to room temperature, aspirate 150 μL / well of the supernatant in the plate, add 100 μL of detection reagent to the plate, let it stand for 5 minutes, and then measure the chemiluminescence value (RLU) using a fluorescence microplate reader.

[0233] The results are shown in Figure 15. After G1 (i.e., wild type) and G6 were immunized with SWE adjuvant, the serum neutralizing antibody titer on the 14th day was compared. G6 was significantly higher than G1, and also higher than Vero cell freeze-dried vaccine and human diploid freeze-dried vaccine, indicating that G6 has better immunogenicity.

[0234] Example 4: Animal Immunization Experiment 2

[0235] (1) Experimental materials

[0236] Mice: 5-6 week-old female BALB / c mice (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.);

[0237] Adjuvant A: Contains the following components: squalene 4.2%, Span 85 0.5%, Tween-80 0.5%, citric acid 0.264%, sodium citrate 0.016% (W / W);

[0238] Pseudovirus: Zhongyan Guobang;

[0239] Cells: 293T cells;

[0240] Control vaccine: marketed freeze-dried human rabies vaccine (Vero cells, Liaoning Chengda);

[0241] Other reagents and consumables are commercial conventional reagents and consumables.

[0242] (2) Experimental plan

[0243] As shown in Table 4, animals were immunized with one dose of RABV-G6 vaccine by intramuscular injection on days 0 and 7, at a dose of 5 μg / dose / animal. Each dose was mixed with 25 μg of Adjuvant A. Blood was collected on days 7 and 14 for serum analysis and neutralizing antibody testing. A control vaccine was administered by intramuscular injection on days 0, 7, 14, and 21, at a dose of 1 / 10 the human dose. Blood was collected on days 7, 14, 21, and 35 for serum analysis and neutralizing antibody testing.

[0244] Table 4: Animal Immunization Scheme 2

[0245] (3) Neutralizing antibody detection:

[0246] 1. Sample preparation: Inactivate mouse serum in a dry-well incubator at 56°C for 30 min.

[0247] Virus preparation: Take out the pseudovirus from -80℃, thaw at 4℃, and dilute the virus to 5×10 4 TCID50 / mL;

[0248] 2. The first well of serum samples was diluted with a dilution factor of 1:100 (i.e., 1.5 μL sample + 148.5 μL culture medium for dilution, the final dilution factor in the well was 1:250), and each serum sample was diluted with two replicates, and then a 3-fold serial dilution was performed, for a total of 7 concentrations (the last 6 gradients were used for reading);

[0249] 3. Add 50 μL of diluted virus to each well to a concentration of 5×10 4 The virus with TCID50 / mL and blank wells were sealed with sterile liquid, 200 μL / well, and incubated in a cell culture incubator at 37°C and 5% CO2 for 1 h;

[0250] 4. After the sample and virus have been incubated for 1 hour, add 100 μL of a 0.5×10 6 / mL of 293T cells, and then cultured in a 37°C, 5% CO2 cell culture incubator for 24 h;

[0251] 7. Detection: Remove the 96-well white plate and Bio-Lite detection reagent, equilibrate to room temperature, aspirate and discard 150 μL / well of the supernatant in the white plate, add 100 μL of detection reagent to the plate, let it stand for 5 minutes, and then measure the chemiluminescence value (RLU) using a fluorescence microplate reader.

[0252] The results are shown in Figure 16, which show that on days 21 and 35, the neutralizing antibody titers of the RABV-G6 vaccine were higher than those of the control vaccine, indicating that the immune effect of two doses of RABV-G6 vaccine was better than that of four doses of the control vaccine.

[0253] Example 5: Animal Immunization Experiment 3

[0254] (1) Experimental materials

[0255] Mice: 5-6 week-old female BALB / c mice (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.);

[0256] Adjuvant A: Contains the following components: squalene 4.2%, Span 85 0.5%, Tween-80 0.5%, citric acid 0.264%, sodium citrate 0.016% (W / W);

[0257] Pseudovirus: Zhongyan Guobang;

[0258] Cells: 293T cells;

[0259] Control vaccine: marketed Vero cell-based human rabies vaccine;

[0260] Other reagents and consumables are commercial conventional reagents and consumables.

[0261] (2) Experimental plan

[0262] As shown in Table 5, the animals were immunized with one dose of vaccine on days 0 and 7, respectively, by intramuscular injection at a dose of 5 μg / dose / animal, and each dose was mixed with 25 μg of adjuvant A. Blood was collected on days 7 and 14 for serum separation and neutralizing antibody detection.

[0263] Table 5: Animal Immunization Scheme 3

[0264] (3) Neutralizing antibody detection:

[0265] 1. Sample preparation: Inactivate mouse serum in a dry-well incubator at 56°C for 30 min.

[0266] Virus preparation: Take out the pseudovirus from -80℃, thaw at 4℃, and dilute the virus to 5×10 4 TCID50 / mL;

[0267] 2. The first well of serum samples was diluted with a dilution factor of 1:100 (i.e., 1.5 μL sample + 148.5 μL culture medium for dilution, the final dilution factor in the well was 1:250), and each serum sample was diluted with two replicates, and then a 3-fold serial dilution was performed, for a total of 7 concentrations (the last 6 gradients were used for reading);

[0268] 3. Add 50 μL of diluted virus to each well to a concentration of 5×10 4 The virus with TCID50 / mL and blank wells were sealed with sterile liquid, 200 μL / well, and incubated in a cell culture incubator at 37°C and 5% CO2 for 1 h;

[0269] 4. After the sample and virus have been incubated for 1 hour, add 100 μL of a 0.5×10 6 / mL of 293T cells, and then cultured in a 37°C, 5% CO2 cell culture incubator for 24 h;

[0270] 7. Detection: Remove the 96-well white plate and Bio-Lite detection reagent, equilibrate to room temperature, aspirate and discard 150 μL / well of the supernatant in the white plate, add 100 μL of detection reagent to the plate, let it stand for 5 minutes, and then measure the chemiluminescence value (RLU) using a fluorescence microplate reader.

[0271] The results are shown in Figure 17. Compared with the serum neutralizing antibody titers of immunized mice on the 14th day, all mutants were better than the control vaccine, and G6, G26, G28, G30, G32, G33, and G36 were better than G1 (wild type).

[0272] In summary, the above embodiments and drawings are only 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 in the scope of protection of the present invention.

Claims

1. A rabies virus G protein mutant, characterized in that: The G protein mutant comprises a mutated wild-type rabies virus G protein extracellular domain, and the mutated wild-type rabies virus G protein extracellular domain can improve the stability of the pre-fusion conformation of the G protein. Preferably, the G protein mutant can induce the production of neutralizing antibodies.

2. The G protein mutant according to claim 1, wherein the wild-type rabies virus G protein is derived from the G protein of the following strains: Pasteur Virus (GenBank accession number is AAA47218.1), Pitman Moore (GenBank accession number is CAI43218.1), HEP-Flury (GenBank accession number is BAC53868.1), MOR1-DG (GenBank accession number is AAK92057.1), MOR3-HM (GenBank accession number is AAK92058.1), NeiMeng1025C (GenBank accession number is ABY19509.2), FRA1-FX (GenBank accession number is AAK92050.1), CNX8601 (GenBank accession number is AAG34722.1), CHI1-BK ( The wild-type rabies virus G protein is derived from the G protein of the Pasteur Virus strain (PV strain).

3. The G protein mutant according to claim 2, wherein the amino acid sequence of the extracellular domain of the wild-type rabies virus G protein is derived from amino acids 20-459 of the G protein of the PV strain, or has a sequence corresponding to or aligned with amino acids 20-459 of the G protein of the PV strain; Preferably, the amino acid sequence of the extracellular domain of the wild-type rabies virus G protein is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1; more preferably, the amino acid sequence of the extracellular domain of the wild-type rabies virus G protein is as shown in SEQ ID NO:

1.

4. The G protein mutant according to any one of claims 1 to 3, wherein the G protein mutant has one, two or more amino acid mutations or substitutions at positions 261, 266, 270, 271, 384 and 424 of the extracellular domain of the wild-type rabies virus G protein; Preferably, the mutation or substitution of the two amino acids is selected from the following combinations: H261+H270; D266+H270.

5. The G protein mutant according to claim 4, wherein the residue at the site to be mutated or substituted is mutated to proline or substituted by proline; alternatively, the residue at the site to be mutated or substituted is mutated to leucine or substituted by leucine.

6. The G protein mutant according to claim 4 or 5, wherein the G protein mutant has one, two or more amino acid mutations or substitutions among H261L, D266P, H270P, L271P, H384P and H424L in SEQ ID NO: 1; Preferably, the two mutations or substitutions present in SEQ ID NO: 1 are selected from the following combinations: H261L+H270P; D266P+H270P.

7. The G protein mutant according to any one of claims 4 to 6, wherein the G protein mutant has an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 9; Preferably, the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2-9; more preferably, the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2, 4-9.

8. The G protein mutant according to any one of claims 1 to 7, wherein a signal peptide or a 6×His tag is introduced at one end of the G protein mutant; preferably, a signal peptide is introduced at the N-terminus of the G protein mutant, and / or a 6×His tag is introduced at the C-terminus; optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:

18.

9. A rabies virus G protein trimer or polymer, characterized in that: The trimer or polymer comprises the rabies virus G protein mutant according to any one of claims 1-8.

10. The G protein trimer or multimer as described in claim 9, wherein the trimer or multimer is formed by self-assembly of the rabies virus G protein mutant according to any one of claims 1 to 8; preferably, the multimer includes a hexamer, a nonamer and a dodecamer.

11. The G protein trimer or multimer according to any one of claims 9 to 10, wherein the trimer or multimer is prepared by the following method, which comprises the following steps: (1) recombinantly expressing the rabies virus G protein mutant according to any one of claims 1 to 8 in CHO cells by conventional molecular cloning techniques, (2) After expressing in the expression medium for a certain period of time, the cell supernatant is harvested, centrifuged and the supernatant is filtered through a membrane. (3) Purify the filtered supernatant by passing it through a nickel column, (4) Collect samples containing the target protein, concentrate by ultrafiltration, and then purify by molecular sieve. (5) Using a buffer to dilute the purified target protein, soluble G protein trimers or polymers with uniform particle size can be obtained; in, The buffer used for molecular sieve purification in step (4) is TBS. Optionally, in step (4), the product of the first molecular sieve purification is purified by anion exchange column and then subjected to a second molecular sieve purification. The buffer in step (5) is TBS or Tween.

12. The G protein trimer or multimer according to claim 11, wherein the step (5) comprises adding 0.02-1% Tween to the purified target protein; Optionally, Tween is selected from Tween-20 or Tween-80; Preferably, the Tween is Tween-80.

13. A method for preparing a G protein trimer or multimer according to any one of claims 9 to 12, comprising the following steps: (1) recombinantly expressing the rabies virus G protein mutant according to any one of claims 1 to 8 in CHO cells by conventional molecular cloning techniques, (2) After expressing in the expression medium for a certain period of time, the cell supernatant is harvested, centrifuged and the supernatant is filtered through a membrane. (3) Purify the filtered supernatant by passing it through a nickel column, (4) Collect samples containing the target protein, concentrate by ultrafiltration, and then purify by molecular sieve. (5) Using a buffer to dilute the purified target protein, soluble G protein trimers or polymers with uniform particle size can be obtained; in, The buffer used for molecular sieve purification in step (4) is TBS. Optionally, in step (4), the product of the first molecular sieve purification is purified by anion exchange column and then subjected to a second molecular sieve purification. The buffer in step (5) is TBS or Tween.

14. The preparation method according to claim 13, wherein the step (5) comprises adding 0.02-1% Tween to the purified target protein; Optionally, Tween is selected from Tween-20 or Tween-80; Preferably, the Tween is Tween-80.

15. An immunogenic complex comprising: (1) An antigen component comprising a rabies virus G protein mutant selected from any one of claims 1 to 8, or a rabies virus G protein trimer or multimer according to any one of claims 9 to 12; (2) a granule protein component, which comprises nanoparticle protein; The immunogenic complex formed by the covalent binding reaction between the antigen component and the granule protein component; Preferably, the antigen component further comprises binding peptide 1, the particle protein component further comprises binding peptide 2, the G protein mutant forms a fusion protein with binding peptide 1, the nanoparticle protein forms a fusion protein with binding peptide 2, the antigen component and the particle protein component are covalently bound to each other via binding peptide 1 and binding peptide 2 to form an immunogenic complex; the G protein mutant and binding peptide 1 form a fusion protein and then assemble into a trimer or a multimer; Preferably, the binding peptide 1 contains the amino acid sequence shown in SEQ ID NO:19, and the binding peptide 2 contains the amino acid sequence shown in SEQ ID NO:

20.

16. The immunogenic complex according to claim 15, characterized in that The antigen component further comprises a connecting peptide 1, and the particle protein component further comprises a connecting peptide 2; the antigen component is formed by fusing a G protein mutant with a binding peptide 1 at the C-terminus through the connecting peptide 1; the particle protein component is formed by fusing a nanoparticle protein with a binding peptide 2 at the N-terminus through the connecting peptide 2; the G protein mutant is assembled into a trimer or a multimer after being fused with the binding peptide 1 at the C-terminus through the connecting peptide 1; Optionally, the connecting peptide 1 is selected from (GGGGS) n 、(EAAAK) n 、(GSGGSG) n 、(GGS) n or (GSG) n The amino acid sequence shown, n can be an integer greater than 0 and less than or equal to 5; the connecting peptide 2 is selected from (GGS) n 、(GGGGS) n 、(EAAAK) n 、(GSGGSG) n An amino acid sequence of, n may be an integer greater than 0 and less than or equal to 10; Optionally, both the antigen component and the particle protein component comprise a histidine tag.

17. The immunogenic complex according to claim 15 or 16, wherein the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2-9; preferably, the amino acid sequence of the G protein mutant is shown in any one of SEQ ID NOs: 2, 4-9.

18. The immunogenic complex according to any one of claims 15 to 17, wherein the nanoparticle protein is NPM, AP205 or Ferritin protein; preferably, the amino acid sequence of NPM is as shown in SEQ ID NO:

21.

19. A method for preparing an immunogenic complex: (1) Connecting the antigen component and granule protein component encoding genes according to any one of claims 15 to 18 into expression vectors to construct expression recombinant plasmids; (2) constructing a recombinant host cell capable of expressing the antigen component and the granule protein component in the host cell; (3) using recombinant host cells to express the fusion protein and purify the recombinant fusion protein; (4) The antigen component and the granule protein component are subjected to a covalent binding reaction to obtain the immunogenic complex.

20. The preparation method according to claim 19, wherein the plasmid expressing the antigen component in step (1) is selected from pcDNA3.4, and the vector expressing the granule protein is selected from pET-28a(+) or pET-30a(+); optionally, the host cell expressing the antigen component in step (2) is CHO, and the host cell expressing the granule protein is E.coli.

21. An immune composition comprising the rabies virus G protein mutant according to any one of claims 1 to 8, or the rabies virus G protein trimer or multimer according to any one of claims 9 to 12, or the immunogenic complex according to any one of claims 15 to 18; optionally, the immune composition of the present invention further comprises a pharmaceutically acceptable carrier.

22. An immune composition according to claim 21, characterized in that: The pharmaceutically acceptable carrier includes a stabilizer, an excipient, a surfactant, a buffer, and a pH adjuster; The stabilizer is preferably sucrose or arginine, the excipient is preferably mannitol, the surfactant is preferably Tween-80, the buffer is preferably disodium hydrogen phosphate dihydrate or sodium dihydrogen phosphate dihydrate, and the pH adjuster is preferably hydrochloric acid.

23. A rabies vaccine, characterized in that: It contains the immune composition according to any one of claims 21-22 and an adjuvant, wherein the adjuvant enhances the immune response through multiple mechanisms, including lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages, and the vaccine includes a human vaccine or a veterinary vaccine.

24. The vaccine according to claim 23, wherein the adjuvant is selected from at least one of aqueous adjuvants, mineral adjuvants, oil emulsion adjuvants, microbial adjuvants, liposome adjuvants, cytokine adjuvants, nucleic acid adjuvants, Chinese herbal adjuvants, and chemical adjuvants.

25. The vaccine according to claim 23, wherein the adjuvant is selected from Toll-like receptor agonists, composite adjuvants, nanoadjuvants, synthetic derivatives of trehalose, genetically engineered toxin-reducing agents or biodegradable polymer microspheres. Preferably, the adjuvant is selected from poly(I:C), GLA-LSQ, flagellin, CpG1018, AS01, AS03, AS04, Covaxin, AS37, PIKACA, BC adjuvant system, SWE, MF59.

26. The vaccine according to claim 23, wherein the component contents (w / w) of the adjuvant are as follows: Squalene 4.2%, Span 85 0.5%, Tween-80 0.5%, citric acid 0.264%, sodium citrate 0.016%.

27. A kit, characterized in that: The invention comprises the vaccine according to any one of claims 23 to 26, and the instruments and containers required for inoculating the vaccine, including needle and syringe instruments, powder-carrying containers, and solvent-carrying containers.

28. Use of the rabies virus G protein mutant according to any one of claims 1 to 8, the rabies virus G protein trimer or multimer according to any one of claims 9 to 13, the immunogenic complex according to any one of claims 15 to 18, the immune composition according to any one of claims 21 to 22, the vaccine according to any one of claims 23 to 26, and the kit according to claim 27 for the preparation of drugs for preventing or treating related diseases caused by rabies virus infection, preferably for the preparation of drugs for pre-exposure prevention and post-exposure prevention of rabies virus infection.

Images