Immune composition product for preventing or treating diseases related to neisseria meningitidis group b, and preparation method therefor

Abstract

An immune composition product for preventing or treating diseases related to Neisseria meningitidis group B, and a preparation method therefor. The immune composition product comprises an fHbp fusion protein, wherein the fusion protein is constructed by means of disassembling two domains of three fHbp variants and then recombining same to construct a chimeric protein, and then on the basis of the chimeric protein, constructing a fusion protein containing characteristic amino acid sequences of the three fHbp variants. Furthermore, a nanoparticle vaccine is prepared by means of using such fusion protein as a vaccine antigen. The fusion protein has better immunological effects than those brought about by simply mixing the three fHbp variants.

Description

An immune composition product for preventing or treating group B Neisseria meningitidis-related diseases and its preparation method Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an immune composition product for preventing or treating diseases associated with group B Neisseria meningitidis and a production method thereof. Background Art

[0002] Epidemic meningitis (EMM) is a respiratory infectious disease caused by Neisseria meningitidis, characterized by acute meningitis and sepsis. It is classified as a Category B infectious disease in my country. Based on the characteristics of their capsular polysaccharides, N. meningitidis can be divided into 13 serogroups: A, B, C, Y, and W135. Currently available quadrivalent meningococcal polysaccharide or polysaccharide-protein conjugate vaccines are effective in preventing infections caused by serogroups A, C, Y, and W135, reducing the incidence of invasive meningococcal disease.

[0003] Neisseria meningitidis group B (MenB), commonly known as serogroup B meningococcus, currently dominates in China due to a lack of effective prevention and control measures. Unlike the capsular polysaccharides of serogroups A, C, Y, and W135, the main component of the MenB capsular polysaccharide is structurally similar to the human central nervous system antigen N-acetylneuraminic acid polymer. This can lead to immune tolerance in the human body and pose the risk of inducing autoimmune diseases. Therefore, the MenB capsular polysaccharide is not suitable as a candidate vaccine antigen.

[0004] Current MenB vaccine development focuses primarily on non-capsular polysaccharide immunogens. Factor H binding protein (fHBP) is one of the most promising vaccine antigens. As a lipoprotein, it is expressed on the cell membrane of nearly all MenB strains. A key function of fHBP is to bind to human complement regulatory factor H, enabling Neisseria meningitidis to evade host bactericidal responses and enhance survival in the bloodstream. Antibodies elicited by fHBP as a MenB vaccine antigen can directly activate bacteriolysis through the classical complement pathway and also block fH binding to the bacterial surface. fHBP is a highly diverse surface antigen that can be divided into three antigenic variant groups: variant 1 (V1), variant 2 (V2), and variant 3 (V3), each of which contains numerous subvariants. A phylogenetic tree of the fHBP protein reveals that variant V1 accounts for approximately 70% of isolates, while variants V2 and V3 account for approximately 30%. There is no significant cross-protection between variant V1 and the other two variants, V2 and V3. Furthermore, different determination methods can lead to different definitions of fHBP protein structure. MASCIONI et al. determined the full-length molecular structure of non-esterified fHBP in aqueous solution using nuclear magnetic resonance (NMR), revealing that fHBP consists of two domains: a C-terminal antiparallel β-barrel structure and a "taco-shaped" N-terminal β-pleated sheet, connected by a 5' residue flexible linker.

[0005] There are currently two main MenB vaccines on the market overseas, Bexsero (GSK) and Trumenba (Pfizer). Bexsero (also known as C4MenB) contains outer membrane vesicles (OMVs) from the prevalent strain of serogroup B Neisseria meningitidis NZ98 / 254 prevalent in New Zealand, as well as a preparation of five meningococcal antigens: Neisserial heparin-binding protein A (NHBA), factor H binding protein (fHBP) variant 1.1, Neisserial adhesion protein A (NadA), and accessory proteins GNA1030 and GNA2091. Trumenba contains two lipidated MenB fHBP antigens A05 and B01 adsorbed on aluminum phosphate. In the two MenB protein vaccines currently on the market, the fHBP protein is expressed as a single variant or in fusion with non-fHBP proteins. The products target MenB strains prevalent in Europe and the United States, but do not have broad protection against other variant strains. The fHBP protein sequences in MenB protein vaccines available internationally primarily target strains VI and V3, while the fHBP type of strains prevalent in China is primarily V2. This suggests that currently available MenB vaccines internationally fail to cover the strains prevalent in my country.

[0006] Nanoparticle vaccines use nanomaterials as antigen carriers. Compared to traditional vaccines, nanoparticle vaccines offer superior antigen encapsulation and structural stability, offering advantages in antigen assembly and presentation. Nanoparticle vaccines primarily fall into four categories: virus-like particle nanovaccines, self-assembling protein nanovaccines, polymer particle nanovaccines, and inorganic particle nanovaccines. In the field of self-assembling protein nanovaccines, researchers recently discovered, based on computational design of icosahedral nanocages, that the nanoparticle protein mi3 can spontaneously form highly ordered 60-subunit dodecahedral nanoparticles. SpyCatcher was displayed on the nanoparticle surface and linked to the antigen protein. Results confirmed that this computationally designed protein nanoparticle vaccine also elicited a strong antibody response, demonstrating its potential application in the preparation of novel self-assembling protein nanovaccines.

[0007] There is currently no serogroup B meningococcal vaccine available in China, and very few serogroup B Neisseria meningitidis nanoparticle vaccines are under development. The two currently available vaccines primarily target MenB strains prevalent in Europe and the United States. Therefore, developing a MenB vaccine that offers broad protection, is safer and more effective, and reduces production costs is crucial.

[0008] Summary of the Invention

[0009] In order to solve the current situation of insufficient technology and supply types of serogroup B Neisseria meningitidis vaccines, especially the basic blank supply of serogroup B Neisseria meningitidis nanoparticle vaccine, the present invention provides a serogroup B Neisseria meningitidis nanoparticle vaccine and a preparation method thereof.

[0010] The present invention structurally alters the sequence of the fHBP antigen protein of MenB. Specifically, two domains from three fHBP variants are disassembled and reassembled to create chimeric proteins. This chimeric protein is then used as a basis for constructing a fusion protein containing the characteristic amino acid sequences of the three fHBP variants. This fusion protein is used as a vaccine antigen to prepare a MenB nanoparticle vaccine that is broadly protective and effective.

[0011] The nanoparticle vaccine provided by the present invention is a vaccine formed based on nanoparticle protein, and the nanoparticle protein is mainly used to display antigens.

[0012] A. Factor H Binding Protein (fHBP) Variants

[0013] Factor H binding protein (fHBP), also referred to in the literature as GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086), and "741," refers to a class of Neisseria meningitidis polypeptides that are found in nature as lipoproteins on the surface of the bacteria. Based on the variability and immunological cross-reactivity of the amino acid sequence, Neisseria meningitidis strains have been subdivided into three fHBP variant groups: variant 1 (V1), variant 2 (V2) and variant (V3), which are further divided into subvariants fHbp-1.x, fHbp-2.x and fHbp-3.x, where x represents a specific peptide subvariant. Chimeric variants such as v1-2,3.x also exist (see "Structural characterization of a cross-protective natural chimera of factor H binding protein from meningococcal serogroup B strain NL096", "Computational and Structural Biotechnology Journal", vol. 20, 2070-2081, 18 April 2022; US9266942 B2).

[0014] The present invention provides an fHBP variant 1, referred to as fHBP V1, whose amino acid sequence is amino acids 1 to 259 of SEQ ID NO: 7.

[0015] The present invention provides an fHBP variant 2, referred to as fHBP V2, whose amino acid sequence is amino acids 1-253 of SEQ ID NO: 8.

[0016] The present invention provides an fHBP variant 3, referred to as fHBP V3, whose amino acid sequence is amino acids 1-260 of SEQ ID NO: 9.

[0017] B. fHBP chimeric protein

[0018] The two fHBP domains involved in the present invention are named domain 1 and domain 2, respectively, wherein domain 1 corresponds to the N-terminal domain of fHBP known in the art, and domain 2 corresponds to the C-terminal domain of fHBP known in the art.

[0019] The present invention provides an fHBP chimeric protein, which comprises different domains of different fHBP variants, wherein the domains are selected from domain 1 or domain 2 of fHBP V1, domain 1 or domain 2 of fHBP V2, and domain 1 or domain 2 of fHBP V3.

[0020] The fHBP V1 domain 1 provided by the present invention exists in the following three forms:

[0021] 1. fHBP V1 domain 1:

[0022] Amino acid sequence:

[0023] Nucleotide sequence:

[0024] 2. fHBP V1 domain 1 truncated form 1 (abbreviated as: fHBP V1 domain 1 T1 ):

[0025] Amino acid sequence:

[0026] Nucleotide sequence:

[0027] 3. fHBP V1 domain 1 truncated form 2 (abbreviated as: fHBP V1 domain 1 T2 ):

[0028] Amino acid sequence:

[0029] Nucleotide sequence:

[0030] The amino acid and nucleotide sequences of fHBP V1 domain 2 provided by the present invention are as follows:

[0031] Amino acid sequence:

[0032] Nucleotide sequence:

[0033] The amino acid and nucleotide sequences of fHBP V2 domain 1 provided by the present invention are as follows:

[0034] Amino acid sequence:

[0035] Nucleotide sequence:

[0036] The amino acid and nucleotide sequences of fHBP V2 domain 2 provided by the present invention are as follows:

[0037] Amino acid sequence:

[0038] Nucleotide sequence:

[0039] The fHBP V3 domain 1 provided by the present invention exists in the following four forms:

[0040] 1. fHBP V3 domain 1:

[0041] Amino acid sequence:

[0042] Nucleotide sequence:

[0043] 2. fHBP V3 domain 1 truncated form 1 (abbreviated as: fHBP V3 domain 1 T1 ):

[0044] Amino acid sequence:

[0045] Nucleotide sequence:

[0046] 3. fHBP V3 domain 1 truncated form 2 (abbreviated as: fHBP V3 domain 1 T2 ):

[0047] Amino acid sequence:

[0048] Nucleotide sequence:

[0049] 4. fHBP V3 domain 1 truncated form 3 (abbreviated as: fHBP V3 domain 1 T3 ):

[0050] Amino acid sequence:

[0051] Nucleotide sequence:

[0052] The amino acid and nucleotide sequences of fHBP V3 domain 2 provided by the present invention are as follows:

[0053] Amino acid sequence:

[0054] Nucleotide sequence:

[0055] In some embodiments, the amino acid sequence of fHBP V1 domain 1 of the present invention has 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more identity to SEQ ID NO: 54, 55, or 56, and is derived from a subvariant of fHBP V1.

[0056] In some embodiments, the amino acid sequence of fHBP V1 domain 2 of the present invention has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% identity to SEQ ID NO: 57 and is derived from a subvariant of fHBP V1.

[0057] Preferably, the fHBP V1 subvariant is selected from v1.1, v1.4, v1.13, v1.15, v1.14, v1.10, v1.260, v1.510, v1.90, v1.275, v1.697, v1.226, v1.110, v1.249, v1.108, v1.227, v1.215 and v1-2,3.x, or other known fHBPV1 subvariants.

[0058] In some embodiments, the amino acid sequence of domain 1 of fHBP V2 of the present invention has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% identity to SEQ ID NO: 58 and is derived from a subvariant of fHBP V2.

[0059] In some embodiments, the amino acid sequence of fHBP V2 domain 2 of the present invention has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% identity to SEQ ID NO: 59 and is derived from a subvariant of fHBP V2.

[0060] Preferably, the fHBP V2 subvariant is selected from v2.16, v2.19, v2.21, v2.22, v2.24 and v1-2,3.x, or other known fHBPV2 subvariants.

[0061] In some embodiments, the amino acid sequence of fHBP V3 domain 1 of the present invention has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% identity to SEQ ID NO: 60, 61, 62, or 63, and is derived from a subvariant of fHBP V3.

[0062] In some embodiments, the amino acid sequence of fHBP V3 domain 2 of the present invention has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% identity to SEQ ID NO: 64 and is derived from a subvariant of fHBP V3.

[0063] Preferably, the fHBP V3 subvariant is selected from v3.116, v3.28, v3.31, v3.45, v3.42 and v1-2,3.x, or other known fHBPV3 subvariants.

[0064] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V3 domain 1 and fHBP V1 domain 2 in sequence from N-terminus to C-terminus.

[0065] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V1 domain 1 and fHBP V2 domain 2 in sequence from N-terminus to C-terminus.

[0066] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V2 domain 1 and fHBP V1 domain 2 in sequence from N-terminus to C-terminus.

[0067] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V1 domain 1 and fHBP V3 domain 2 in sequence from N-terminus to C-terminus.

[0068] In some embodiments, the fHBP chimeric protein provided by the present invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0069] In some embodiments, the fHBP chimeric protein provided by the present invention is used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0070] The neutralizing antibodies, protective antibodies or bactericidal antibodies mentioned above refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0071] C.fHBP fusion protein

[0072] The present invention provides an fHBP fusion protein, which comprises two different fHBP chimeric proteins. The two fHBP chimeric proteins can be connected in series through a linker. The fHBP fusion protein can simultaneously induce antibodies against fHBP V1, V2 and V3.

[0073] In some embodiments, the fHBP fusion protein provided by the present invention is composed of two fHBP chimeric proteins connected in series, and the specific structure is fHBP V3 domain 1-fHBP V1 domain 2-fHBP V1 domain 1-fHBP V2 domain 2.

[0074] Preferably, the structure of the fHBP fusion protein is fHBP V3 domain 1-fHBP V1 domain 2-linker-fHBP V1 domain 1-fHBP V2 domain 2.

[0075] In some embodiments, the fHBP fusion protein provided by the present invention is composed of two fHBP chimeric proteins connected in series, and the specific structure is fHBP V1 domain 1-fHBP V2 domain 2-fHBP V3 domain 1-fHBP V1 domain 2.

[0076] Preferably, the structure of the fHBP fusion protein is fHBP V1 domain 1-fHBP V2 domain 2-linker-fHBP V3 domain 1-fHBP V1 domain 2.

[0077] In some embodiments, the fHBP fusion protein provided by the present invention is composed of two fHBP chimeric proteins connected in series, and the specific structure is fHBP V2 domain 1-fHBP V1 domain 2-fHBP V1 domain 1-fHBP V3 domain 2.

[0078] Preferably, the structure of the fHBP fusion protein is fHBP V2 domain 1-fHBP V1 domain 2-linker-fHBP V1 domain 1-fHBP V3 domain 2.

[0079] The above-mentioned linker is any commonly used linker peptide in the art, such as a flexible linker peptide, a rigid linker peptide, a semi-rigid linker peptide, including but not limited to G n or GSGGGG or (EAAAK) n or an amino acid sequence of GGSGGEAAAK, wherein n can be an integer greater than 0 and less than or equal to 10, preferably n is 1, 2, 3 or 4.

[0080] In some embodiments, the fHBP fusion protein provided by the present invention comprises the amino acid sequence shown in any one of the following (1)-(9):

[0081] (1) amino acid sequence 1-520 of SEQ ID NO: 1;

[0082] (2) amino acid sequence from positions 1 to 514 of SEQ ID NO: 2;

[0083] (3) amino acid sequence 1-518 of SEQ ID NO: 17;

[0084] (4) amino acid sequence 1-507 of SEQ ID NO: 18;

[0085] (5) amino acid sequence 1-523 of SEQ ID NO: 19;

[0086] (6) amino acid sequence 1-528 of SEQ ID NO: 20;

[0087] (7) amino acid sequence 1-511 of SEQ ID NO: 21;

[0088] (8) amino acid sequence 1-516 of SEQ ID NO: 22; or

[0089] (9) Amino acid sequence from position 1 to position 521 of SEQ ID NO: 23.

[0090] In some embodiments, the fHBP fusion protein provided by the present invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0091] In some embodiments, the fHBP fusion protein provided by the present invention is used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0092] The neutralizing antibodies, protective antibodies or bactericidal antibodies mentioned above refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0093] D. Immunogenic complex

[0094] The present invention provides an immunogenic complex, which comprises a protein formed by a covalent binding reaction between an antigen component and a particle protein.

[0095] The antigen component in the immunogenic complex of the present invention comprises fHBP protein, wherein the fHBP protein is selected from the above-mentioned fHBP chimeric protein, fHBP fusion protein or fHBP variant, and the fHBP variant is selected from fHBP V1, fHBP V2 or fHBP V3.

[0096] The present invention provides an immunogenic complex comprising:

[0097] (1) an antigen component comprising fHBP protein;

[0098] (2) A granular protein component comprising nanoparticle protein.

[0099] The present invention provides an immunogenic complex comprising:

[0100] (1) an antigen component comprising fHBP protein and binding peptide 1;

[0101] (2) A particle protein component comprising nanoparticle protein and binding peptide 2.

[0102] The present invention provides an immunogenic complex comprising:

[0103] (1) an antigen component comprising fHBP protein, connecting peptide 1 and binding peptide 1;

[0104] (2) A particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2.

[0105] In some embodiments, in any immunogenic complex provided by the present invention, the antigen component is formed by fusing the fHBP protein at the C-terminus via a connecting peptide 1 and a binding peptide 1.

[0106] 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.

[0107] 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.

[0108] In some embodiments, in any one of the immunogenic complexes provided herein, the binding peptide 1 comprises an amino acid sequence as shown in AHIVMVDAYKPTK (SEQ ID NO: 47), hereinafter referred to as "4T".

[0109] 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: 48), hereinafter referred to as "4C".

[0110] In some embodiments, both the antigen component and the granule protein component comprise a histidine tag.

[0111] 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.

[0112] 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.

[0113] In some embodiments, in any one of the immunogenic complexes provided by the invention, the fHBP protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-23.

[0114] Preferably, in any one of the immunogenic complexes provided by the invention, the fHBP protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-2, 5-9, 12-13, and 17-33.

[0115] Table 1: Structure of the antigen component (including control molecules) and granule protein component of the present invention

[0116] The nucleic acid sequence corresponding to the amino acid sequence of any one of SEQ ID NOs: 1-23 is shown in any one of SEQ ID NOs: 24-46, and the amino acid sequences of SEQ ID NOs: 1-23 are listed in Table 2. The nucleic acid sequences of SEQ ID NOs: 24-46 are listed in Table 3.

[0117] Table 2: Amino acid sequences shown in SEQ ID NOs: 1-23 (RD012-1 to RD012-9, RD012-13 to RD012-26 molecules)

[0118] Table 3: Nucleic acid sequences shown in SEQ ID NO: 24-46

[0119] (Encoding molecules RD012-1 to RD012-9, RD012-13 to RD012-26)

[0120] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 1 comprises (GGGGS) n 、(EAAAK) n 、(GSGGSG) n 、(GGS) nwherein 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 connecting peptide 1 is preferably GSGGSG (SEQ ID NO: 53).

[0121] In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 2 comprises an amino acid sequence of (GGS)n, (GGGGS)n, (EAAAK)n, or (GSGGSG)n, where n is an integer greater than 0 and less than or equal to 10. In some embodiments, in any one of the immunogenic complexes provided herein, the connecting peptide 2 is preferably GGSGGSGGS (SEQ ID NO: 49) or GGSGGSGGSGGS (SEQ ID NO: 50).

[0122] 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 a 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 (designated "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; the resulting particle protein components are designated NPM-4C, AP205-4C, or Ferritin-4C.

[0123] 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: 1-2, 17-23, and the amino acid sequence of the granule protein component is shown in SEQ ID NO: 52.

[0124] Specifically, in some alternative embodiments, under suitable reaction conditions, such as room temperature, any of the aforementioned antigen components is subjected to a conjugation reaction with the particle protein component, and the binding peptide 1 of the antigen component is coupled to the binding peptide 2 of the particle protein component via a covalent bond, thereby forming the immunogenic complex. Immunogenic complexes formed using different nanoparticle proteins, such as NPM, AP205, or Ferritin, are designated fHBP-NPM, fHBP-AP205, or fHBP-Ferritin, respectively.

[0125] In some embodiments, the present invention provides a method for the covalent binding reaction of an antigen component and 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 reaction is incubated at 22°C for 24 hours. Endotoxin levels are detected to be less than 100 EU / ml, meeting the requirements for large-scale production.

[0126] In some embodiments, the present invention provides an immunogenic complex comprising:

[0127] (1) an antigen component comprising fHBP protein, connecting peptide 1 and binding peptide 1;

[0128] (2) a particle protein component comprising nanoparticle protein, connecting peptide 2 and binding peptide 2;

[0129] 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 (SEQ ID NO: 53).

[0130] 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, where n can be an integer greater than 0 and less than or equal to 10, preferably GGSGGSGGS (SEQ ID NO:49) or GGSGGSGGSGGS (SEQ ID NO:50); the nanoparticle protein is NPM, AP205 or ferritin.

[0131] 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:51.

[0132] Preferably, in any immunogenic complex provided by the present invention, the particle protein component comprises NPM-4C, as shown in SEQ ID NO:52, and NPM-4C comprises a fusion protein obtained by connecting the binding peptide 2 as shown in SEQ ID NO:48 to the nanoparticle protein NPM as shown in SEQ ID NO:51 via the connecting peptide 2.

[0133] Furthermore, the present invention provides a method for preparing an immunogenic complex:

[0134] (1) The fHBP antigen component and granule protein component encoding genes were respectively connected into expression vectors to construct expression recombinant plasmids;

[0135] (2) constructing a recombinant strain capable of expressing the fHBP antigen component and granule protein component in host cells;

[0136] (3) using the recombinant strain to express the fusion protein and purifying the recombinant fusion protein;

[0137] (4) The above antigen component and the granule protein component are subjected to a covalent binding reaction to obtain an immunogenic complex.

[0138] Preferably, the immunogenic complex obtained in the above step (4) is purified to obtain a vaccine stock solution.

[0139] Preferably, in the method for preparing an immunogenic complex for preventing or treating diseases related to group B Neisseria meningitidis, in step (1), the plasmid for expressing the group B Neisseria meningitidis antigen component can be pcDNA3.4, and the vector for expressing the nanoparticle can be pET-28a(+) or pET-30a(+).

[0140] In some embodiments, the immunogenic complexes provided by the present invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0141] In some embodiments, the immunogenic complexes provided by the present invention are used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0142] The neutralizing antibodies, protective antibodies or bactericidal antibodies mentioned above refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0143] In the method for preparing an immunogenic complex for preventing or treating group B Neisseria meningitidis-related diseases described in the present invention, in step (2), the host cell expressing the group B Neisseria meningitidis antigen is CHO, and the host cell expressing the granule protein carrier is E. coli.

[0144] In step (3) of the method for preparing an immunogenic complex for preventing or treating group B Neisseria meningitidis-related diseases described in the present invention, the method for purifying the granule protein can be referred to patent CN 114395015 B.

[0145] E. Nucleic acid

[0146] The present invention provides the encoding nucleotides of the fHBP protein and antigen components. The specific nucleotide sequences can be easily obtained by those skilled in the art through conventional means such as codon tables.

[0147] 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.

[0148] Preferably, the nucleotide sequence of the fHBP protein provided by the present invention is shown in SEQ ID NO: 24-46, see Table 3 for details.

[0149] 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.

[0150] In some embodiments, the vectors used in the present invention include pcDNA3.4, pET-28a(+), and pET-30a(+).

[0151] 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.

[0152] F. Immunization Composition

[0153] The present invention provides an immune composition comprising the fHBP protein or immunogenic complex of the present invention.

[0154] Optionally, the immunogenic composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0155] Preferably, the pharmaceutically acceptable carrier includes a stabilizer, an excipient, a surfactant, a buffer, and a pH regulator. 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 regulator is hydrochloric acid.

[0156] In some embodiments, the immune composition provided by the present invention is an injection or a lyophilized preparation, preferably a lyophilized preparation.

[0157] In some embodiments, the immune composition of the present invention comprises one or more fHBP proteins as described above.

[0158] In some embodiments, the present invention provides an immune composition obtained by mixing the above-mentioned fHBP chimeric proteins, wherein the mixture has better immunogenicity than a simple mixture of three fHBP variants (ie, V1, V2, and V3).

[0159] Preferably, the immune composition of the present invention is obtained by mixing the above-mentioned fHBP chimeric proteins in equal weight ratios.

[0160] In some embodiments, the immune composition provided by the present invention comprises a chimeric protein fHBP V3 domain 1-fHBP V1 domain 2 and a chimeric protein fHBP V1 domain 1-fHBP V2 domain 2.

[0161] In some embodiments, the immune composition provided by the present invention comprises a chimeric protein fHBP V2 domain 1-fHBP V1 domain 2 and a chimeric protein fHBP V1 domain 1-fHBP V3 domain 2.

[0162] In some embodiments, the immunogenic composition of the present invention comprises the aforementioned fHBP V1, fHBP V2, and fHBP V3.

[0163] Preferably, the immune composition is obtained by mixing the above-mentioned fHBP V1, fHBP V2 and fHBP V3 in equal mass ratios.

[0164] In some embodiments, the immune composition of the present invention comprises one or more of the above-mentioned immunogenic complexes. Preferably, the immune composition of the present invention comprises the following two immunogenic complexes: (1) an immunogenic complex comprising an antigen component set forth in SEQ ID NO:5 and a granule protein component set forth in SEQ ID NO:52; and (2) an immunogenic complex comprising an antigen component set forth in SEQ ID NO:6 and a granule protein component set forth in SEQ ID NO:52.

[0165] Preferably, the immune composition of the present invention comprises the following three immunogenic complexes: (1) an immunogenic complex formed by the antigen component shown in SEQ ID NO: 7 and the granule protein component shown in SEQ ID NO: 52; (2) an immunogenic complex formed by the antigen component shown in SEQ ID NO: 8 and the granule protein component shown in SEQ ID NO: 52; (3) an immunogenic complex formed by the antigen component shown in SEQ ID NO: 9 and the granule protein component shown in SEQ ID NO: 52.

[0166] In some embodiments, the immunological compositions of the present invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0167] In some embodiments, the immunological compositions of the present invention are used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0168] The neutralizing antibodies, protective antibodies or bactericidal antibodies mentioned above refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0169] The protective effect for Neisseria meningitidis can be measured by the conventional means of this area, for example, in clinical trials, epidemiological measurements are carried out, or indirect measures are used to confirm that the immunogenic composition causes serum antibacterial antibodies (SBA) to be replied in the receptor. In SBA, in the presence of complement (preferably human complement, although often using baby rabbit complement on the contrary), the serum of the receptor from the composition is hatched with target bacteria (Neisseria meningitidis in the present invention), and the killing of bacteria is evaluated to measure SBA activity under the various dilutions of serum. The results observed in the SBA assay can be strengthened by implementing competitive SBA to provide further indirect evidence of the immunogenic activity of the target antigen. In competitive SBA, the serum of the receptor containing one or more antigens is pre-incubated with the one or more antigens, and subsequently in the presence of human complement with the target bacteria. The killing of bacteria is then evaluated, and if the antibacterial antibodies in the serum of the receptor combine the target antigen and therefore cannot combine the surface antigens on the bacteria during the pre-incubation stage, the killing of bacteria will be reduced or abolished.

[0170] G. Vaccine

[0171] The present invention provides a vaccine comprising the immune composition of the present invention and an adjuvant.

[0172] In some embodiments, the vaccine of the present invention contains an adjuvant selected from at least one of: aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvant, water-oil adjuvant, cytokine, CpG DNA, flagellin, genetically engineered attenuated toxin, immunostimulatory complex, liposome, saponin, and Poly (I: C) adjuvant.

[0173] The aluminum salt adjuvant in the present invention is aluminum hydroxide adjuvant, specifically Alhydrogel.

[0174] 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 a water-in-oil 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.

[0175] The squalene water-oil adjuvant used in the specific embodiment of the present invention comprises the following ingredients: Span 85 0.5%, Tween 80 0.5%, squalene 4.2%, sodium citrate 0.264%, and citric acid 0.016%.

[0176] In some embodiments, the vaccines provided by the present invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0177] In some embodiments, the vaccines provided by the present invention are used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0178] The neutralizing antibodies, protective antibodies or bactericidal antibodies mentioned above refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0179] The present invention provides a method for preparing a vaccine for preventing or treating group B Neisseria meningitidis-related diseases, wherein the purified antigen component and the granule protein component are covalently bound to each other through binding peptide 1 and binding peptide 2, and then mixed 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.

[0180] The present invention provides a method for preparing a vaccine for preventing or treating diseases associated with serogroup B Neisseria meningitidis, wherein the aqueous 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.

[0181] The present invention further provides a kit comprising a serogroup B Neisseria meningitidis vaccine and the necessary instruments and containers for administering the vaccine, specifically including a needle and syringe, a powder container, and a solvent container. Preferably, the vaccine of the present invention is marketed as a pre-filled syringe containing the immune composition and adjuvant.

[0182] H. Medical use

[0183] The present invention provides medical uses of the above-mentioned serogroup B Neisseria meningitidis fHBP protein, immunogenic complex, immune composition, and vaccine, including the prevention or treatment of serogroup B Neisseria meningitidis-related diseases.

[0184] The present invention provides pharmaceutical preparations of the above-mentioned serogroup B Neisseria meningitidis fHBP protein, immunogenic complex, immune composition, and vaccine, and the pharmaceutical preparations are used for preventing or treating diseases related to serogroup B Neisseria meningitidis.

[0185] Diseases associated with serogroup B Neisseria meningitidis in the present invention include, but are not limited to, cerebrospinal meningitis, sepsis, septic shock, arthritis, myocarditis, pericarditis, endophthalmitis, meningitis, hemorrhagic skin diseases, activated fibrinolysis and blood coagulation, organ dysfunction, such as renal, pulmonary and heart failure, adrenal hemorrhage and muscle infarction, capillary leak, edema, peripheral limb ischemia, and respiratory distress syndrome.

[0186] Preferably, the disease associated with group B Neisseria meningitidis is cerebrospinal meningitis.

[0187] I. Beneficial Effects

[0188] Compared with the prior art, the present invention has the following beneficial effects:

[0189] (1) It was found that the fusion protein of the characteristic amino acid sequences of the three variants of fHBP may have a technical problem of broken bands, and the molecular sizes of the two bands are close, which is not conducive to production and purification. This technical problem was solved through structural optimization, while ensuring effective immunogenicity, reducing the complexity that may be brought to the production process from the source, improving production efficiency, and saving the cost of large-scale production.

[0190] (2) In the first aspect, the fusion protein (e.g., RD012-1) comprising the characteristic amino acid sequences of the three fHBP variants of the present invention has a superior immunological effect on inducing anti-fHBP than the immunological effect of a simple mixture of the three fHBP variants of the present invention (e.g., RD012-7, RD012-8, and RD012-9). Furthermore, the fusion protein (e.g., RD012-1) comprising the characteristic amino acid sequences of the three fHBP variants of the present invention has a superior immunological effect on inducing anti-fHBP than the immunological effect of a simple mixture of the molecules RD012-13 and RD012-14 (used as control molecules in the present invention) known in the art.

[0191] Secondly, the immunogenicity of the nanoparticle vaccine antigen constructed by combining the antigen components with NPM particles is also better than that of the simple recombinant protein antigen.

[0192] Thirdly, they discovered that a nanoparticle immunogenic complex (RD012-1 NPM) constructed by binding a fusion protein containing the characteristic amino acid sequences of the three fHBP variants to NPM particles achieved the same immunogenicity as that elicited by combining the three fHBP variants (RD012-7, RD012-8, and RD012-9) with NPM to form nanoparticle immunogenic complexes. This approach streamlines the preparation process, reduces production costs, and improves production efficiency.

[0193] (3) The nanoparticle vaccine product provided by the present invention can induce better cellular immunity and antibody immune responses compared with the two MenB vaccines Bexsero and Trumenba that have been on the market, and can achieve similar effects with significantly reduced dosage.

[0194] (4) The granular protein of the present invention is prepared by fermentation with E. coli and chromatography purification, and the fHBP antigen is prepared by culture in a BL21 (DE3) cell reactor and chromatography purification. Both are suitable for industrial large-scale production and have the advantages of high expression, stable process and yield, and simple operation. The amount of granular protein components in one batch can be combined with multiple batches of fHBP antigen, thereby improving production efficiency. Compared with conventional recombinant protein vaccines, the nanoparticle vaccine of the present invention has the advantage of higher immune protection level at the same or lower dose, which can save the cost of large-scale production.

[0195] (5) The preparation method of the recombinant granule protein product provided by the present invention is suitable for industrial production, can reduce the cost of large-scale industrial production, is simple to operate, and reduces the amount of organic solvent used in subsequent chromatography purification; the product prepared using the recombinant granule protein provided by the present invention effectively reduces the side effects caused by the residues of impurities, host proteins, organic solvents, exogenous DNA, antibiotics, bacterial endotoxins and other substances in the granules, thereby improving safety.

[0196] (6) The present invention also studies the effects of linkers with different structures on the expression of fusion proteins.

[0197] Fourthly, the fHBP fusion protein of the present invention can trigger an effective bactericidal response against group B Neisseria meningitidis strains, and the bactericidal activity of the nanoparticle vaccine obtained after connection to NPM is further improved, that is, the vaccine of the present invention can induce the production of high levels of antibodies with broad-spectrum bactericidal activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0198] 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.

[0199] Figures 1A-1E: Figure 1A shows the SDS-PAGE identification results of the purified fusion proteins RD012-1 and RD012-2; Figure 1B shows the SDS-PAGE identification results of the purified RD012-1M and RD012-2M; Figure 1C shows the particle size identification results of RD012-1M and RD012-2M; Figure 1D shows the negative staining electron microscopy results of RD012-1M and RD012-2M; Figure 1E shows the SDS-PAGE identification results of RD012-3, RD012-4, and RD012-4M;

[0200] Figures 2A-2F: Figures 2A-2C are graphic representations of the antibody titers against fHBP V1, V2, and V3 induced by vaccines RD012-1, RD012-1M, RD012-2, RD012-2M, RD012-5 / 6admix, RD012-5M / M6 admix, RD012-7 / 8 / 9admix, and RD012-7M / 8M / 9M admix; Figures 2D-2F are graphic representations of the antibody titers against fHBP V1, V2, and V3 induced by vaccines RD012-1, RD012-2, RD012-5 / 6admix, RD012-7 / 8 / 9admix, and a control molecule;

[0201] Figure 3A-Figure 3B: Figure 3A shows the SDS-PAGE identification results of the purified fusion proteins RD012-15, RD012-16, and RD012-17; Figure 3B shows the SDS-PAGE identification results of the purified fusion proteins RD012-15M and RD012-16M;

[0202] Figures 4A-4C: Graphical representation of antibody titers against fHBP V1, V2, and V3 induced by vaccines RD012-1M, RD012-2M, RD012-7M / 8M / 9M admix, RD012-15, RD012-15M, RD012-16, and RD012-16M;

[0203] Figures 5A-5C: Figure 5A shows the SDS-PAGE identification results of the purified fusion proteins RD012-19, RD012-20, RD012-22, and RD012-23; Figure 5B shows the SDS-PAGE identification results of the purified fusion proteins RD012-21, RD012-24, RD012-25, and RD012-26; Figure 5C shows the SDS-PAGE identification results of the purified fusion proteins RD012-20M, RD012-21M, RD012-22M, RD012-23M, RD012-24M, RD012-25M, and RD012-26M;

[0204] Figures 6A-6C are graphic representations of the antibody titers against fHBP V1, V2, and V3 induced by vaccines RD012-1M, RD012-2M, RD012-20M, RD012-21M, RD012-22M, RD012-23M, RD012-24M, RD012-25M, RD012-26M, Trumenba, and Bexsero. DETAILED DESCRIPTION

[0205] 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.

[0206] Example 1: Expression and purification of fHBP fusion protein molecules

[0207] 1. Experimental Materials:

[0208] Capsule filter (Bricap C01: 180 cm2) was purchased from Cobetter, membrane package was purchased from Millipore, HisTrap excel was purchased from Cytiva, and molecular sieve (Superdex 200 pg 10 / 300 GL) was purchased from Cytiva.

[0209] 2. Experimental methods:

[0210] 2.1. The fHBP protein molecules shown in Table 1 were prepared using conventional molecular cloning techniques.

[0211] 2.2. Induction conditions: Inoculate RD012 BL21 (DE3) monoclonal bacteria into 800 mL LB (Amp+) medium and culture at 37°C and 220 rpm for 4-5 h. When the OD600 of the culture solution is approximately 0.6-0.8, transfer the culture solution to 18°C ​​and add IPTG to a final concentration of 0.5 mM. Induce protein expression at 200 rpm for 16 h.

[0212] 2.3. Harvest the bacteria by centrifugation: Collect the cultured cells by centrifugation at 7000g at room temperature, discard the culture medium, and resuspend the cells in 80 mL of 150 mM NaCl, 20 mM Tris 7.4 solution.

[0213] 2.4 Ultrasonic disruption: Place the resuspended bacterial solution in an ice-water bath for ultrasonic disruption. Use horn #2, 50% power, sonicate for 3 seconds, pause for 7 seconds, for a total ultrasonic duration of 12 minutes.

[0214] 2.5. Collect the target protein by centrifugation: 13000g, 4℃, 30min, and collect the supernatant of cell disruption.

[0215] 2.6. Purify the target protein using Histrap: Wash buffer: 20mM Tris-HCl, 150mM NaCl, pH 7.4; Elution buffer: 20mM Tris-HCl, 150mM NaCl, 500mM Imidazole, pH 7.4. Purify using a Histrap Excel-5ml NI column. Equilibrate the Histrap Excel-5ml column with Wash buffer for 10CV before loading the sample. Rinse the column with Wash buffer for 10CV after loading. Wash with 2% Elution buffer for 10CV to remove impurities. Elute the target protein linearly for 15CV using 2%-100% Elution buffer. After elution, check protein purity by SDS-PAGE.

[0216] 2.7 SEC Purification of Target Protein: After nickel column purification, the target protein was collected and concentrated to 1 ml using a concentrator. The protein was then separated and purified using a molecular sieve (Superdex 200pg 10 / 300GL) in a purification buffer of 20mM Tris-HCl, 150mM NaCl, pH 7.4. After elution, protein purity was assessed by SDS-PAGE. SDS-PAGE analysis of purified RD012-1 and RD012-2 is shown in Figure 1A. Protein concentration was determined by BCA assay and stored at an appropriate temperature for subsequent binding reactions.

[0217] Example 2: Expression and purification of binding peptide 2-NPM fusion protein

[0218] The nanoparticle protein NPM (as shown in SEQ ID NO: 51) is connected to the binding peptide 2 (as shown in SEQ ID NO: 48) at the N-terminus through the connecting peptide 2 (as shown in SEQ ID NO: 50), thereby forming the binding peptide 2-NPM fusion protein, namely NPM-4C (as shown in SEQ ID NO: 52), as the particle protein component. The relevant sequences of NPM and NPM-4C are shown in Table 4.

[0219] Table 4: NPM, NPM-4C fusion protein sequences in the examples of this application

[0220] The coding gene of the fusion protein is expressed in Escherichia coli. After the bacteria are harvested, they need to be crushed by high-pressure homogenization to release the target protein and clarify the liquid. The main purpose is to remove bacterial fragments and impurity proteins. The liquid clarification is mainly completed by heat treatment. A two-step heating method is used for heat treatment. The supernatant after E. coli crushing is subjected to the first step heating and the second step heating (ie, "two-step heating"), and the impurity removal effect of the two-step heating step and the purity of the recombinant granular protein component are measured.

[0221] 60 g of wet E. coli cells collected by centrifugation were resuspended in 240 ml of buffer (20 mM Tris-HCl, 2 mM PMSF, pH 9.0) and disrupted using a high-pressure homogenizer at 1000 bar. After centrifugation, 280 ml of supernatant was collected, of which 40 ml was subjected to a two-step heating procedure. The supernatant after disruption, the supernatant from the first heating centrifugation step, and the resuspension of the pellet from the second heating centrifugation step were analyzed by SDS-PAGE.

[0222] The specific method is shown in Table 5. In the first heating step, adjust the pH to 9.0, heat in an 80°C water bath for 1 hour, return to room temperature, and centrifuge to collect approximately 35 ml of supernatant. In the second heating step, add 35 ml of 100 mM Tris-HCl, 5 mM EDTA, 4% Triton, pH 7.4 buffer, followed by 7 ml of 1 M Tris-HCl, pH 7.4, and mix thoroughly. Heat in a 60°C water bath for 10 minutes, immediately centrifuge to collect the precipitate, and re-dissolve the precipitate in 20 mM Tris-HCl, 5 mM EDTA, pH 9.0 buffer.

[0223] Table 5: Two-step heating extraction method for recombinant granule protein component products

[0224] After the two-step heating process, adding different concentrations of urea and sodium chloride before chromatographic purification can significantly reduce the presence of unidentified substances near the target recombinant granule protein band. The optimal process conditions for pretreatment of recombinant granule protein samples before Fractogel DEAE M chromatography are soaking in 8M urea and 50-200mM sodium chloride.

[0225] The recombinant granule protein fraction sample was purified using ion exchange and hydrophobic chromatography. The first chromatographic purification step was performed using a Fractogel DEAE M chromatography process. Specific steps and parameters are shown in Table 6. The Fractogel DEAE M elution pool was first diluted with buffer and stabilized with 50% (w / v) sucrose to prevent precipitation of the recombinant granule protein during the next chromatography step. Specific parameters are shown in Table 7. The fraction was then purified using a hydrophobic Octyl Bestarose 4FF chromatography process (second chromatographic purification step). Specific steps and parameters are shown in Table 8.

[0226] First step chromatography method: chromatography filler - Fractogel DEAE M, retention time - 12.5min

[0227] Table 6: First step chromatography method

[0228] Table 7: Sample dilution method before the second step chromatography

[0229] Second step chromatography method: Chromatographic filler - Octyl Bestarose 4FF, retention time - 12.5min

[0230] Table 8: Second step chromatography method

[0231] Results and Analysis:

[0232] Purity testing revealed that after further purification using the above chromatographic medium combination, the purity of the obtained product could reach over 99.0%.

[0233] Example 3: Binding of fHBP and NPM, and Particle Characterization

[0234] 1. Binding of fHBP and NPM and product purification:

[0235] 1. Experimental methods:

[0236] MenB antigens RD012-1 and RD012-2 were mixed with carrier NPM-4C at a BCA protein ratio of 6:1. A 50% sucrose stock solution was added to a final concentration of approximately 25%. A 1M Tris-HCl pH 7.4 stock solution was added at 10% of the total reaction volume to stabilize the pH. The binding reaction was performed at 22°C for 24 hours. The binding product was separated and purified using molecular sieves (purification buffer: 20mM Tris-HCl, 150mM NaCl, 25% sucrose, pH 7.4). The RD012-NPM fraction was collected. The purified RD012-NPM was identified by SDS-PAGE, and its particle size was determined and negatively stained by electron microscopy.

[0237] The specific experimental operations are as follows:

[0238] (1) TEM detection

[0239] Negatively stained samples were prepared using the flotation method. A 400-mesh support film was used, pre-treated for hydrophilicity, and deionized water and 2% uranyl formate negative staining solution were prepared. 3 μL of the prepared protein sample (0.12 mg / mL) was dripped directly onto one side of the support film. After a one-minute timer, excess liquid was removed from the edge of the support film using clean filter paper. After drying slightly, the sample was quickly rinsed twice with a drop of deionized water. Then, 5 μL of negative staining solution was added, followed by a final rinse with 5 μL of negative staining solution. Finally, 5 μL of negative staining solution was added dropwise for one minute. After the sample was removed, the sample was lifted with tweezers and the stain removed using filter paper. A thin layer was allowed to air dry before examination. Examination was performed under a 120 kV transmission electron microscope (FERRITINI Tecnai Spirit). The overall staining of the support film was observed at low magnification, with wells of appropriate thickness selected for observation. Under high magnification, a suitable area was photographed and stored.

[0240] (2) SDS-PAGE method

[0241] The SDS-PAGE test sample was prepared with LDS sample loading buffer (4x) plus reducing agent DTT, heated at 70°C for 5min, cooled to room temperature, centrifuged at 10,000rpm for 20s, vortexed to mix, and the final loading amount was 5μg. The test sample and non-prestained protein molecular weight standard were loaded onto a 4-12% Bis-Tris gel, matched with MES electrophoresis buffer (1 swim), set the voltage to 150V, and electrophoresis lasted for about 60 minutes. After the electrophoresis is completed, take out the gel, place the gel in a clean container, add an appropriate amount of Coomassie Brilliant Blue staining solution to cover the gel, and stain on a shaker for 2h. After the staining is completed, pour out the staining solution, soak in purified water for decolorization, continue decolorization on a shaker until the gel background color is completely removed, and use a GelDoc Go gel imager to take pictures of the gel.

[0242] (3)DLS method

[0243] Dilute the purified test sample to a concentration of 0.25 mg / mL. Using a Zetasizer Lab instrument, inject ≥1 mL of the sample into the sample cell. Run the instrument for detection. Analyze the data based on the Z-Average (nm) and Polydispersity Index (PI) values, as well as the Size Distribution by Intensity / Volume curve, and report the results.

[0244] 2. Experimental results:

[0245] SDS-PAGE analysis of the purified NPM VLPs of RD012-1 and RD012-2 was performed, as shown in Figure 1B. The particle size and negative staining results of the NPM VLPs of RD012-1 and RD012-2 are shown in Figures 1C and 1D. The results showed that the expression level of the RD012-1 fusion protein was significantly reduced by removing the 4T sequence and switching the His tag from the C-terminus to the N-terminus, i.e., RD012-3, as shown in Figure 1E. Furthermore, placing the 4T sequence between the two chimeric proteins, i.e., RD012-4, significantly reduced its binding rate to the VLP vector NPM-4C, as shown in Figure 1E. Therefore, the RD012-1 and RD012-2 molecules were designed as the optimal fusion protein molecules.

[0246] Example 4: Animal Immunity Test

[0247] (1) Experimental materials

[0248] Mice: 5-6 weeks old female BALB / c mice (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.)

[0249] Adjuvant: aluminum hydroxide adjuvant

[0250] Other reagents and consumables are commercial conventional reagents and consumables.

[0251] (2) Experimental steps

[0252] As shown in Table 9, the same vaccine construct was administered intramuscularly on days 0 and 14, with a dose of 9 μg / dose / animal, mixed with 75 μg of aluminum hydroxide adjuvant. Blood was collected on day 28 for serum analysis of binding antibodies.

[0253] Table 9: Animal Immunization Scheme 1

[0254] (3) Combined antibody detection:

[0255] ELISA plates were first coated with the three fHBP variants and blocked in Blocker Casein in PBS (ThermoFisher) for 1-4 hours. Serum samples collected from mice in each experimental group at the end of immunization (as listed in Table 9) were serially diluted threefold from 1:300 to 656:100 and added to each well. The negative control consisted of the sample dilutions. The plates were incubated for 2-3 hours. The plates were then incubated with an HRP-conjugated goat anti-mouse secondary antibody for 1 hour. The plates were then developed using TMB substrate. After development, the reaction was terminated with 1M hydrochloric acid and the absorbance (OD) was measured using a microplate reader at a dominant wavelength of 450 nm and a reference wavelength of 620 nm. Sample OD = OD450 - OD620. The results were measured within 5 minutes of termination. See Figures 2A-F.

[0256] The results of the mouse immunization test found that the RD012-1 / 2 fusion protein induced antibody titers against fHBP variants V1, V2, and V3 that were approximately 10.5-fold, 3.6-fold, and 10.3-fold higher than the equivalent mixture of the three fHBP variants (RD012-7 / 8 / 9admix). Therefore, the fusion protein containing the characteristic amino acid sequences of the three fHBP variants induced better antibody titers against fHBP than the simple mixture of the three fHBP variants, as shown in Figures 2A-F. In addition, the immunogenicity of the nanoparticle vaccine RD012-1M / 2M antigen constructed by binding RD012-1 / 2 to NPM particles was also better than that of the simple recombinant protein antigen (i.e., RD012-1 / 2), as shown in Figures 2A-2C.

[0257] Example 5: Optimization of RD012 fusion protein molecular design

[0258] 1. Experimental methods:

[0259] The results of Example 4 above show that the fusion protein antigen constructed by the present invention, comprising the characteristic amino acid sequences of three fHBP variants, has a broad spectrum and is effective. However, the results of Figure 1A show that the RD012-1 protein has broken bands, which would complicate the product production process. Based on previous research data, we interchanged the tandem order of the two chimeric proteins of RD012-1 / 2 to obtain two molecules of RD012-15 / 16, and truncated the N-terminal amino acid portion of RD012-1 to obtain the RD012-17 molecule. The recombinant protein antigens and VLP particle antigens of RD012-15 / 16 / 17 were prepared according to the methods of Examples 1-2. As shown in Figure 3, neither the RD012-15 / 16 recombinant protein antigens nor the VLP particle antigens produced broken bands, while the RD012-17 fusion protein still produced broken bands. The immunogenicity of RD012-15 / 16 and RD012-1 / 2 was compared according to the method of Example 4. The animal immunization scheme is shown in Table 10. The results of the ELISA binding antibody titer test are shown in Figures 4A-C.

[0260] Table 10: Animal Immunization Scheme 2

[0261] 2. Experimental results:

[0262] The experimental results show that although swapping the tandem sequence of the two chimeric proteins in the fusion protein can solve the problem of RD012-1 molecular breakage, the antibody titer induced against fHBP variants V2 and V3 is also reduced compared with the previous method.

[0263] Example 6: Optimization of RD012 fusion protein molecular design

[0264] 1. Experimental methods:

[0265] To address the issue of fragmentation bands in the RD012-1 fusion protein, we used LC-MS to determine the intact molecular weight of the target molecule and the fragmented molecule. We also compared the peptide maps of the two molecules using MS to identify the fragmented amino acid sequence. Previous research data indicated that the fragmentation sequence did not affect immunogenicity, so we directly truncated the fragmented amino acids to generate the molecule RD012-18. Recombinant protein antigens derived from RD012-18 were generated, revealing a single target band, but protein expression levels were more than halved. Subsequently, the tandem flexible linker between the two chimeric proteins of the fusion protein molecule was replaced with a semi-rigid linker (GGSGGEAAAK) and rigid linkers of varying lengths (EAAAK)n to generate molecules RD012-19, 20, 22, and 23. The results, as shown in Figure 5A, show that the rigid linker was virtually eliminated, eliminating the fragmentation bands. In addition, the fHBP type of the prevalent strains of group B meningococci in China is mainly V2. To further optimize the antigen-induced antibody titer against the fHBP variant V2, we replaced the fHBP V3 domain 1 and fHBP V2 domain 2 in the fusion protein molecule RD012-1 chimeric protein with fHBP V2 domain 1 and fHBP V3 domain 2 in the same tandem order to obtain RD012-21 / 24 / 25 / 26 fusion protein antigen molecules. The prepared recombinant protein antigen is shown in Figure 5B. The VLP particle antigen of the above fusion protein molecule was prepared according to the method of Example 1, and the results are shown in Figure 5C. The immunogenicity of the above optimized molecules was compared with that of RD012-1 / 2 according to the experimental method of Example 4, and the animal immunization scheme is shown in Table 11.

[0266] Preparation method of Trumenba vaccine: 60μg lipid-modified fHBP V1 protein and 60μg lipid-modified fHBP V3 protein are adsorbed on aluminum phosphate adjuvant.

[0267] Bexsero vaccine preparation method: 50μg fHBP V1 fusion protein, 50μg NHBA fusion protein, 50μg NadA protein and 25μg outer membrane vesicles (OMV) adsorbed on aluminum hydroxide adjuvant.

[0268] Table 11. Animal immunization plan 3

[0269] 2. Experimental results:

[0270] The results are shown in Figures 6A-6C: Compared with RD012-1M / 2M, RD012-20M / 22M / 23M induced increased antibody titers against fHBPV1, V2, and V3 variants, indicating that replacing the tandem flexible linker between the two chimeric proteins with a rigid linker can improve the immunogenicity of the fusion protein. In addition, the RD012-21M / 24M / 25M / 26M fusion protein antigen molecules, which replaced the fHBP V3 domain 1 and fHBP V2 domain 2 in the fusion protein molecule RD012-1 chimeric protein with fHBP V2 domain 1 and fHBP V3 domain 2 in the same tandem order, also induced significantly higher antibody titers against fHBP compared to RD012-1M / 2M. In general, the immune effect of the vaccine RD012-1M / 2M / 20M / 21M / 22M / 23M / 24M / 25M / 26M of the present invention is better than that of the control commercially available vaccine.

[0271] Example 7: Serum bactericidal activity assay (SBA)

[0272] First, the MenB strain was spread on a chocolate plate and cultured overnight at 37°C and 5% CO2. A single colony was inoculated into Mueller-Hinton medium and the initial OD value of the bacterial solution was 600 Controlled at 0.05-0.08, cultured in a shaking incubator at 37°C until OD 600 Reach 0.23-0.24, and measure bacterial activity. The test serum of the immune groups RD012-24M, Trumenba and Bexsero in Example 6 was heat-inactivated at 56°C for 1 hour. The total volume in each well was 50 μL, including 25 μL of test serum diluted two-fold, 12.5 μL of bacterial working solution and 12.5 μL of rabbit complement. The controls included: serum incubated with complement serum, immune serum incubated with bacteria, and inactivated complement. After shaking and mixing, culture on a shaker at 37°C for 2-4 hours. Take 10 μL of each sample and add it to a Mueller-Hinton agar plate and culture it at 37°C overnight. The overnight cultured agar plate was developed with TTC, the bacterial colonies were counted, and the bactericidal titer was calculated.

[0273] As shown in Table 12, the vaccine RD012-24M of the present invention demonstrated superior bactericidal activity against subtype V2 strains compared to the commercial vaccine, and comparable bactericidal activity against subtype V1 and V3 strains. This suggests that RD012-24M exhibits a broader spectrum of immune responses and bactericidal activity than the commercial vaccine.

[0274] Table 12. Bactericidal activity of sera from immune mice against different fHBP variant strains

[0275] Based on a comprehensive analysis of the above experimental results, it can be concluded that the RD012 series of proteins, immunogenic complexes, immune compositions and vaccines have good immunogenicity and bactericidal activity, and also have a broad-spectrum immune effect.

[0276] 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 fHBP protein, characterized in that: The fHBP protein is as shown in any one of the following (1) or (2): (1) an fHBP chimeric protein, wherein the chimeric protein comprises different domains of different fHBP variants, wherein the domains are selected from domain 1 or domain 2 of fHBP variant 1 (fHBP V1), domain 1 or domain 2 of fHBP variant 2 (fHBP V2), and domain 1 or domain 2 of fHBP variant 3 (fHBP V3); (2) fHBP fusion protein, wherein the fHBP fusion protein comprises two different fHBP chimeric proteins, the two fHBP chimeric proteins can be connected in series via a linker, and the fHBP fusion protein can simultaneously induce antibodies against fHBP V1, V2 and V3; The fHBP V1 is selected from v1.1, v1.4, v1.13, v1.15, v1.14, v1.10, v1.260, v1.510, v1.90, v1.275, v1.697, v1.226, v1.110, v1.249, v1.108, v1.227, v1.215 and v1-2,3.x; the fHBP V2 is selected from v2.16, v2.19, v2.21, v2.22, v2.24 and v1-2,3.x; the fHBP V3 is selected from v3.116, v3.28, v3.31, v3.45, v3.42 and v1-2,3.x.

2. The fHBP protein according to claim 1, wherein the fHBP chimeric protein is as shown in any one of the following (A)-(D): (A) includes fHBP V3 domain 1 and fHBP V1 domain 2; (B) includes fHBP V1 domain 1 and fHBP V2 domain 2; (C) comprises fHBP V2 domain 1 and fHBP V1 domain 2; and / or (D) includes fHBP V1 domain 1 and fHBP V3 domain 2; Preferably, the fHBP fusion protein comprises the fHBP chimeric proteins shown in (A) and (B), and / or comprises the fHBP chimeric proteins shown in (C) and (D).

3. The fHBP protein according to claim 1 or 2, characterized in that: The amino acid sequence of fHBP V1 domain 1 has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% identity with SEQ ID NO: 54, 55 or 56; The amino acid sequence of fHBP V1 domain 2 has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% identity to SEQ ID NO: 57; The amino acid sequence of fHBP V2 domain 1 is 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more identical to SEQ ID NO: 58; The amino acid sequence of fHBP V2 domain 2 has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% identity to SEQ ID NO: 59; The amino acid sequence of fHBP V3 domain 1 has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% identity with SEQ ID NO: 60, 61, 62 or 63; And / or the amino acid sequence of fHBP V3 domain 2 has greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90% identity with SEQ ID NO:

64.

4. The fHBP protein according to any one of claims 1 to 3, characterized in that The amino acid sequence of the fHBP V1 domain 1 is selected from SEQ ID NO: 54, 55 or 56, the amino acid sequence of the fHBP V1 domain 2 is selected from SEQ ID NO: 57, the amino acid sequence of the fHBP V2 domain 1 is selected from SEQ ID NO: 58, the amino acid sequence of the fHBP V2 domain 2 is selected from SEQ ID NO: 59, the amino acid sequence of the fHBP V3 domain 1 is selected from SEQ ID NO: 60, 61, 62 or 63, and the amino acid sequence of the fHBP V3 domain 2 is selected from SEQ ID NO: 64; optionally, the linker is selected from G n 、GSGGGG、(EAAAK) n Or an amino acid sequence of GGSGGEAAAK, wherein n can be an integer greater than 0 and less than or equal to 10, preferably n is 1, 2, 3 or 4.

5. An immunogenic complex comprising: (1) an fHBP antigen component, comprising the fHBP protein according to any one of claims 1 to 4 or comprising an fHBP variant, wherein the fHBP variant is selected from fHBP V1, fHBP V2 or fHBP V3; (2) a granule protein component, which comprises nanoparticle protein; Preferably, the antigen component further comprises binding peptide 1, the particle protein component further comprises binding peptide 2, the fHBP protein and binding peptide 1 form a fusion protein, the nanoparticle protein and binding peptide 2 form a fusion protein, and 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; Preferably, the binding peptide 1 contains the amino acid sequence shown in SEQ ID NO:53, and the binding peptide 2 contains the amino acid sequence shown in SEQ ID NO:

54.

6. The immunogenic complex according to claim 5, 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 the fHbp protein at the C-terminus through the connecting peptide 1 and the binding peptide 1; the particle protein component is formed by fusing the nanoparticle protein at the N-terminus through the connecting peptide 2 and the binding peptide 2; 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.

7. The immunogenic complex according to any one of claims 5-6, wherein the amino acid sequence of the fHBP antigen component is selected from any one of SEQ ID NOs: 1-9, 12-23; preferably, the amino acid sequence of the fHBP antigen component is selected from any one of SEQ ID NOs: 1-2, 5-9, 20-23.

8. The immunogenic complex according to any one of claims 5 to 7, wherein the nanoparticle protein is NPM, AP205 or Ferritin protein; preferably, the amino acid sequence of NPM is as shown in SEQ ID NO:

52.

9. A method for preparing an immunogenic complex for preventing or treating diseases associated with group B Neisseria meningitidis: (1) Connecting the encoding genes of the fHbp antigen component and the granule protein component according to any one of claims 5 to 8 into expression vectors to construct expression recombinant plasmids; (2) constructing a recombinant strain capable of expressing the fHbp antigen component and the granule protein component in a host cell; (3) using the recombinant strain to express the fusion protein and purifying the recombinant fusion protein; (4) subjecting the antigen component purified in step (3) and the granule protein component to a covalent binding reaction to obtain the immunogenic complex; Preferably, in step (1), the plasmid for expressing the antigen component is selected from pcDNA3.4, and the vector for expressing the granule protein is selected from pET-28a(+) or pET-30a(+); Optionally, in step (2), the host cell expressing the antigen component is CHO, and the host cell expressing the granule protein is E. coli.

10. An immune composition, characterized in that The immune composition is as shown in any of the following: (1) containing one or more fHBP proteins as described in claims 1 to 4; or (2) containing one or more immunogenic complexes as described in claims 5-8; Optionally, the immune composition further comprises a pharmaceutically acceptable carrier.

11. An immune composition according to claim 10, comprising any two fHBP chimeric proteins of (A)-(D) according to claim 2; Alternatively, the immunogenic composition comprises three variants of fHBP: fHBP V1, fHBP V2, and fHBP V3; Optionally, the amino acid sequence of fHBP V1 is amino acids 1-259 of SEQ ID NO:7, the amino acid sequence of fHBP V2 is amino acids 1-253 of SEQ ID NO:8, and the amino acid sequence of fHBP V3 is amino acids 1-260 of SEQ ID NO:

9.

12. An immune composition according to any one of claims 10-11, characterized in that: The pharmaceutically acceptable carrier comprises a stabilizer, an excipient, a surfactant, a buffer, and a pH regulator. 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 regulator is hydrochloric acid.

13. A vaccine for preventing or treating diseases associated with group B Neisseria meningitidis, characterized in that: It contains the immune composition according to any one of claims 10 to 12 and an adjuvant.

14. The vaccine according to claim 13, wherein the adjuvant is selected from at least one of aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvant, water-oil adjuvant, cytokine, CpG DNA, flagellin, genetically engineered attenuated toxin, immunostimulatory complex, liposome, saponin, and Poly (I: C) adjuvant; preferably, the aluminum salt adjuvant is aluminum hydroxide, the water-oil adjuvant is squalene, and the saponin adjuvant comprises QS-21; Optionally, the adjuvant contains 3%-5% squalene, 0.4%-1% Span 85, 0.4%-1% Tween 80, 10mM citrate buffer or 0.1-0.5% sodium citrate, 0.01-0.05% citric acid (w / v), 3.5%-4.5% (w / v) squalene, and the squalene adjuvant may be MF59 or SWE; preferably, the adjuvant ingredients are: 0.5% Span 85, 0.5% Tween 80, 4.2% squalene, 0.264% sodium citrate, and 0.016% citric acid.

15. Use of the fHBP protein according to any one of claims 1 to 4, the immunogenic complex according to any one of claims 5 to 8, the immune composition according to any one of claims 10 to 12, and the vaccine according to any one of claims 13 to 14 for preparing a drug for preventing or treating diseases related to group B Neisseria meningitidis.

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