Immune composition product for preventing or treating group B neisseria meningitidis related diseases and preparation method thereof

Abstract

The invention provides an immune composition product for preventing or treating group B neisseria meningitidis related diseases and a preparation method thereof, the immune composition product comprises fHbp fusion protein, and the fusion protein is chimeric protein constructed by disassembling two structural domains of three fHbp variants and recombining, and constructing a fusion protein containing three fHbp variant characteristic amino acid sequences on the basis of the chimeric protein. Furthermore, the fusion protein is used as a vaccine antigen to prepare a nanoparticle vaccine. The fusion protein provided by the invention has an immunological effect better than that brought by simply mixing three fHbp variants, and compared with two MenB vaccines Bexseo and Trumenba which are sold on the market, the nano-particle vaccine provided by the invention has better cellular immunity and antibody immune response.

Description

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 group B Neisseria meningitidis related diseases and a production method thereof. Background Art

[0002] Epidemic cerebrospinal meningitis (EMM) is an infectious disease caused by Neisseria meningitidis, which is transmitted through the respiratory tract and has acute cerebrospinal meningitis and sepsis as the main symptoms. It is a Class B infectious disease stipulated by my country. According to the characteristics of capsular polysaccharides, Neisseria meningitidis can be divided into 13 serogroups, including A, B, C, Y, and W135. The currently available quadrivalent meningococcal polysaccharide or polysaccharide-protein conjugate vaccine can effectively prevent pathogenic serogroups A, C, Y, and W135, and reduce the occurrence of invasive meningococcal disease.

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

[0004] At present, the development of MenB vaccine mainly focuses on non-capsular polysaccharide immunogens. Factor H binding protein (fHBP) is the most promising vaccine antigen. As a lipoprotein, it is expressed on the cell membrane of almost all MenB strains. An important function of fHBP is to bind to human complement regulatory factor H, allowing Neisseria meningitidis to escape the bactericidal effect of the host and improve its survival ability in the blood. When fHBP is used as a MenB vaccine antigen, the anti-fHBP antibodies elicited can directly activate the classical complement pathway for bacteriolysis and also prevent fH from binding to the bacterial surface. fHBP is a surface antigen with highly diverse sequences and can be divided into three antigenic variant groups: variant 1 (V1), variant 2 (V2) and variant 3 (V3), which include a large number of subvariants. The phylogenetic tree of fHBP protein shows that the V1 variant accounts for about 70% of the isolated bacteria, and variants V2 and V3 account for about 30%. There is no obvious cross-protection reaction between variant V1 and the other two variants V2 and V3. In addition, different determination methods may have different definitions of fHBP protein structure. MASCIONI et al. determined the full-length molecular structure of non-esterified fHBP protein in aqueous solution by nuclear magnetic resonance, revealing that fHBP protein consists of two domains: C-terminal antiparallel β-barrel structure and "Taco-shaped" N-terminal β-pleated sheet, which are connected by a 5' residue flexible linker.

[0005] There are currently two main MenB vaccines on the market abroad, Bexsero (GSK) and Trumenba (Pfizer). Bexsero (also known as C4MenB) contains outer membrane vesicles (OMVs) from the epidemic strain of group B Neisseria meningitidis NZ98 / 254 prevalent in New Zealand, as well as preparations of five meningococcal antigens: Neisserial heparin-binding protein A (NHBA), factor H binding protein (fHBP) variant 1.1, Neisserial adhesion protein A (NadA), and auxiliary proteins GNA1030 and GNA2091. Trumenba contains two lipidated MenB fHBP antigens A05 and B01 adsorbed on aluminum phosphate. In the above two MenB protein vaccines currently on the market, the fHBP protein is expressed in the form of a single variant or fused with a non-fHBP protein. The products target the MenB strains prevalent in Europe and the United States, but do not have broad protection against other variant strains. The fHBP protein sequences in the MenB protein vaccines marketed overseas are mainly targeted at VI and V3, while the fHBP type of the domestic prevalent strains is mainly V2. It can be seen that the MenB vaccines currently marketed overseas cannot cover the prevalent strains in my country.

[0006] Nanoparticle vaccines use nanomaterials as antigen carriers. Compared with traditional vaccines, nanoparticle vaccines have good antigen encapsulation and stable structure, and have advantages in antigen assembly and antigen presentation. Nanoparticle vaccines mainly include four types: virus-like particle nanovaccines, self-assembled protein nanovaccines, polymer particle nanovaccines, and inorganic particle nanovaccines. In the field of self-assembled protein nanovaccines, researchers recently discovered that nanoparticle protein mi3 can spontaneously form highly ordered 60-subunit dodecahedral nanoparticles based on the computational design of icosahedral nanocages, display SpyCatcher on the surface of nanoparticles and connect with antigen proteins. The results confirmed that this protein nanoparticle vaccine completely based on computational design can also induce a strong antibody response, and is expected to be used in the preparation of new self-assembled protein nanovaccines.

[0007] There is currently no serogroup B meningococcal vaccine on the market in China, and there are very few serogroup B meningococcal nanoparticle vaccines under development. The two vaccines currently on the market are mainly targeted at MenB strains prevalent in Europe and the United States, so it is of great significance to develop a MenB vaccine that is widely protective, safer and more effective, and reduces production costs. Summary of the invention

[0008] In order to solve the current situation that the technology and supply types of group B Neisseria meningitidis vaccines are insufficient, especially the basic blank supply of group B Neisseria meningitidis nanoparticle vaccine, the present invention provides a group B Neisseria meningitidis nanoparticle vaccine and a preparation method thereof.

[0009] The present invention structurally changes the sequence of the fHBP antigen protein of MenB, specifically, disassembles the two structural domains of the three variants of fHBP and reassembles them to construct a chimeric protein, and then constructs a fusion protein containing the characteristic amino acid sequences of the three variants of fHBP based on the chimeric protein. The fusion protein is used as a vaccine antigen to prepare a MenB nanoparticle vaccine with broad protectiveness and effectiveness.

[0010] The nanoparticle vaccine provided by the present invention is a vaccine formed based on nanoparticle protein, and the nanoparticle protein is mainly used for displaying antigens.

[0011] A. Factor H Binding Protein (fHBP) Variants

[0012] 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 naturally present as lipoproteins on the surface of the bacteria. Based on the variability and immune 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. There are also chimeric variants such as v1-2,3.x (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; US9266942B2).

[0013] The present invention provides an fHBP variant 1, referred to as fHBPV1, whose amino acid sequence is amino acids 1 to 259 of SEQ ID NO: 7.

[0014] The present invention provides an fHBP variant 2, referred to as fHBPV2, whose amino acid sequence is amino acids 1 to 253 of SEQ ID NO: 8.

[0015] The present invention provides an fHBP variant 3, referred to as fHBPV3, whose amino acid sequence is amino acids 1 to 260 of SEQ ID NO:9.

[0016] B. fHBP chimeric protein

[0017] 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.

[0018] 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 fHBPV3.

[0019] The fHBP V1 domain 1 provided by the present invention exists in the following three forms:

[0020] 1. fHBP V1 domain 1:

[0021] Amino Acid Sequence:

[0022] SSGGGGSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIA(SEQID NO: 54);

[0023] Nucleotide sequence:

[0024] TCTAGCGGAGGTGGTGGTTCCGGTGGTGGCGGTGTTACCGCGGACATCGGTACCGGTTTGGCGGATGCGCTTACGGCACCGCTGGATCACAAAGACAAGGGTCTGAAGTCCTTGACCTTGGAGGACTCCATTTCGCAAAACGGCACCCTGACGCTTAGCGCTCAAGGCGCAGAAAAGACCTACGGCAATGGTGACTCACTGAACACCGGT AAACTGAAGAACGACAAAGTTAGCCGCTTCGACTTTATCCGTCAGATTGAGGTTGATGGTCAGCTGATTACCCTGGAAAGCGGGGAGTTTCAGGTATATAAGCAATCCCATTCCGCGCTGACTGCCCTGCAGACCGAACAAGAACAGGACCCGGAGCACTCCGAAAAAATGGTTGCAAAGCGCCGTTTTCGTATTGGTGACATTGCG(SEQ ID NO: 65);

[0025] 2. fHBP V1 domain 1 truncated form 1 (abbreviated as: fHBP V1 domain 1 T1 ):

[0026] Amino Acid Sequence:

[0027] GSGGGGVTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIA(SEQ ID NO: 55);

[0028] Nucleotide sequence:

[0029] GGCAGCGGAGGTGGCGGTGTTACCGCGGACATTGGTACTGGTTTGGCGGACGCTCTGACTGCGCCACTGGACCACAAAGATAAGGGCTTGAAGTCATTGACCTTGGAGGATAGCATTAGCCAAAATGGCACCCTGACACTGTCTGCGCAGGGTGCCGAGAAGACCTACGGTAATGGGGACTCTCTGAACACTGGCAAACTTAAAAACGATAAGGTGAGCCGCTTCGATTTTATCCGTCAAATTGAAGTGGACGGTCAGCTGATTACGCTGGAGAGCGGCGAGTTTCAGGTTTATAAACAAAGCCATTCCGCTCTGACGGCTTTGCAAACCGAACAAGAACAGGACCCGGAGCACTCTGAAAAGATGGTGGCTAAACGTCGTTTCCGTATTGGCGACATTGCA(SEQ ID NO: 66);

[0030] 3. fHBP V1 domain 1 truncation 2 (abbreviation: fHBP V1 domain 1 T2 ):

[0031] Amino acid sequence:

[0032] VTADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSEKMVAKRRFRIGDIA(SEQ ID NO: 56);

[0033] Nucleotide sequence:

[0034] GTGACCGCGGACATCGGCACTGGTCTGGCTGATGCCCTCACTGCGCCTCTGGACCACAAGGATAAGGGTTTGAAGTCCCTCACCCTGGAGGACAGCATCAGCCAAAATGGTACGCTGACCCTGAGCGCTCAGGGCGCGGAGAAGACCTACGGTAATGGTGATTCTCTGAATACCGGTAAGCTGAAGAACGACAA GGTGTCTCGCTTCGACTTCATCCGTCAGATTGAGGTTGACGGCCAGCTGATTACCTTGGAAAGCGGTGAGTTCCAGGTTTATAAACAAAGCCATTCCGCGCTGACCGCGCTGCAAACCGAACAGGAACAGGATCCGGAGCACAGCGAAAAAATGGTCGCGAAGCGCCGCTTTCGTATCGGTGATATCGCA(SEQ ID NO: 67).

[0035] The amino acid and nucleotide sequences of fHBP V1 domain 2 provided by the present invention are as follows:

[0036] Amino Acid Sequence:

[0037] GEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAVAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGEKAQEVAGSAEVETANGIHHIGLAAKQ (SEQ ID NO: 57);

[0038] Nucleotide sequence:

[0039] GGCGAACACACCAGCTTTGATAAGCTGCCAAAAGATGTCATGGCAACCTACCGTGGTACGGCGTTCGGCTCGGACGACGCGGGTGGCAAATTAACTTACACCATTGACTTTGCAGCGAAGCAGGGCCATGGCAAAATCGAGCACCTGAAGTCCCCGGAACTGAACGTGGACCTGGCAGTGGC GTACATCAAACCGGACGAAAAGCATCATGCTGTGATCAGCGGTTCGGTGCTGTATAACCAAGATGAGAAAGGCAGCTATAGCCTGGGTATTTTTGGCGAGAAGGCGCAGGAGGTGGCGGGTTCCGCCGAGGTTGAAACCGCGAATGGCATCCATCACATTGGGTTGGCGGCAAAACAG(SEQ ID NO: 68).

[0040] The amino acid and nucleotide sequences of fHBP V2 domain 1 provided by the present invention are as follows:

[0041] Amino Acid Sequence:

[0042] SSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLG (SEQ ID NO: 58);

[0043] Nucleotide sequence:

[0044] AGTTCAGGTGGAGGGGGAGTAGCAGCTGACATCGGTGCAGGCCTCGCAGACGCGCTGACCGCGCCGCTTGATCATAAGGATAAGAGCCTGCAGAGCCTGACATTGGATCAGTCCGTGCGTAAAAATGAAAAGTTGAAGCTAGCGGCCCAAGGTGCGGAAAAGACCTACGGCAACGGCGACTCCTTGAACACCGGTAAGCTGAA GAACGACAAGGTGAGCAGATTCGACTTCATCCGCCAGATCGAAGTCGACGGTCAGCTGATTACCTTGGAATCTGGCGAGTTTCAGATCTATAAGCAGGACCACAGCGCGGTCGTGGCCCTGCAGATTGAGAAGATCAACAACCCGGATAAGATCGATAGCTTGATCAATCAACGTAGCTTCCTGGTTAGTGGTTTGGGC(SEQ ID NO: 69).

[0045] The amino acid and nucleotide sequences of fHBP V2 domain 2 provided by the present invention are as follows:

[0046] Amino Acid Sequence:

[0047] GEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ (SEQ ID NO: 59);

[0048] Nucleotide sequence:

[0049] GGCGAGCACACCGCGTTCAACCAGCTGCCTGATGGTAAAGCCGAGTATCACGGCAAGGCCTTCAGCAGCGATGACGCCGGGGGTAAGTTAACGTACACCATTGACTTTGCCGCGAAGCAAGGTCACGGGAAAATCGAGCACCTGAAGACCCGGAGCAAAATGTCGAGTTGGCTGCGCA GAGCTGAAGGCTGACGAAAAGAGCCATGCAGTCATCCTGGGCGATACCCGTTATGGTTCTGAAAAAAGGGTACGTACCACCTGGCCTTGTTCGGTGATCGTGCCCAAGAGATCGCGGGCTCTGCTACCGTTAAAATCGGCGAAAAAGTGCATGAAATCGGTATCGCGGGTAAACAG(SEQ ID NO:70).

[0050] The fHBP V3 structure provided by the present invention is present in the following four kinds of format:

[0051] 1.fHBP V3 structure 1:

[0052] amino acid sequence:

[0053] SSGSGSGGGGVAADIGTGLADALTAPLDHKDCGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLG(SEQID NO:60);

[0054] Nucleus sequence:

[0055] TCTAGCGGTTCAGGAAGTGGCGGAGGGGGGGTGGCAGCTGACATCGGCACTGGTCTGGCGGACGCTCTGACCGCTCCGCTGGACCACAAGGACAAAGGTTTGAAAAGCCTGACTTTGGAAGATTCGATCTCTCAGAATGGTACGCTGACCCTCAGCGCGCAAGGCGCGGAGAAGACCTTTAAAGTTGGTGATAAGGACAACAGCTTGAACACCGGTAAACTGAAAAACGATAAGATTAGCCGCTTCGACTTCGTGCAGAAAATCGAAGTGGACGGTCAGACCATTACCCTTGCGAGCGGCGAGTTCCAGATTTACAAGCAGGATCATAGTGCGGTTGTCGCATTACAAATCGAAAAAATCAATAATCCGGATAAGATTGATTCTCTCATCAACCAACGTAGCTTCTTGGTTAGCGGCCTGGGT(SEQ ID NO: 71).

[0056] 2. fHBP V3 domain 1 truncated form 1 (abbreviation: fHBP V3 domain 1 T1 ):

[0057] Amino acid sequence:

[0058] GSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLG(SEQ ID NO: 61);

[0059] Nucleotide sequence:

[0060] GGTTCGGGTGGTGGCGGGGTGGCAGCCGATATCGGCACCGGGCTTGCTGACGCGCTCACGGCTCCGCTGGACCACAAAGACAAGGGTTTAAAGAGCCTGACCCTGGAAGATAGCATTTCCCAGAACGGCACCCTGACCCTGTCTGCACAAGGTGCGGAGAAAACCTTTAAAGTAGGTGATAAGGACAACAGCCTAAACACCGGTAAGCTGAAAAATGACAAGATCTCTCGTTTCGACTTTGTTCAGAAAATCGAGGTGGATGGTCAGACCATTACCTTGGCTAGCGGCGAGTTCCAGATCTATAAACAAGACCATAGCGCGGTCGTGGCGTTACAGATCGAGAAGATTAACAACCCGGATAAGATCGATTCGCTGATTAACCAGCGTAGCTTTCTGGTTTCCGGACTCGGT(SEQ ID NO:72).

[0061] 3. fHBP V3 domain 1 truncation 2 (abbreviation: fHBP V3 domain 1 T2 ):

[0062] Amino acid sequence:

[0063] VAADIGTGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAEKTFKVGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLG(SEQ ID NO: 62);

[0064] Nucleotide sequence:

[0065] GTGGCAGCTGACATCGGCACTGGTCTGGCGGACGCTCTGACCGCTCCGCTGGACCACAAGGACAAAGGTTTGAAAAGCCTGACTTTGGAAGATTCGATCTCTCAGAATGGTACGCTGACCCTCAGCGCGCAAGGCGCGGAGAAGACCTTTAAAGTTGGTGATAAGGACAACAGCTTGAACACCGGTAAACTGAAAAAC GATAAGATTAGCCGCTTCGACTTCGTGCAGAAAATCGAAGTGGACGGTCAGACCATTACCCTTGCGAGCGGCGAGTTCCAGATTTACAAGCAGGATCATAGTGCGGTTGTCGCATTACAAATCGAAAAAATCAATAATCCGGATAAGATTGATTCTCTCATCAACCAACGTAGCTTCTTGGTTAGCGGCCTGGGT(SEQ ID NO: 73).

[0066] 4. fHBP V3 domain 1 truncated form 3 (abbreviated as: fHBP V3 domain 1 T3 ):

[0067] Amino Acid Sequence:

[0068] LNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLG (SEQ ID NO: 63);

[0069] Nucleotide sequence:

[0070] TTGAACACCGGTAAACTGAAAAACGATAAGATTAGCCGCTTCGACTTCGTGCAGAAAATCGAAGTGGACGGTCAGACCATTACCCTTGCGAGCGGCGAGTTCCAGATTTACAAGCAGGATCATAGTGCGGTTGTCGCATTACAAATCGAAAAAATCAATAATCCGGATAAGATTGATTCTCTCATCAACCAACGTAGCTTCTTGGTTAGCGGCCTGGGT (SEQ ID NO: 74).

[0071] The amino acid and nucleotide sequences of fHBP V3 domain 2 provided by the present invention are as follows:

[0072] Amino acid sequence: GEHTAFNQLPSGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGIAGKQ (SEQ ID NO: 64);

[0073] Nucleotide sequence:

[0074] GGCGAACACACCGCGTTCAACCAGCTGCCGAGCGGTAAGGCTGAGTATCACGGCAAGGCGTTTAGCAGCGACGACGCAGGTGGTAAGCTGACGTACACCATTGACTTCGCCGCGAAACAAGGTCATGGTAAGATTGAGCATCTGAAAACTCCGGAGCAAAATGTTGAATTGGCGAGCGCT GAGCTGAAGGCAGATGAAAAGAGCCACGCAGTGATTCTGGGTGACACCCGTTATGGTTCGGAGGAAAAAGGCACCTACCATCTGGCATTGTTTGGCGACCGCGCACAGGAGATCGCGGGTTCGGCGACCGTTAAAATTCGTGAAAAAGTTCATGAAATCGGCATCGCTGGCAAGCAG(SEQ ID NO: 75).

[0075] 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 with SEQ ID NO: 54, 55, or 56, and is derived from a subvariant of fHBP V1.

[0076] In some embodiments, the amino acid sequence of domain 2 of fHBPV1 of the present invention has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 57, and is derived from a subvariant of fHBPV1.

[0077] Preferably, the fHBPV1 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.

[0078] In some embodiments, the amino acid sequence of domain 1 of fHBPV2 of the present invention has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 58, and is derived from a subvariant of fHBPV2.

[0079] In some embodiments, the amino acid sequence of domain 2 of fHBPV2 of the present invention has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 59, and is derived from a subvariant of fHBPV2.

[0080] 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.

[0081] In some embodiments, the amino acid sequence of fHBP V3 domain 1 of the present invention has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 60, 61, 62 or 63, and is derived from a subvariant of fHBPV3.

[0082] In some embodiments, the amino acid sequence of domain 2 of fHBPV3 of the present invention has more than 40%, more than 50%, more than 60%, more than 70%, more than 80% or more identity with SEQ ID NO: 64, and is derived from a subvariant of fHBPV3.

[0083] 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.

[0084] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V3 domain 1 and fHBPV1 domain 2 from N-terminus to C-terminus.

[0085] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V1 domain 1 and fHBPV2 domain 2 from N-terminus to C-terminus.

[0086] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V2 domain 1 and fHBPV1 domain 2 from N-terminus to C-terminus.

[0087] In some embodiments, the fHBP chimeric protein provided by the present invention includes fHBP V1 domain 1 and fHBPV3 domain 2 in sequence from N-terminus to C-terminus.

[0088] In some embodiments, the fHBP chimeric protein provided by the present invention can induce the production of neutralizing antibodies, protective antibodies or bactericidal antibodies.

[0089] In some embodiments, the fHBP chimeric protein provided by the present invention is used to prepare neutralizing antibodies, protective antibodies or bactericidal antibodies.

[0090] The above-mentioned neutralizing antibodies, protective antibodies or bactericidal antibodies refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0091] C. fHBP fusion protein

[0092] 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, and the fHBP fusion protein can simultaneously induce antibodies against fHBPV1, V2 and V3.

[0093] 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-fHBPV1 domain 2-fHBPV1 domain 1-fHBPV2 domain 2.

[0094] Preferably, the structure of the fHBP fusion protein is fHBP V3 domain 1-fHBPV1 domain 2-linker-fHBP V1 domain 1-fHBP V2 domain 2.

[0095] 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-fHBPV2 domain 2-fHBPV3 domain 1-fHBPV1 domain 2.

[0096] Preferably, the structure of the fHBP fusion protein is fHBP V1 domain 1-fHBPV2 domain 2-linker-fHBP V3 domain 1-fHBP V1 domain 2.

[0097] 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-fHBPV1 domain 2-fHBPV1 domain 1-fHBPV3 domain 2.

[0098] Preferably, the structure of the fHBP fusion protein is fHBP V2 domain 1-fHBPV1 domain 2-linker-fHBP V1 domain 1-fHBP V3 domain 2.

[0099] 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.

[0100] 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):

[0101] (1) amino acid sequence from position 1 to position 520 of SEQ ID NO: 1;

[0102] (2) amino acid sequence from position 1 to position 514 of SEQ ID NO: 2;

[0103] (3) amino acid sequence 1-518 of SEQ ID NO: 17;

[0104] (4) amino acid sequence 1-507 of SEQ ID NO: 18;

[0105] (5) amino acid sequence 1-523 of SEQ ID NO: 19;

[0106] (6) amino acid sequence 1-528 of SEQ ID NO: 20;

[0107] (7) amino acid sequence 1-511 of SEQ ID NO: 21;

[0108] (8) amino acid sequence 1 to 516 of SEQ ID NO: 22; or

[0109] (9) Amino acid sequence from positions 1 to 521 of SEQ ID NO: 23.

[0110] In some embodiments, the fHBP fusion protein provided by the present invention can induce the production of neutralizing antibodies, protective antibodies or bactericidal antibodies.

[0111] In some embodiments, the fHBP fusion protein provided by the present invention is used to prepare neutralizing antibodies, protective antibodies or bactericidal antibodies.

[0112] The above-mentioned neutralizing antibodies, protective antibodies or bactericidal antibodies refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0113] D. Immunogenic complex

[0114] 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.

[0115] 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 or fHBP fusion protein or fHBP variant, and the fHBP variant is selected from fHBPV1, fHBPV2 or fHBPV3.

[0116] The present invention provides an immunogenic complex comprising:

[0117] (1) an antigen component comprising fHBP protein;

[0118] (2) A granular protein component, which comprises nanoparticle protein.

[0119] The present invention provides an immunogenic complex comprising:

[0120] (1) an antigen component comprising fHBP protein and binding peptide 1;

[0121] (2) A particle protein component, which comprises nanoparticle protein and binding peptide 2.

[0122] The present invention provides an immunogenic complex comprising:

[0123] (1) an antigen component comprising fHBP protein, connecting peptide 1 and binding peptide 1;

[0124] (2) A particle protein component, which comprises nanoparticle protein, connecting peptide 2 and binding peptide 2.

[0125] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the antigen component is formed by fusing the fHBP protein with the binding peptide 1 via the connecting peptide 1 at the C-terminus.

[0126] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the particle protein component is formed by fusing the N-terminus of the nanoparticle protein with the binding peptide 2 via the connecting peptide 2.

[0127] 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.

[0128] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the binding peptide 1 contains an amino acid sequence as shown in AHIVMVDAYKPTK (SEQ ID NO: 47), hereinafter referred to as "4T".

[0129] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the binding peptide 2 contains an amino acid sequence as shown in DSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYL YPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 48), hereinafter referred to as "4C".

[0130] In some embodiments, both the antigen component and the granule protein component comprise a histidine tag.

[0131] 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 be combined to self-assemble to form a particle structure.

[0132] In some embodiments, the self-assembled 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 bacterial phage capsid protein AP205, etc.

[0133] 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.

[0134] 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, 17-33.

[0135] Table 1: Structure of the antigen component (including control molecules) and granule protein component of the present invention

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] The nucleic acid sequence corresponding to the amino acid sequence shown in any one of the above SEQ ID NOs: 1-23 is shown in any one of SEQ ID NOs: 24-46, and the amino acid sequences shown in SEQ ID NOs: 1-23 are shown in Table 2. The nucleic acid sequences shown in SEQ ID NOs: 24-46 are shown in Table 3.

[0142] Table 2: Amino acid sequences shown in SEQ ID NO: 1-23 (RD012-1 to RD012-9, RD012-13 to RD012-26 molecules)

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] Table 3: Nucleic acid sequences shown in SEQ ID NO: 24-46 (encoding RD012-1 to RD012-9, RD012-13 to RD012-26 molecules)

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the connecting peptide 1 comprises (GGGGS) n 、(EAAAK) n 、(GSGGSG) n 、(GGS) n The amino acid sequence of 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).

[0167] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the connecting peptide 2 comprises an amino acid sequence of (GGS)n, (GGGGS)n, (EAAAK)n, (GSGGSG)n, and 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 (SEQ ID NO: 49) or GGSGGSGGSGGS (SEQ ID NO: 50).

[0168] In some embodiments, in any of the above immunogenic complexes provided by the invention, the particle protein component is a fusion protein formed by connecting peptide 2 and binding peptide 2 at the N-terminus of the nanoparticle protein; preferably, the nanoparticle protein is NPM, AP205 capsidprotein3 (AP205) or Ferritin protein. Specifically, in some optional schemes, the binding peptide 2 (the binding peptide 2 is named "4C") is connected to the coding gene of the nanoparticle protein through the connecting peptide 2, inserted into a prokaryotic expression vector (such as pET-28a (+), pET-30a (+)), and expressed in E. coli cells to obtain a fusion protein of binding peptide 2 and nanoparticle protein, and the fusion protein can be purified by chromatography, for example, by anion exchange chromatography, hydrophobic chromatography, to obtain a product. The nanoparticle protein is preferably NPM, AP205 or Ferritin; the formed particle protein component is named NPM-4C, AP205-4C, Ferritin-4C.

[0169] 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.

[0170] Specifically, in some optional schemes, under appropriate reaction conditions, such as at room temperature, any of the above antigen components is subjected to a conjugation reaction with the particle protein component, and the binding peptide 1 of the antigen component is coupled with the binding peptide 2 of the particle protein component through covalent bonding, thereby forming the immunogenic complex. The immunogenic complexes formed by using different nanoparticle proteins, such as NPM, AP205 or Ferritin, are named fHBP-NPM, fHBP-AP205 or fHBP-Ferritin, respectively.

[0171] In some embodiments, the present invention provides a method for the covalent binding reaction of an antigen component and a granule protein component, wherein the antigen component and the granule protein component are mixed at a protein concentration ratio of 6:1 determined by the BCA method, 50% sucrose mother solution is added to a final sucrose concentration of about 25%, and 10% of the total reaction volume of 1M Tris-HCl pH7.4 mother solution is added to stabilize the pH. The reaction is carried out at 22°C for 24 hours. The endotoxin detection is less than 100EU / ml, which meets the requirements of large-scale production.

[0172] In some embodiments, the present invention provides an immunogenic complex comprising:

[0173] (1) an antigen component comprising fHBP protein, connecting peptide 1 and binding peptide 1;

[0174] (2) a particle protein component, which comprises a nanoparticle protein, a connecting peptide 2 and a binding peptide 2;

[0175] 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).

[0176] 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.

[0177] In some embodiments, in any one of the immunogenic complexes provided by the present invention, the nanoparticle protein NPM comprises the amino acid sequence shown in SEQ ID NO:51.

[0178] 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 and the nanoparticle protein NPM as shown in SEQID NO:51 via the connecting peptide 2.

[0179] Furthermore, the present invention provides a method for preparing an immunogenic complex:

[0180] (1) connecting the fHBP antigen component and the granule protein component encoding genes into expression vectors respectively to construct expression recombinant plasmids;

[0181] (2) constructing a recombinant strain capable of expressing the fHBP antigen component and the granule protein component in a host cell;

[0182] (3) using the recombinant strain to express the fusion protein and purify the recombinant fusion protein;

[0183] (4) The above antigen component and the granule protein component are subjected to a covalent binding reaction to obtain an immunogenic complex.

[0184] Preferably, the immunogenic complex obtained in the above step (4) is purified to obtain a vaccine stock solution.

[0185] 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 expressing the group B Neisseria meningitidis antigen component may be pcDNA3.4, and the vector expressing the nanoparticle may be pET-28a(+) or pET-30a(+).

[0186] In some embodiments, the immunogenic complexes provided by the present invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0187] In some embodiments, the immunogenic complexes provided by the present invention are used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0188] The above-mentioned neutralizing antibodies, protective antibodies or bactericidal antibodies refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0189] 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.

[0190] 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 refer to patent CN 114395015 B.

[0191] E. Nucleic Acids

[0192] The present invention provides the encoding nucleotides of the above-mentioned fHBP protein and antigen components, and the specific nucleotide sequences can be easily obtained by those skilled in the art through conventional means such as codon tables.

[0193] The present invention also provides the coding nucleotides of 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.

[0194] 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.

[0195] 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.

[0196] In some embodiments, the vectors used in the present invention include pcDNA3.4, pET-28a(+), and pET-30a(+).

[0197] 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.

[0198] F. Immunization Composition

[0199] The present invention provides an immune composition comprising the fHBP protein or the immunogenic complex of the present invention.

[0200] Optionally, the immunological composition of the present invention further comprises a pharmaceutically acceptable carrier.

[0201] 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.

[0202] In some embodiments, the immune composition provided by the present invention is an injection or a lyophilized preparation, preferably a lyophilized preparation.

[0203] In some embodiments, the immune composition described herein comprises one or more fHBP proteins described above.

[0204] 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).

[0205] Preferably, the immune composition of the present invention is obtained by mixing the above-mentioned fHBP chimeric proteins in equal weight ratios.

[0206] In some embodiments, the immune composition provided by the present invention comprises a chimeric protein fHBPV3 domain 1-fHBPV1 domain 2 and a chimeric protein fHBPV1 domain 1-fHBP V2 domain 2.

[0207] In some embodiments, the immune composition provided by the present invention comprises a chimeric protein fHBPV2 domain 1-fHBPV1 domain 2 and a chimeric protein fHBPV1 domain 1-fHBP V3 domain 2.

[0208] In some embodiments, the immune composition described in the present invention comprises the above-mentioned fHBPV1, fHBPV2 and fHBPV3.

[0209] Preferably, the immune composition is obtained by mixing the above-mentioned fHBPV1, fHBP V2 and fHBPV3 in equal mass ratios.

[0210] 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 formed by an antigen component shown in SEQ ID NO:5 and a granule protein component shown in SEQ ID NO:52; (2) an immunogenic complex formed by an antigen component shown in SEQ ID NO:6 and a granule protein component shown in SEQ ID NO:52.

[0211] Preferably, the immune composition of the present invention comprises the following three immunogenic complexes: (1) an immunogenic complex formed by an antigen component shown in SEQ ID NO:7 and a granule protein component shown in SEQ ID NO:52; (2) an immunogenic complex formed by an antigen component shown in SEQ ID NO:8 and a granule protein component shown in SEQ ID NO:52; (3) an immunogenic complex formed by an antigen component shown in SEQ ID NO:9 and a granule protein component shown in SEQ ID NO:52.

[0212] In some embodiments, the immunological compositions of the invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0213] In some embodiments, the immunological compositions of the invention are used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0214] The above-mentioned neutralizing antibodies, protective antibodies or bactericidal antibodies refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0215] The protective effect for Neisseria meningitidis can be measured by conventional means in this area, such as epidemiological measurements in clinical trials, or indirect measures are used to confirm that the immunogenic composition causes serum antibacterial antibodies (SBA) responses in the recipient. In the SBA assay, in the presence of complement (preferably human complement, although often using baby rabbit complement), the serum from the recipient of the composition is incubated with the target bacteria (Neisseria meningitidis in the present invention), and the killing of bacteria is evaluated to measure the SBA activity under various dilutions of the serum. The results observed in the SBA assay can be strengthened by implementing competitive SBA assays to provide further indirect evidence of the immunogenic activity of the target antigen. In the competitive SBA assay, the serum of the recipient of the immunogenic composition containing one or more antigens is pre-incubated with the one or more antigens, and then incubated with the target bacteria in the presence of human complement. Then the killing of bacteria is evaluated, and if the antibacterial antibodies in the serum of the recipient bind to the target antigen during the pre-incubation stage and therefore cannot bind to the surface antigens on the bacteria, the killing of bacteria will be reduced or abolished.

[0216] G. Vaccine

[0217] The present invention provides a vaccine comprising the immune composition of the present invention and an adjuvant.

[0218] 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.

[0219] The aluminum salt adjuvant in the present invention is an aluminum hydroxide adjuvant, specifically Alhydrogel.

[0220] 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, 0.01-0.05% citric acid (w / v). Squalene is preferably 3.5%-4.5%, more preferably 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.1%, 4.2%, 4.3%, 4.4%, 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.

[0221] The squalene water-oil adjuvant used in the specific embodiment of the present invention comprises: Span 85 0.5%, Tween 80 0.5%, squalene 4.2%, sodium citrate 0.264%, and citric acid 0.016%.

[0222] In some embodiments, the vaccines provided by the present invention can induce the production of neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0223] In some embodiments, the vaccines provided by the present invention are used to prepare neutralizing antibodies, protective antibodies, or bactericidal antibodies.

[0224] The above-mentioned neutralizing antibodies, protective antibodies or bactericidal antibodies refer to antibodies that can bind to corresponding live bacteria, activate the complement system, and cause the bacteria to dissolve and die, preferably IgG.

[0225] 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 mixed with an available 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.

[0226] The method for preparing a vaccine for preventing or treating diseases related to group B Neisseria meningitidis of the present invention comprises the following steps: 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 volume ratio of 1:1.

[0227] The present invention further provides a complete kit, characterized in that it comprises a group B Neisseria meningitidis vaccine, and instruments and containers required for inoculating the vaccine, specifically including needle and syringe instruments, powder-carrying containers, and solvent-carrying containers. Preferably, the vaccine of the present invention is marketed in the form of a pre-filled needle in which the immune composition and adjuvant are mixed.

[0228] H. Medical Use

[0229] The present invention provides medical uses of the above-mentioned group B Neisseria meningitidis fHBP protein, immunogenic complex, immune composition, and vaccine, including preventing or treating group B Neisseria meningitidis related diseases.

[0230] The present invention provides pharmaceutical preparations of the above-mentioned group B Neisseria meningitidis fHBP protein, immunogenic complex, immune composition, and vaccine, and the medicine is used for preventing or treating diseases related to group B Neisseria meningitidis.

[0231] The diseases associated with group 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 leakage, edema, peripheral limb ischemia, and respiratory distress syndrome.

[0232] Preferably, the disease associated with group B Neisseria meningitidis is cerebrospinal meningitis.

[0233] I. Beneficial Effects

[0234] Compared with the prior art, the present invention has the following beneficial effects:

[0235] (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 of the production process from the source, improving production efficiency, and saving the cost of large-scale production.

[0236] (2) In the first aspect, the immunological effect of inducing anti-fHBP by the fusion protein (such as RD012-1) containing the characteristic amino acid sequences of the three variants of fHBP in the present invention is better than the immunological effect brought about by the simple mixture of the three variants of fHBP (such as RD012-7, RD012-8, and RD012-9) in the present invention. At the same time, the immunological effect of inducing anti-fHBP by the fusion protein (such as RD012-1) containing the characteristic amino acid sequences of the three variants of fHBP in the present invention is better than the immunological effect brought about by the simple mixture of the molecules RD012-13 and RD012-14 (used as control molecules in the present invention) known in the art.

[0237] 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.

[0238] Thirdly, it was found that the immunogenicity of the nanoparticle immunogenic complex (RD012-1 NPM) constructed by combining the fusion protein containing the characteristic amino acid sequence of the three variants of fHBP with the NPM particles can reach the immunogenicity effect caused by the three variants of fHBP (RD012-7, RD012-8, RD012-9) separately forming the nanoparticle immunogenic complex with NPM and then mixing them. Thus, the preparation process steps are saved, the production cost is reduced, and the production efficiency is improved.

[0239] (3) The nanoparticle vaccine product provided by the present invention can induce better cellular immunity and antibody immune response than the two MenB vaccines Bexsero and Trumenba that have been on the market, and can achieve similar effects with significantly reduced usage.

[0240] (4) In the present invention, the granular protein is prepared by Escherichia coli fermentation and chromatography purification, and the fHBP antigen is prepared by BL21 (DE3) cell reactor culture and chromatography purification, both of which are suitable for industrial large-scale production and have the advantages of high expression, stable process and yield, and simple operation. The amount of a batch of granular protein components can be combined with multiple batches of fHBP antigens to improve production efficiency. Compared with ordinary recombinant protein vaccines, the nanoparticle vaccine of the present invention has the advantage of a higher immune protection level at the same or lower dose, which can save the cost of large-scale production.

[0241] (5) The method for preparing 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.

[0242] (6) The present invention also studies the effects of linkers with different structures on the expression of fusion proteins.

[0243] Fourthly, the fHBP fusion protein of the present invention can trigger an effective bactericidal response to group B Neisseria meningitidis strains, and the bactericidal activity of the nanoparticle vaccine obtained after connecting 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

[0244] In order to more clearly illustrate the specific implementation manner of the present invention or the technical solution in the prior art, the drawings used in the description of the specific implementation manner or the examples are briefly introduced below.

[0245] Figure 1A-1E :in Figure 1A The results of SDS-PAGE identification of the purified fusion proteins RD012-1 and RD012-2; Figure 1B The results of SDS-PAGE identification of RD012-1M and RD012-2M after purification; Figure 1C The particle size identification results of RD012-1M and RD012-2M; Figure 1D The negative staining results of electron microscopy for RD012-1M and RD012-2M; Figure 1E The SDS-PAGE identification results of RD012-3, RD012-4, and RD012-4M;

[0246] Figure 2A-2F :in Figure 2A-2C It is a graphical representation of the antibody titers against fHBPV1, 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; Figure 2D-2F The graph shows the antibody titers against fHBP V1, V2, and V3 induced by vaccines RD012-1, RD012-2, RD012-5 / 6admix, RD012-7 / 8 / 9admix, and control molecules;

[0247] Figure 3A-3B :in Figure 3A The results of SDS-PAGE identification of the purified fusion proteins RD012-15, RD012-16 and RD012-17; Figure 3B The SDS-PAGE identification results of RD012-15M and RD012-16M after purification;

[0248] Figure 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;

[0249] Figure 5A-Figure 5C :in Figure 5A The results of SDS-PAGE identification of the purified fusion proteins RD012-19, RD012-20, RD012-22 and RD012-23; Figure 5B The results of SDS-PAGE identification of the purified fusion proteins RD012-21, RD012-24, RD012-25 and RD012-26; Figure 5C The SDS-PAGE identification results of RD012-20M, RD012-21M, RD012-22M, RD012-23M, RD012-24M, RD012-25M and RD012-26M after purification;

[0250] Figure 6A-6C : Graphical representation of 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

[0251] The principles and features of the present invention are described below in conjunction with examples. The examples are only used to explain the present invention and are not used 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 specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, rather than to limit the scope of protection of the present invention. The test methods for which specific conditions are not indicated in the following examples are usually carried out under conventional conditions or under conditions recommended by various manufacturers. When the examples give a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the examples, according to the technical personnel in the art's grasp of the prior art and the records of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to the methods, equipment, and materials described in the examples of the present invention can also be used to implement the present invention. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional reagent companies.

[0252] Example 1: Expression and purification of fHBP fusion protein molecules

[0253] 1. Experimental materials:

[0254] Capsule filter (Bricap C01: 180 cm2) was purchased from Cobetter, membrane package was purchased from Millipore, HisTrapexcel was purchased from Cytiva, and molecular sieve (Superdex 200 pg 10 / 300 GL) was purchased from Cytiva.

[0255] 2. Experimental methods:

[0256] 2.1. The fHBP protein molecules shown in Table 1 were prepared according to conventional molecular cloning techniques.

[0257] 2.2. Induced expression conditions: inoculate RD012 BL21 (DE3) monoclonal bacteria into 800 mL LB (Amp+) medium, culture at 37°C, 220 rpm for 4-5 h. When the OD600 of the culture solution is about 0.6-0.8, transfer the culture solution to 18°C, add IPTG to a final concentration of 0.5 mM, and induce protein expression at 200 rpm for 16 h.

[0258] 2.3. Collect bacteria by centrifugation: Collect the cultured bacterial solution by centrifugation at 7000g at room temperature, discard the culture solution, and resuspend the bacteria in 80mL of 150mM NaCl, 20mM Tris 7.4 solution.

[0259] 2.4. Ultrasonic disruption: Place the resuspended bacterial solution in an ice-water bath for ultrasonic disruption. Use horn No. 2, 50% power, ultrasonic for 3 seconds, stop for 7 seconds, and the total ultrasonic duration is 12 minutes.

[0260] 2.5. Collect the target protein by centrifugation: 13000g, 4℃, 30min, collect the supernatant of cell disruption.

[0261] 2.6. Purify the target protein by Histrap: Wash buffer is 20mM Tris-HCl, 150mM NaCl, pH 7.4; Elution buffer is 20mM Tris-HCl, 150mM NaCl, 500mM Imidazole, pH 7.4. Use Histrapexcel-5ml NI column for purification; After equilibrating the Histrapexcel-5ml with Wash buffer for 10 CV, load the sample; After loading, rinse the column with Wash buffer for 10CV; Rinse with 2% Elution buffer for 10CV to wash away impurities; Use 2%-100% Elution buffer to linearly elute the target protein for 15CV; After elution, detect the protein purity by SDS-PAGE.

[0262] 2.7. Purification of target protein by SEC: The target protein after purification by nickel column was collected and concentrated to 1 ml using a concentrator tube, and then separated and purified by molecular sieve (Superdex 200pg 10 / 300GL). The purification buffer was 20mM Tris-HCl, 150mM NaCl, pH7.4. After elution, the protein purity was detected by SDS-PAGE. The purified RD012-1 and RD012-2 were identified by SDS-PAGE. Figure 1A The protein concentration was determined by BCA method and stored at an appropriate temperature for subsequent binding reactions.

[0263] Example 2: Expression and purification of binding peptide 2-NPM fusion protein

[0264] 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) to form 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.

[0265] Table 4: NPM, NPM-4C fusion protein sequences in the examples of this application

[0266]

[0267] 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 clarification of the liquid is mainly completed by heating. The two-step heating method is used for heating treatment. The supernatant after E. coli crushing is heated in the first step and the second step (ie, "two-step heating"), and the impurity removal effect of the two-step heating steps and the purity of the recombinant granule protein components are measured.

[0268] 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), crushed using a high-pressure homogenizer at a pressure of 1000 bar, and 280 ml of supernatant was collected after centrifugation, of which 40 ml was taken for two-step heating operation. SDS-PAGE analysis was performed on the supernatant after crushing, the supernatant of the first step of heating centrifugation, and the resuspended precipitate of the second step of heating centrifugation.

[0269] The specific method is shown in Table 5. In the first heating step, adjust pH=9.0, 80°C, heat in a water bath for 1 hour, return to room temperature and centrifuge to collect about 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, then add 7 ml of 1 M Tris-HCl pH 7.4, and mix well. Heat in a 60°C water bath for 10 min, immediately centrifuge to collect the precipitate, and use 20 mM Tris-HCl, 5 mM EDTA, pH=9.0 buffer to re-dissolve the precipitate.

[0270] Table 5: Two-step heating extraction method for recombinant granular protein component products

[0271]

[0272]

[0273] After the two-step heating process, adding different concentrations of urea and sodium chloride before chromatography purification can significantly reduce the presence of unknown 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.

[0274] The above-mentioned recombinant granule protein component sample solution was refined by ion exchange and hydrophobic chromatography. The first step of chromatographic purification was performed by Fractogel DEAE M chromatography process. The specific steps and parameters are shown in Table 6. The Fractogel DEAEM elution collection solution sample was first diluted with buffer, and 50% (w / v) sucrose stabilizer was added to prevent the precipitation of recombinant granule protein during the next step of chromatography. The specific parameters are shown in Table 7. Then, the hydrophobic chromatography Octyl Bestarose 4FF chromatography process was used for purification (second step of chromatographic purification). The specific steps and parameters are shown in Table 8.

[0275] First step chromatography method: Chromatographic filler - Fractogel DEAE M, retention time - 12.5min

[0276] Table 6: First step chromatography method

[0277] Chromatography steps Chromatography buffer / conditions parameter Equilibration buffer 20mMTris-HCl,5mMEDTA,8MUrea,50mMNaCl,pH9.0 6CV pH after equilibrium 8.8±0.05 8.80 Wash 1 Buffer 20mMTris-HCl,5mMEDTA,8MUrea,50mMNaCl,pH9.0 1.5CV Wash 2 Buffer 20mM Tris-HCl, 5mMEDTA, 8MUrea, 2% Triton, pH9.0 5CV Wash 3 Buffer 20mM Tris-HCl, 8MUrea, pH 9.0 5CV Wash 4 Buffer 20mM Tris-HCl, 4M Urea, pH 9.0 5CV Elution buffer 20mM Tris-HCl, 4MUrea, 150mM, pH9.0 2CV Collection interval 50mAU-50mAU Light diameter 2mm

[0278] Table 7: Sample dilution method before the second step chromatography

[0279]

[0280]

[0281] Second step chromatography method: Chromatographic filler-Octyl Bestarose 4FF, retention time-12.5min

[0282] Table 8: Second step chromatography method

[0283] Chromatography steps Chromatography buffer / conditions parameter Equilibration buffer 20 mM Tris-HCl, 1 M NaCl, 25% (w / v) sucrose, pH 9.0 2CV Wash buffer 20 mM Tris-HCl, 1 M NaCl, 25% (w / v) sucrose, pH 9.0 1.5CV Elution buffer 20 mM Tris-HCl, 25% (w / v) sucrose, pH 9.0 3CV Collection interval 50mAU-50mAU Light diameter 2mm

[0284] Results and Analysis:

[0285] Through purity testing, it was found that after further refining by the above chromatographic medium combination, the purity of the obtained product can reach more than 99.0%.

[0286] Example 3: Binding of fHBP and NPM, particle characterization

[0287] 1. Binding of fHBP and NPM and product purification:

[0288] 1. Experimental methods:

[0289] RD012-1 and RD012-2 as MenB antigens were mixed with NPM-4C as a carrier at a BCA protein concentration ratio of 6:1, and 50% sucrose mother solution was added to a final concentration of about 25% sucrose, and 10% of the total reaction volume of 1M Tris-HCl pH7.4 mother solution was added to stabilize the pH. The binding reaction was carried out at 22°C for 24 hours. The combined product was separated and purified by molecular sieve (purification buffer was 20mM Tris-HCl, 150mM NaCl, 25% Sucrose, pH7.4), and the RD012-NPM component was collected. The purified RD012-NPM was identified by SDS-PAGE, and the particle size of RD012-NPM and the negative staining results of electron microscopy were detected.

[0290] The specific experimental operations are as follows:

[0291] (1) TEM inspection

[0292] Use the floating method to prepare negative staining samples. Select a 400-mesh grid with a support membrane, pre-treat it hydrophilically, and prepare deionized water and 2% uranium formate negative staining solution. Take the prepared 3uL protein sample (0.12mg / ml) and drop it directly on one side of the grid of the support membrane; after timing for 1 minute, use clean filter paper to absorb excess liquid from the edge of the grid; after drying slightly, rinse it twice quickly with deionized water droplets in sequence; then take 5ul negative staining solution and rinse once, and finally add 5ul negative staining solution and time for 1 minute. After the end, use tweezers to pick up the grid and use filter paper to absorb the staining solution, leaving a thin layer to let it dry naturally for testing. Check under a 120kV transmission electron microscope (FERRITINI Tecnai Spirit), observe the overall staining of the grid at low magnification, select holes with appropriate thickness for observation, and select appropriate areas under high magnification for photography and preservation.

[0293] (2) SDS-PAGE method

[0294] 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 10000rpm 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 run), the voltage was set to 150V, and the electrophoresis lasted for about 60 minutes. After the electrophoresis, the gel was removed, placed in a clean container, an appropriate amount of Coomassie Brilliant Blue staining solution was added to cover the gel, and the gel was stained on a shaker for 2h. After the staining was completed, the staining solution was poured out, and the gel was destained by soaking in purified water. The decolorization was continued on a shaker until the gel base color was completely removed, and the gel was photographed using a GelDoc Go gel imager.

[0295] (3) DLS method

[0296] Dilute the purified test sample to 0.25 mg / mL. Use the Zetasizer Lab instrument to inject ≥1 mL of the test sample into the sample pool. Run the instrument for detection. Combine the Z-Average (nm) and Polydispersity Index (PI) values, as well as the Size Distribution by Intensity / Volume distribution curve for data analysis and report the results.

[0297] 2. Experimental results:

[0298] The purified NPM VLPs of RD012-1 and RD012-2 were identified by SDS-PAGE. Figure 1B As shown, the particle size and electron microscopy negative staining results of NPM VLPs of RD012-1 and RD012-2 are as follows Figure 1C , Figure 1D The results showed that the expression level of 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. Figure 1E In addition, when the 4T sequence was placed between the two chimeric proteins, namely RD012-4, its binding rate to the VLP vector NPM-4C was significantly reduced, as shown in the results. Figure 1E Therefore, RD012-1 and RD012-2 molecules were designed as the optimal fusion protein molecules.

[0299] Example 4: Animal Immunization Test

[0300] (1) Experimental Materials

[0301] Mice: 5-6 weeks old female BALB / c mice (purchased from Guangdong Weitonglihua Experimental Animal Technology Co., Ltd.)

[0302] Adjuvant: Aluminum hydroxide adjuvant

[0303] Other reagents and consumables are commercial conventional reagents and consumables.

[0304] (2) Experimental procedures

[0305] As shown in Table 9, the same vaccine designed by construct was immunized on day 0 and day 14, respectively, by intramuscular injection, with a dose of 9 μg / dose / animal, and each dose of vaccine was mixed with 75 μg of aluminum hydroxide adjuvant. Blood was collected on day 28 to separate serum for the detection of binding antibodies.

[0306] Table 9: Animal Immunization Scheme 1

[0307]

[0308] (3) Combined antibody detection:

[0309] First, the ELISA plate was coated with three variant proteins of fHBP, and the coated ELISA plate was blocked in Blocker Casein in PBS blocking solution (purchased from ThermoFisher) for 1-4 hours; the serum samples collected from the mice in each experimental group at the end of immunization in Table 9 were diluted three times from 1:300 to 656100, and the diluent was added to each well. The negative control was the sample diluent, incubated for 2-3 hours, incubated with HRP-coupled goat anti-mouse secondary antibody for 1 hour, and then developed with TMB substrate. After the color development was completed, the reaction was terminated with 1M hydrochloric acid, and the absorbance (OD) was measured with an enzyme reader. The main wavelength was 450nm, the reference wavelength was 620nm, and the sample OD value = OD450-OD620. The measurement was completed within 5 minutes after termination. The experimental results are shown in Figure 2A -F.

[0310] The results of the mouse immunization test showed that the antibody titers induced by the RD012-1 / 2 fusion protein against fHBP variants V1, V2 and V3 were about 10.5 times, 3.6 times and 10.3 times higher than the equal 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. Figure 2A -F; In addition, the immunogenicity of the nanoparticle vaccine RD012-1M / 2M antigen constructed by combining RD012-1 / 2 with NPM particles is also better than that of the simple recombinant protein antigen (ie, RD012-1 / 2). Figure 2A-2C shown.

[0311] Example 5: Optimization of RD012 fusion protein molecule design

[0312] 1. Experimental methods:

[0313] The results of Example 4 above show that the fusion protein antigen comprising the characteristic amino acid sequences of the three variants of fHBP constructed by the present invention has a broad spectrum and effectiveness. Figure 1A The results showed that the RD012-1 protein had broken bands, which would complicate the product production process. Based on previous research data, we swapped 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 method of Example 1-2. The results are shown in Figure 3. Neither the RD012-15 / 16 recombinant protein antigen nor the VLP particle antigen produced broken bands, but 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, and the results of the ELISA detection binding antibody titer experiment are shown in Table 10. Figure 4A -C.

[0314] Table 10: Animal Immunization Scheme 2

[0315]

[0316] 2. Experimental results:

[0317] The experimental results show that although exchanging the tandem order 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 analysis.

[0318] Example 6: Optimization of RD012 fusion protein molecule design

[0319] 1. Experimental methods:

[0320] In order to solve the problem of broken bands of RD012-1 fusion protein, we used LC-MS to detect the intact molecular weight of the target molecule and the broken molecule, and compared the peptide maps of the two by MS to clarify the broken amino acid sequence. Previous research data found that the broken sequence did not affect the immunogenicity, so the broken amino acid was directly cut off to obtain the molecule RD012-18. The recombinant protein antigen of RD012-18 was prepared and found to be a single target band, but the protein expression level was reduced by more than half. 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 different lengths: (EAAAK)n to obtain the molecules RD012-19 / 20 / 22 / 23. The results are as follows Figure 5AAs shown in the figure, almost no broken bands were generated after the rigid linker was replaced. In addition, the fHBP type of the domestic group B meningococcal epidemic strains is mainly V2. In order to further optimize the antigen-induced antibody titer against 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 fHBPV3 domain 2 in the same tandem order to obtain RD012-21 / 24 / 25 / 26 fusion protein antigen molecules. The prepared recombinant protein antigens are 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 result was as 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.

[0321] Preparation method of Trumenba vaccine: 60μg lipid-modified fHBP V1 protein and 60μg lipid-modified fHBPV3 protein, adsorbed on aluminum phosphate adjuvant.

[0322] Preparation of Bexsero vaccine: 50 μg fHBP V1 fusion protein, 50 μg NHBA fusion protein, 50 μg NadA protein and 25 μg outer membrane vesicles (OMV) adsorbed on aluminum hydroxide adjuvant.

[0323] Table 11. Animal immunization scheme 3

[0324]

[0325] 2. Experimental results:

[0326] The results are as follows Figure 6A-6C As shown: Compared with RD012-1M / 2M, RD012-20M / 22M / 23M induced an increase in antibody titers against three variants of fHBPV1, V2, and V3, 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 obtained by replacing 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 significantly increased the antibody titers induced 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.

[0327] Example 7: Serum bactericidal activity assay (SBA)

[0328] First, the MenB strain was spread on chocolate plates and cultured overnight at 37°C and 5% CO2. A single colony was inoculated into Mueller-Hinton medium and the initial OD 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 group RD012-24M, Trumenba and Bexsero in Example 6 was heated and inactivated at 56°C for 1 hour. The total volume in each well was 50 μL, including 25 μL of test serum diluted twice continuously, 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 at 37°C for 2-4 hours. Take 10 μL of each sample and add it to the Mueller-Hinton agar plate and culture it at 37°C overnight. Use TTC to color the agar plate cultured overnight, count the bacterial colonies, and calculate the bactericidal titer.

[0329] The experimental results are shown in Table 12. The bactericidal activity of the vaccine RD012-24M of the present invention against the V2 subtype strain is better than that of the commercial vaccine, and the bactericidal activity against the V1 and V3 subtype strains is comparable to that of the commercial vaccine. It can be seen that RD012-24M has a broader spectrum of immune effects and bactericidal activity than the commercial vaccine.

[0330] Table 12. Bactericidal activity of sera from immune mice against different fHBP variant strains

[0331]

[0332] 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.

[0333] To sum up, the above embodiments and drawings are only preferred embodiments of the present invention and are not intended to limit the protection scope 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 protection scope 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 fHBPV2 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 fHBPV2 domain 2; (C) comprises fHBP V2 domain 1 and fHBPV1 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 fHBPV1 domain 1 is selected from SEQ ID NO: 54, 55 or 56, the amino acid sequence of the fHBPV1 domain 2 is selected from SEQ ID NO: 57, the amino acid sequence of the fHBPV2 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 immune composition comprises three variants of fHBP: fHBPV1, fHBPV2 and fHBPV3; 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 fHBPV3 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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