Design and application of group B meningococcal multi-subtype fHbp-NHBA fusion antigen

Abstract

This application relates to the field of protein engineering. This application provides the design and use of a group B meningococcal multi-subtype fHbp-NHBA fusion antigen, which includes fHbp-NHBA fusion antigens of different subtypes. The fusion antigen designed in this application can be administered in combination with one or more different subtypes to produce a broader spectrum of immune effects, while simultaneously improving antigen stability and simplifying the production process and reducing costs.

Description

Technical Field

[0001] This application relates to the field of protein engineering, specifically to the design and application of group B meningococcal multi-subtype fHbp-NHBA fusion antigen. Background Technology

[0002] Epidemic cerebrospinal meningitis (abbreviated as meningococcal) is caused by Gram-negative meningococcal bacteria (Neisseria meningitidis). Neisseria meningitidis Meningococcal meningitis, primarily caused by infection with Neisseria meningitidis (N. m.), is a respiratory infectious disease that mainly manifests as cerebrospinal meningitis and bacteremia. It often occurs worldwide in an endemic manner, primarily affecting infants and adolescents. Neisseria meningitidis can colonize the nasopharyngeal mucosa, causing local infection, or cross the mucosal barrier, causing invasive sepsis or meningococcal meningitis. Although antibiotic treatment and vaccination are available, the global mortality rate for meningococcal meningitis remains as high as 10%-15%, accompanied by a high rate of disability, resulting in a severe disease burden worldwide.

[0003] Based on the chemical structure of the capsular polysaccharide, Neisseria meningitidis can be divided into 13 serogroups, among which A, B, C, Y, W, and X are considered the main pathogenic serogroups causing meningococcal meningitis, accounting for over 95% of meningococcal infections. With the successful development and widespread use of meningococcal polysaccharide vaccines for serogroups A, C, Y, and W, the prevalence of meningococcal meningitis in these serogroups has been effectively controlled. The incidence of serogroup B meningitis in my country is showing a year-on-year increasing trend, mainly concentrated in infants under 2 years old, followed by adolescents. Group B meningitidis (group B...) N. meningitidis The main components of Meningococcal B capsular polysaccharide are similar in structure to the human central nervous system antigen N-acetylneuraminic acid polymer, which makes capsular polysaccharide vaccines against Meningococcal B unable to produce an effective immune response and can cause cross-reactivity.

[0004] Vaccine development against serogroup B meningococci mainly focuses on outer membrane vesicle (OMV) vaccines and recombinant protein vaccines. OMV vaccines are internationally used for meningitis against a specific circulating strain of serogroup B, with their main component being meningeal porin A (PorA). Due to the complex protein typing of PorA and low cross-protection between serotypes and subtypes, it is difficult to obtain an internationally universally applicable vaccine. Currently, two recombinant protein vaccines against serogroup B meningitis are available in Europe and the United States: Bexsero / 4CMenB developed by GSK and Trumenba developed by Pfizer. Bexsero was approved in 2013, and its main component is GNA2091-fHbp. v1.1 Fusion protein, NHBA p2The vaccine contains the GNA1030 fusion protein, NadAv3.1, and OMV extracted from the NZ98 / 254 strain. The vaccine composition is complex, requiring separate expression and purification of antigens. Furthermore, the OMV production process is complex, and residual endotoxins can easily cause significant side effects, posing potential safety risks. Pfizer's group B meningitis vaccine, Trumenba, received FDA approval in 2014. Its main antigens are two subgroups of fHbp (V1.55 / B01 and V3.45 / A05), with fHbp undergoing lipid modification to enhance immunogenicity. Because the expression level of fHbp varies significantly among different strains, particularly in some Variant 2 and Variant 3 strains where fHbp expression is low, vaccines containing only the fHbp antigen offer limited protective coverage against different prevalent strains.

[0005] It is noteworthy that the serogroup B meningitis antigen gene sequence exhibits polymorphism, with significant differences in antigen genotypes between the prevalent strains in China and those abroad: the fHbp gene subtypes in the prevalent Chinese strains are predominantly Variant2 family; the NHBA gene subtypes are scattered, and the cross-protective effects between different subtypes are unclear; while the NadA gene is almost absent in the prevalent strains in China. Therefore, the protective efficacy of the Bexsero and Trumenba vaccines against the prevalent serogroup B Nm strains in my country is not ideal. Secondly, existing serogroup B meningitis vaccines have complex compositions, requiring lipid modification or containing OMV components, resulting in complex production processes, high costs, and significant side effects. Therefore, there is an urgent need to develop a domestically produced serogroup B meningitis vaccine that ensures antigen stability while improving the vaccine's immunogenicity and broad-spectrum efficacy. Summary of the Invention

[0006] In order to address the problems existing in the prior art, the purpose of this application is to provide a fusion antigen of group B meningococcus and its use.

[0007] To solve the above technical problems, this application adopts the following technical solution: In one aspect, this application provides an fHbp-NHBA fusion antigen, wherein the fusion antigen comprises two groups of different subtypes of fHbp and different subtypes of NHBA truncated variants to form a fusion antigen of different subtypes.

[0008] More specifically, the fusion antigen comprises: i) one or more subtypes of fHbp antigen, wherein the fHbp antigen is selected from fHbp-V1.1 or its mutant, fHbp-V1.13 or its mutant, fHbp-V2.22 or its mutant, fHbp-V2.18 or its mutant, fHbp-V2.16 or its mutant, fHbp-V3.31 or its mutant; ii) One or more NHBA antigens, wherein the NHBA antigens are selected from NHBA-P3 truncated variant, NHBA-P669 truncated variant, NHBA-P688 truncated variant, NHBA-P503 truncated variant, NHBA-P20 truncated variant, and NHBA-P945 truncated variant.

[0009] The fHbp antigen uses the full-length sequence. The NHBA antigen lacks secondary structure at its N-terminus, which affects its stability to some extent; therefore, in this fusion antigen design, the NHBA antigen is truncated by removing the unstable N-terminal region to enhance the overall structural stability and immunogenicity of the fusion antigen. To further improve the stability of NHBA, the sequence lacking secondary structure between its inter domain and C-terminal domain is deleted and replaced with a peptide linker, constructing the fHbp-NHBA (Inter+C domain) fusion antigen.

[0010] On the other hand, this application provides an immunogenic composition comprising one or more of the aforementioned fusion proteins.

[0011] Preferably, the immunogenic composition may further contain an antigen fragment having at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 46-90.

[0012] In another aspect, this application provides a method for preventing and / or treating group B meningitis, the method comprising administering to a subject an effective amount of the aforementioned fusion antigen and / or the aforementioned immunogenic composition.

[0013] On the other hand, this application provides a method for inducing an individual to produce an antigen-specific immune response, the method comprising administering to a subject an effective amount of the aforementioned fusion antigen and / or the aforementioned immunogenic composition.

[0014] On the other hand, this application provides the use of the aforementioned fusion antigen and / or the aforementioned immunogenic composition in the preparation of vaccines and / or medicaments for the prevention and / or treatment of group B meningitis. The beneficial effects of this application are at least as follows:

[0015] (1) The fHbp-NHBA fusion antigen designed in this application contains the two most important antigenic components of group B meningococci. In this design, the unstable random sequence at the N-terminus of NHBA is removed, and only the domain with a stable three-dimensional structure is retained, thereby maximizing the retention of key antigenic epitopes while ensuring the overall stability of the antigen. This strategy helps to simultaneously improve the stability and immunogenicity of the antigen.

[0016] (2) The immunogenic composition of group B meningococcus designed in this application covers different subtypes of fHbp-NHBA fusion antigen, which can be used in combination with one or more to produce a broader spectrum of immune protection against the prevalent strains in different regions.

[0017] (3) The immunogenic composition prepared in this application has simple components, the antigen does not need to be lipid modified, and it does not contain OMV components, which simplifies the production process and reduces costs, and improves the stability and safety of the antigen. Attached Figure Description

[0018] Figure 1 This diagram illustrates the three-dimensional structural analysis, truncation, and mutation of fHbp and NHBA based on structural biology. Figure a shows the three-dimensional structural analysis of fHbp, Figure b shows the three-dimensional structural analysis of NHBA, and Figure c shows the fusion antigen design strategy.

[0019] Figure 2 The results show the purity and uniformity characterization of different isotypes of fHbp-NHBA (inter+C domain) recombinant proteins.

[0020] Figure 3 The results show the purity and uniformity of different subtypes of fHbp-NHBA (inter+C domain) mutant proteins.

[0021] Figure 4 The aggregation temperature T of different subtypes of fHbp-NHBA fusion antigen agg266 Test results.

[0022] Figure 5 The melting temperature T of the fHbp-NHBA fusion antigen containing the fHbp-V1.13 subtype. m Test results Figure 6 The melting temperature T of the fHbp-NHBA fusion antigen containing the fHbp-V2.16 subtype. m Test results Figure 7 The melting temperature T of the fHbp-NHBA fusion antigen containing the fHbp-V2.18 subtype. m Test results Figure 8 The melting temperature T of the fHbp-NHBA fusion antigen containing the fHbp-V2.22 subtype. m Test results Figure 9 Results of detection of total IgG antibody levels in immune sera containing different subtypes of fHbp-NHBA fusion antigens formulated with Freund's adjuvant Figure 10Results of detection of total IgG antibody levels in immune sera containing different subtypes of fHbp-NHBA fusion antigens formulated with aluminum adjuvant Figure 11 The results show the serum bactericidal titers of immune sera containing different subtypes of fHbp-NHBA fusion antigen.

[0023] Figure 12 To detect the broad-spectrum protection of different subtypes of fHbp-NHBA fusion antigen immune serum mixtures against multiple target bacteria.

[0024] Figure 13 To evaluate the in vivo protective effect of different subtypes of fHbp-NHBA fusion antigen mixtures against MenB-infected mice. Detailed Implementation I. Definition

[0025] In this application, unless otherwise stated, the scientific and technical terms used herein have meanings commonly understood by those skilled in the art. Furthermore, the relevant terms and laboratory procedures used herein are widely used terms and routine procedures in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below.

[0026] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0027] The terms "comprising" and "having," and any variations thereof, in this disclosure are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device. The term "multiple" as used in this disclosure means two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist; for example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship.

[0028] In the description herein, references to “some embodiments,” “some implementations,” or “some implementation schemes” describe a subset of all possible embodiments. However, it is understood that “some embodiments” may be the same subset or different subsets of all possible embodiments and may be combined with each other without conflict.

[0029] In this disclosure, polypeptides such as "antigen" and "fusion antigen" include polypeptides having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology with it; preferably, it includes polypeptides having at least 80% homology with it, specifically, for example, polypeptides having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology with it.

[0030] The term "fHbp" refers to factor H binding protein (fHbp). fHbp binds to human complement factor H and helps Neisseria meningitidis evade complement-dependent immune responses by downregulating complement-mediated bactericidal activity. fHbp is the most important antigen in meningitis, expressed on the cell membrane of most MenB strains, and is a lipoprotein. fHbp sequences exhibit high diversity and can be classified into three variants based on their amino acid sequences. Variant V1 belongs to subfamily B, while variants V2 and V3 belong to subfamily A. Variant variants within the same subfamily show high homology (over 90% amino acid sequence homology) and possess cross-protective immunogenicity. Variant variants from different subfamilies show lower homology (as low as 62.8%) and do not possess cross-protective immunogenicity. Vaccine antigen design was conducted using the most prevalent fHbp genotypes V2.22, V2.18, V2.16, V3.31, and V1.13 among the prevalent serogroup B Neisseria meningitidis strains in China, aiming to improve vaccine coverage. MC58 is the standard strain for serogroup B meningitis, with fHbp genotype V1.1, containing 320 amino acids, including a signal peptide and transmembrane region (1-65), which anchors fHbp to the cell membrane. Mature fHbp contains 255 amino acids, and its three-dimensional structure is mainly divided into two parts: the N-terminal β-sheet (66-202) and the C-terminal β-barrel (203-320).

[0031] The term "NHBA" refers to Neisserial Heparin-binding antigen (NHBA), which binds to heparin to form a complex, recruiting complement inhibitors to prevent complement activation and promote bacterial survival. There are over 400 variants of NHBA in group B meningococci. The most prevalent NHBA genotypes among the circulating strains of group B meningococci in China—Peptide669, Peptide688, Peptide503, Peptide20, and Peptide945—were selected to improve vaccine coverage. The NHBA genotype derived from strain MC58 is Peptide3, containing 488 amino acids, with amino acids 1-26 forming the signal peptide and transmembrane region. NHBA has a relatively flexible N-terminus and lacks a stable three-dimensional domain. AlphaFold predicts that amino acids 210-293 at the N-terminus may form a relatively stable domain containing two α-helices and four β-sheets, which is referred to as the Inter-domain in this application; the C-terminus has a stable domain containing β-sheets (335-364) and β-barrels (365-488), which is named the C-terminal domain in this application.

[0032] The term "antigen" refers to a substance that can stimulate the body to produce a (specific) immune response and can bind to the immune response products, antibodies and sensitized lymphocytes, in vitro to produce an immune effect (specific reaction). Antigens have two basic characteristics: the ability to induce an immune response, i.e., immunogenicity, and the ability to react with the products of the immune response, i.e., antigenicity.

[0033] The terms “joint” and “connector” are used interchangeably and refer to the chemical portion of an atomic chain that is covalently attached to or attached to items such as carrier proteins or polysaccharides.

[0034] The term "fragment" refers to a polypeptide that substantially retains its activity in activating an immune response against meningococcal group B. The protein fragments of this application may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituent groups in one or more amino acid residues, or (iii) polypeptides formed by fusing the protein of this application with another compound (such as a compound that prolongs the half-life of the polypeptide, e.g., polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (derived proteins formed by fusing with a leader sequence, secretory sequence, or tag sequence such as 6X His). In accordance with the teachings herein, these fragments, derivatives, and analogs may be wild-type or mutant, both of which are well known to those skilled in the art.

[0035] The term "inter-domain" refers to the inter-domain structure, also known as the inter-domain structure.

[0036] The term "truncated protein" generally refers to a shortened protein. A truncated protein can be obtained by protein hydrolysis or deletion of a gene sequence to eliminate any N- or C-terminus or intermediate portion of the protein. Alternatively, a truncated protein can be obtained by nonsense mutations that result in the presence of a stop codon in the structural gene, prematurely terminating translation. In this disclosure, a truncated protein is a shortened protein that also possesses biological function.

[0037] The term "mutant" refers to a mutant protein whose amino acid sequence has been altered to obtain a protein that is either functional or nonfunctional. In this disclosure, mutants are mutant proteins that also possess biological functions.

[0038] In this disclosure, the terms "mutant and truncated" refer to new fusion proteins obtained by altering the amino acid sequence of the original fusion protein while truncating certain structures in the protein, which have the same or enhanced biological functions.

[0039] The term "host cell" preferably refers to a bacterial or yeast cell expressing the protein or polypeptide of this application. The bacteria include, but are not limited to, for example, meningococci or Escherichia coli. The bacteria may express the protein or polypeptide, but in some embodiments, expression may be controlled by an inducible promoter.

[0040] The term "subject" refers to a living multicellular vertebrate organism, including humans and non-human mammals. In some implementations, subjects include veterinary subjects, including livestock such as cattle and sheep, rodents (e.g., mice and rats), and non-human primates.

[0041] In the term “prevention and / or treatment of disease,” “prevention” means suppressing the complete occurrence of a disease or condition, for example, suppressing the occurrence of a disease such as meningococcal meningitis in a subject at risk of developing such a disease. In some instances, the term “prevention” refers to reducing or delaying the onset or progression of a disease. “Treatment” refers to a therapeutic intervention that alleviates the signs or symptoms of a disease or pathological condition after the disease has already begun to occur. As used herein, the term “improvement” in relation to a disease, pathological condition, or symptom refers to any observable beneficial effect of treatment. Such beneficial effects can be demonstrated, for example, by delaying the onset of clinical symptoms of the disease in susceptible subjects, reducing the severity of some or all of the clinical symptoms of the disease, slowing disease progression, reducing the number of disease relapses, improving the overall health or well-being of the subject, or by other parameters known in the art that are specific to a particular disease. II. Detailed Description of Implementation Methods

[0042] On the one hand, this application provides an fHbp-NHBA fusion antigen, wherein the fusion antigen comprises one or more fHbp antigens and one or more NHBA antigens.

[0043] Preferably, the fHbp antigen includes fHbp-V1.1 or its mutant, fHbp-V1.13 or its mutant, fHbp-V2.22 or its mutant, fHbp-V2.18 or its mutant, fHbp-V2.16 or its mutant, and fHbp-V3.31 or its mutant.

[0044] Preferably, the NHBA antigen includes NHBA-P3 or a truncated form thereof, NHBA-P669 or a truncated form thereof, NHBA-P688 or a truncated form thereof, NHBA-P503 or a truncated form thereof, NHBA-P20 or a truncated form thereof, and NHBA-P945 or a truncated form thereof.

[0045] Preferably, the amino acid sequence of fHbp-V1.1 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1; more preferably, the amino acid sequence of fHbp-V1.1 is as shown in SEQ ID NO: 1.

[0046] Preferably, the mutant of fHbp-V1.1 has E218A and / or E239A mutations. More preferably, it has both E218A and E239A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 7.

[0047] Preferably, the amino acid sequence of fHbp-V1.13 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 2; more preferably, the amino acid sequence of fHbp-V1.13 is as shown in SEQ ID NO: 2.

[0048] Preferably, the mutant of fHbp-V1.13 has E218A and / or E239A mutations. More preferably, it has both E218A and E239A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 8.

[0049] Preferably, the amino acid sequence of fHbp-V2.22 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 3; more preferably, the amino acid sequence of fHbp-V2.22 is as shown in SEQ ID NO: 3.

[0050] Preferably, the mutant of fHbp-V2.22 has one or more mutations selected from L130R, G133D, E217A, and T238A. Preferably, it has both L130R and G133D mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 13; or, it has both L130R, G133D, E217A, and T238A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 14.

[0051] Preferably, the amino acid sequence of fHbp-V2.18 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 4; more preferably, the amino acid sequence of fHbp-V2.18 is as shown in SEQ ID NO: 4.

[0052] Preferably, the mutant of fHbp-V2.18 has one or more mutations selected from L130R, G133D, E217A, and T238A. Preferably, it has both L130R and G133D mutations; more preferably, the amino acid sequence of the mutant is shown in SEQ ID NO: 11; or, it has both L130R, G133D, E217A, and T238A mutations; more preferably, the amino acid sequence of the mutant is shown in SEQ ID NO: 12.

[0053] Preferably, the amino acid sequence of fHbp-V2.16 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 5; more preferably, the amino acid sequence of fHbp-V2.16 is as shown in SEQ ID NO: 5.

[0054] Preferably, the mutant of fHbp-V2.16 has one or more mutations selected from L130R, G133D, E217A, and T238A. Preferably, it has both L130R and G133D mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 9; or, it has both L130R, G133D, E217A, and T238A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 10.

[0055] Preferably, the amino acid sequence of fHbp-V3.31 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 6; more preferably, the amino acid sequence of fHbp-V3.31 is as shown in SEQ ID NO: 6.

[0056] Preferably, the mutant of fHbp-V3.31 has E225A and / or T246A mutations. More preferably, it has both E225A and T246A mutations; more preferably, the amino acid sequence of the mutant is shown in SEQ ID NO: 15.

[0057] Preferably, the amino acid sequence of NHBA-P3 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 16; preferably, the amino acid sequence of NHBA-P3 is as shown in SEQ ID NO: 16.

[0058] Preferably, the amino acid sequence of NHBA-P669 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 21; preferably, the amino acid sequence of NHBA-P669 is as shown in SEQ ID NO: 21.

[0059] Preferably, the amino acid sequence of NHBA-P688 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 26; preferably, the amino acid sequence of NHBA-P688 is as shown in SEQ ID NO: 26.

[0060] Preferably, the amino acid sequence of NHBA-P503 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 31; preferably, the amino acid sequence of NHBA-P503 is as shown in SEQ ID NO: 31.

[0061] Preferably, the amino acid sequence of NHBA-P20 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 36; preferably, the amino acid sequence of NHBA-P20 is as shown in SEQ ID NO: 36.

[0062] Preferably, the amino acid sequence of NHBA-P945 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 41; preferably, the amino acid sequence of NHBA-P945 is as shown in SEQ ID NO: 41.

[0063] Preferably, the amino acid sequence of the NHBA-P3 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 17; more preferably, the amino acid sequence of the NHBA-P3 truncated form is as shown in SEQ ID NO: 17. Alternatively, the NHBA-P3 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 18-19, either directly or via peptide linkers; preferably, the NHBA-P3 truncated form is composed of SEQ ID NO: 18-19 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P3 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 20; even more preferably, the amino acid sequence of the NHBA-P3 truncated form is as shown in SEQ ID NO: 20.

[0064] Preferably, the amino acid sequence of the NHBA-P669 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 22; more preferably, the amino acid sequence of the NHBA-P669 truncated form is as shown in SEQ ID NO: 22. Alternatively, the NHBA-P669 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 23-24, either directly or via peptide linkers; preferably, the NHBA-P669 truncated form is composed of SEQ ID NO: 23-24 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P669 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 25; even more preferably, the amino acid sequence of the NHBA-P669 truncated form is as shown in SEQ ID NO: 25.

[0065] Preferably, the amino acid sequence of the NHBA-P688 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 27; more preferably, the amino acid sequence of the NHBA-P688 truncated form is as shown in SEQ ID NO: 27. Alternatively, the NHBA-P688 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 28-29, either directly or via peptide linkers; preferably, the NHBA-P688 truncated form is composed of SEQ ID NO: 28-29 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P688 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 30; even more preferably, the amino acid sequence of the NHBA-P688 truncated form is as shown in SEQ ID NO: 30.

[0066] Preferably, the amino acid sequence of the NHBA-P503 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 32; more preferably, the amino acid sequence of the NHBA-P503 truncated form is as shown in SEQ ID NO: 32. Alternatively, the NHBA-P503 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 33-34, either directly or via peptide linkers; preferably, the NHBA-P503 truncated form is composed of SEQ ID NO: 33-34 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P503 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 35; even more preferably, the amino acid sequence of the NHBA-P503 truncated form is as shown in SEQ ID NO: 35.

[0067] Preferably, the amino acid sequence of the NHBA-P20 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 37; more preferably, the amino acid sequence of the NHBA-P20 truncated form is as shown in SEQ ID NO: 37. Alternatively, the NHBA-P20 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 38-39, either directly or via peptide linkers; preferably, the NHBA-P20 truncated form is composed of SEQ ID NO: 38-39 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P20 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 40; even more preferably, the amino acid sequence of the NHBA-P20 truncated form is as shown in SEQ ID NO: 40.

[0068] Preferably, the amino acid sequence of the NHBA-P945 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 42; more preferably, the amino acid sequence of the NHBA-P945 truncated form is as shown in SEQ ID NO: 42. Alternatively, the NHBA-P945 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 43-44, either directly or linked through a peptide linker; preferably, the NHBA-P945 truncated form is composed of SEQ ID NO: 43-44 linked through a first peptide linker; more preferably, the amino acid sequence of the NHBA-P945 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 45; even more preferably, the amino acid sequence of the NHBA-P945 truncated form is as shown in SEQ ID NO: 45.

[0069] Preferably, the first peptide linker comprises (G)n, (GS)n, (GSG)n, (GGGS)n, (GSGGS)n, (GGGGS)n, and (GGGGS)n, wherein n includes 1, 2, 3, 4, 5, 6, 7, or 8. More preferably, the first peptide linker comprises GSGGSGGSGGSG.

[0070] Preferably, the fHbp antigen and NHBA antigen in the fusion antigen are directly linked or linked through a peptide linker; more preferably, they are linked through a second peptide linker.

[0071] More preferably, the connection between the fHbp antigen and the NHBA antigen includes: fHbp antigen or a fragment thereof - second peptide linker - NHBA antigen or a fragment thereof, or NHBA antigen or a fragment thereof - second peptide linker - fHbp antigen or a fragment thereof.

[0072] Preferably, the second peptide linker comprises (G)n, (GS)n, (GSG)n, (GGGS)n, (GSGGS)n, (GGGGS)n, and (GGGGS)n, wherein n includes 1, 2, 3, 4, 5, 6, 7, or 8. More preferably, the second peptide linker comprises GGGSGGGSGGGS.

[0073] Preferably, the amino acid sequence of the fusion antigen has at least 80% homology with any of the amino acid sequences shown in SEQ ID NO: 46-90; more preferably, the amino acid sequence of the fusion antigen is as shown in any of SEQ ID NO: 46-90.

[0074] On the other hand, this application provides an immunogenic composition comprising one or more of the aforementioned fusion antigens.

[0075] Preferably, the immunogenic composition further contains an antigen fragment having at least 80% homology with the amino acid sequences shown in any one of SEQ ID NO: 46-90. Preferably, the amino acid sequence of the antigen fragment is shown in any one of SEQ ID NO: 46-90. Preferably, the immunogenic composition contains a fusion antigen with amino acid sequences shown in SEQ ID NO: 63, 66, 83, 84, 85, or the immunogenic composition contains a fusion antigen with amino acid sequences shown in SEQ ID NO: 86-90.

[0076] Preferably, the immunogenic composition further comprises one or more of an adjuvant, a buffer solution, a stabilizer, and a surfactant.

[0077] Preferably, the adjuvant is at least one selected from aluminum adjuvant, Freund's adjuvant, liposome, MF59, monophospholipid A, flagellin, CpG-ODN, and Poly(I:C).

[0078] Preferably, the final concentration of the fusion antigen or antigen fragment in the immunogenic composition is 0.1-1 mg / mL; for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL; preferably 0.1-0.5 mg / mL; more preferably 0.2-0.4 mg / mL; preferably, the final concentration of the fusion antigen or antigen fragment in the immunogenic composition is 0.2 or 0.4 mg / mL.

[0079] In another aspect, this application provides a method for preventing and / or treating group B meningitis, the method comprising administering to a subject an effective amount of the aforementioned fusion antigen and / or the aforementioned immunogenic composition.

[0080] In some embodiments, the dosage of the fusion antigen or immunogenic composition is 10-200 μg.

[0081] On the other hand, this application provides a method for preventing and / or treating meningitis, the method comprising administering to a subject an effective amount of the aforementioned fusion antigen and / or the aforementioned immunogenic composition and other serotype meningitis vaccines; preferably, the other serotype meningitis vaccines include one or more of group A meningitis, group C meningitis, group W135 meningitis, and group Y meningitis. Specifically, for example, the aforementioned fusion antigen and / or the aforementioned immunogenic composition are used in combination with the ACYW tetravalent polysaccharide-conjugated meningitis vaccine.

[0082] In some embodiments, the fusion protein or immunogenic composition may also be administered in combination with a variety of vaccines that are currently in use or under development and are intended for use in human or non-human subjects. Examples of vaccines for use in human subjects against infectious diseases include combined diphtheria-tetanus toxin vaccines; whole-cell pertussis vaccines; inactivated influenza vaccines; 23-valent pneumococcal vaccines; live measles vaccines; or live mumps vaccines; live rubella vaccines; Bacille Calmette-Guerin I (BCG) tuberculosis vaccines; hepatitis A vaccines; hepatitis B vaccines; hepatitis C vaccines; rabies vaccines (e.g., human diploid cell vaccines); inactivated polio vaccines; meningococcal polysaccharide vaccines (e.g., Sanofi Pasteur); and tetravalent meningococcal conjugate vaccines (e.g., Sanofi Pasteur). Pasteur (or Novartis); Yellow fever live virus vaccine; Typhoid inactivated whole cell vaccine; Cholera vaccine; Japanese encephalitis inactivated virus vaccine; Adenovirus vaccine; Cytomegalovirus vaccine; Rotavirus vaccine; Varicella vaccine; Anthrax vaccine; Smallpox vaccine; and other commercially available and experimental vaccines.

[0083] On the other hand, this application provides a method for inducing an antigen-specific immune response in an individual, the method comprising administering to a subject an effective amount of the aforementioned fusion antigen and / or the aforementioned immunogenic composition. In some embodiments, the dose of the fusion antigen or immunogenic composition is 10-200 μg.

[0084] On the other hand, this application provides the use of the aforementioned fusion antigen and / or the aforementioned immunogenic composition in the preparation of vaccines and / or medicaments for the prevention and / or treatment of group B meningitis.

[0085] On the other hand, this application provides the use of the aforementioned fusion antigen and / or the aforementioned immunogenic composition in combination with other serotype meningitis vaccines in the preparation of vaccines and / or medicaments for the prevention and / or treatment of meningitis.

[0086] Preferably, the other serotype meningitis vaccines include one or more of group A meningitis, group C meningitis, group W135 meningitis, and group Y meningitis.

[0087] In some embodiments, the immunogenic composition can be formulated as an injectable preparation, such as a liquid solution or emulsion; it can also be formulated as a solid form suitable for reconstitution into a solution or suspension, or a liquid excipient, prior to injection. The formulation may also be emulsified or encapsulated in liposomes to enhance adjuvant effects under the aforementioned pharmaceutically acceptable carriers. The conventional method is to administer the immunogenic composition via injection via a parenteral route (subcutaneous or intramuscular). Other formulations suitable for other routes of administration include oral, suppository, and transdermal applications. Therapeutic doses can be administered as a single-dose or multi-dose regimen. The immunogenic composition may be administered in combination with other immunomodulators. Example

[0088] To make the objectives, technical solutions, and advantages of this application clearer, a further detailed description of this application will be provided below. The embodiments described below are merely illustrative of several implementations of this application and should not be considered as limitations on this application. All other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this application. Therefore, the scope of protection of this patent application should be determined by the appended claims.

[0089] The raw materials and equipment used in the specific embodiments of this application are all known products, obtained by purchasing commercially available products. Example 1. Design of Group B meningococcal fHbp-NHBA fusion antigen 1.1 Design of fHbp-NHBA fusion antigen based on structural biology Taking the MC58 standard strain antigen as an example, the three-dimensional structures of fHbp and NHBA proteins were analyzed. While ensuring the immunogenicity and three-dimensional structural stability of the antigen, fHbp... - V1.1 uses the mature full-length antigen sequence (fHbp-V1.1, amino acid sequence as shown in SEQ ID NO: 1). NHBA-P3 is a sequence with no secondary structure deleted at the N-terminus (NHBA). △N (Amino acid sequence as shown in SEQ ID NO:17) fusion mode schematic diagram as shown in... Figure 1 As shown. 1.2 Construction of fHbp-NHBA fusion antigens of different subtypes Multiple antigenic subtypes from the CC4821 clonal group of the prevalent Chinese strain were selected to construct the fHbp-NHBA fusion antigen. The five fHbp antigen genotypes are: fHbp Variant 1.13 (fHbp-V1.13, amino acid sequence as shown in SEQ ID NO: 2), Variant 2.22 (fHbp-V2.22, amino acid sequence as shown in SEQ ID NO: 3), Variant 2.18 (fHbp-V2.18, amino acid sequence as shown in SEQ ID NO: 4), Variant 2.16 (fHbp-V2.16, amino acid sequence as shown in SEQ ID NO: 5), and Variant 3.31 (fHbp-V3.31, amino acid sequence as shown in SEQ ID NO: 6). The five NHBA genotypes are: Peptide 669 (NHBA-P669, amino acid sequence as shown in SEQ ID NO: 21), Peptide 688 (NHBA-P688, amino acid sequence as shown in SEQ ID NO: 26). The amino acid sequences of Peptide 503 (NHBA-P503, amino acid sequence as shown in SEQ ID NO: 31), Peptide 20 (NHBA-P20, amino acid sequence as shown in SEQ ID NO: 36), and Peptide 945 (NHBA-P945, amino acid sequence as shown in SEQ ID NO: 41) are shown in Table 1 below.

[0090] Different subtypes of fHbp are fused pairwise with different subtypes of NHBA to form fusion proteins covering multiple antigen subtypes. Specifically, the N-terminal amino acid of one antigen or its fragment is directly fused to the C-terminal amino acid of another antigen or its fragment, or fused via a peptide linker, such as (G)n, (GS)n, (GSG)n, (GGGS)n, (GSGGS)n, or (GGGGS)n, where n is an integer from 1 to 8, for example, 1, 2, 3, 4, 5, 6, 7, or 8; more preferably, the peptide linker includes GGGSGGGSGGGS or GSGGSGGSGGSG.

[0091] This embodiment provides different isoforms of fHbp-NHBA fusion proteins as shown in Table 2, including but not limited to: fHbp-V1.1+ NHBA-P3 △N fHbp-V1.13+ NHBA-P669 △N fHbp-V2.22+ NHBA-P669 △N fHbp-V1.13+ NHBA-P688 △N fHbp-V2.22+ NHBA-P688△N fHbp-V2.18+ NHBA-P669 △N fHbp-V3.31+NHBA-P669 △N fHbp-V2.18+ NHBA-P688 △N fHbp-V3.31+ NHBA-P688 △N fHbp-V2.16+ NHBA-P669 △N fHbp-V2.16+ NHBA-P688 △N fHbp-V2.16 +NHBA-P503 △N fHbp-V2.18 +NHBA-P20 △N fHbp-V2.22 +NHBA-P945 △N . 1.3 Design and optimization of fHbp-NHBA fusion antigens of different subtypes To further improve the stability and immunogenicity of the antigen, we studied different subtypes of fHbp-NHBA. △N Further truncated variants and point mutations were constructed from the fusion antigen design. Details are as follows: To further improve the stability of NHBA, we deleted the flexible sequence without secondary structure between the inter domain and the C-terminal domain and replaced it with a peptide linker to form fHbp-NHBA(Inter+C domain).

[0092] To address the inherent instability of the fHbp Variant 2 antigen, a point mutation L130R / G133D (simultaneously possessing L130R and G133D mutations) was introduced into the N-terminal domain of the fHbp Variant 2 subtype antigen, based on the fHbp-NHBA (Inter+C domain).

[0093] To reduce the interaction between fHbp and human factor H (FH) and improve the immunogenicity of the vaccine, point mutations E218A / E239A (containing both E218A and E239A mutations) were introduced for the fHbp-NHBA (Inter+C domain) antigen; point mutations E217A / T238A (containing both E217A and T238A mutations) were introduced for the fHbp Variant 1 antigen; and point mutations E225A / T246A (containing both E225A and T246A mutations) were introduced for the fHbp Variant 3 antigen.

[0094] Corresponding truncation, truncation / mutation site design, such as Figure 1 The sequence is shown in Table 2. 1.4 Different subtypes of fHbp-NHBA fusion antigen compositions The aforementioned fHbp-NHBA fusion protein of any one subtype can be combined with other fHbp-NHBA fusion proteins to form immunogenic compositions covering multiple antigen subtypes. For example, the combinations composed of fHbp-V1.13+NHBA-P688, fHbp-V3.31+NHBA-P669, fHbp-V2.16+NHBA-P503, fHbp-V2.18+NHBA-P20, and fHbp-V2.22+NHBA-P945 correspond to VB86T, VB96T, VB129, VB172, and VB174, respectively, with amino acid sequences of SEQ ID NO: 63, 66, 83, 84, and 85, respectively; another example is an immunogenic composition containing VB135, VB136, VB137, VB138, and VB139, with amino acid sequences as shown in SEQ ID NO: 86-90. Different forms of immunogenic compositions all have immune-enhancing effects and provide broad-spectrum immune protection against a variety of prevalent bacterial strains. Table 1. Antigen mutations and truncated fragments

[0095] Table 2. Design and sequences of different isotypes of fHbp-NHBA fusion proteins

[0096] Note: fHbp antigen is directly linked to NHBA antigen or linked through a peptide linker, indicated by "+". Example 2. Construction of prokaryotic expression plasmids

[0097] Based on the antigen amino acid sequence described in Example 1 and the codon preference of *E. coli*, a full-length DNA sequence was designed and synthesized. The full-length DNA sequence was then ligated into the expression vector pET28a or pET30a, a process performed by GenScript Biotech. The constructed expression plasmid was then transformed... E. coli BL21(DE3) was cultured overnight at 37°C in a petri dish containing 50 μg / ml kanamycin to obtain the expression strain. Single clones were picked and inoculated into liquid LB medium, and cultured at 37°C with shaking at 200 rpm until the bacterial density reached OD. 600At approximately 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the mixture was cultured at 37°C with shaking for 4 hours. SDS-PAGE analysis revealed a clear expression band after induction. Example 3. Expression and purification of recombinant proteins 3.1 Mass cultivation The selected bacterial strains were inoculated into LB liquid medium and cultured at 37°C and 200 rpm for 3-6 hours until the OD of the bacterial culture was measured. 600 When the bacterial cell density reaches 0.8–1.0, the temperature is lowered to 16°C, and 0.5 mM isopropyl thiogalactoside (IPTG) is added for induction for 18 hours. Subsequently, the cells are collected by centrifugation at 5000 rpm for 15 min, and the supernatant is discarded. 3.2 Cell disruption Add PBS to the collected bacterial cells, homogenize using high pressure, centrifuge at high speed (15000 rpm, 30 min, 4°C), and collect the supernatant. 3.3 Protein Extraction The supernatant of the centrifuged bacterial lysate was incubated with Ni-NTA at 4°C for 2 hours. The mixture was then added to a purification column, allowing the liquid to flow through the column by gravity. Non-specifically bound protein impurities were washed away using buffer A (20 mM imidazole, 50 mM Tris, 150 mM NaCl, pH 8.0). Eluting was then performed using buffer B (500 mM imidazole, 50 mM Tris, 150 mM NaCl, pH 8.0). 3.4 Protein purification The protein purified by the nickel column was concentrated to 1 mL and then subjected to gel filtration chromatography. A suitable molecular sieve model (Superdex 200 or Superdex 75) was selected based on the protein's molecular weight. The molecular sieve was connected to the ÄKTA high-performance chromatography system and pre-equilibrated using a buffer solution containing 50 mM Tris, 150 mM NaCl, and pH 8.0, with an equilibration volume of one column volume (approximately 24 mL). The concentrated protein sample was then introduced into the molecular sieve through a loading loop, and the protein was eluted at a flow rate of 0.5 mL / min. The elution volume was one column volume. 3.5 SDS-PAGE Identification The collected samples were validated by SDS-PAGE to ensure that the purity of the samples reached more than 95%. Example 4. Drug-likeness analysis of recombinant protein 4.1 Analysis of the purity, uniformity, and yield of recombinant proteins The purity and homogeneity of fHbp-NHBA recombinant proteins from different design schemes were identified by molecular gel filtration chromatography and SDS-PAGE. Specific procedures are detailed in Example 3, Protein Purification. The purification results of the fHbp-NHBA (Inter+C domain) series of recombinant proteins are shown below. Figure 2 As shown, the purification results of the recombinant proteins designed from the fHbp-NHBA (Inter+C domain) mutant series are as follows: Figure 3 As shown. 4.2 Thermal stability analysis of recombinant proteins The stability of the antigen determines the conditions for vaccine production, storage, and transportation. Melting temperature (T0) is a key factor. m ) and polymerization temperature (T) agg The thermostability of recombinant proteins reflects antigen stability and is an important indicator of vaccine drugability. The thermostability of recombinant proteins was identified using an Uncle Analysis instrument. The specific procedure was as follows: the purified test protein was uniformly diluted to a concentration of 1 mg / mL, centrifuged at 14000 rpm for 10 minutes before loading, and then 9 μL of the supernatant was added to the sample well for testing. The Tg of each subtype of fHbp-NHBA fusion antigen was determined. agg The test result at 266 (°C) is as follows: Figure 4 As shown, T m The test results (°C) are shown in Figure 5-8.

[0098] The experimental results are as follows: 1) fHbp-NHBA △N The results of thermostability analysis of the series of recombinant proteins are shown in Table 3. Different isotypes of fHbp-NHBA △N The thermal stability of fusion antigens varies; for example, the fusion antigen containing fHbp variant 2 exhibits higher thermal stability. m and T agg The value was significantly lower than that of other fHbp subtype fusion antigen constructions. Table 3. Thermal stability analysis of some fHbp-NHBA△N designs

[0099] 2) The thermostability analysis results of the fHbp-NHBA (Inter+C domain) series of recombinant proteins are shown in Table 4. The further truncated fHbp-NHBA (Inter+C domain) designs showed better thermostability than the same isotype of fHbp-NHBA. △N The antigen exhibits higher temperature tolerance, as reflected in T... m Value and T agg266All values ​​increased. Table 4. Thermal stability analysis of the fHbp-NHBA (Inter+Cdomain) design

[0100] 3) The thermostability analysis results of the fHbp-NHBA (Inter+C domain) mutant series of recombinant proteins are shown in Table 5. Experiments revealed that the series of mutant-designed T... m Value and T agg266 All values ​​increased. Among them, the stability of the fHbp variant2 antigen design containing the L130R / G133D mutation was significantly increased. Table 5. Thermal stability analysis of the fHbp-NHBA(Inter+Cdomain) mutation design

[0101] Example 5. Vaccine Preparation

[0102] The purified protein was detoxified using a Thermo Fisher endotoxin removal column (catalog number 88276), and the residual endotoxin content was then detected using Limulus amebocyte lysate (LAL) reagent, confirming that the endotoxin content in each injection was less than 10 EU.

[0103] Protein sample concentrations were determined using the BCA method, followed by antigen and adjuvant preparation. In the aluminum adjuvant immunization protocol, filtered and sterilized antigen was incubated with either aluminum hydroxide or aluminum phosphate adjuvant at room temperature for 1 hour before mouse immunization. The working antigen concentration was 0.4 mg / ml, and the working aluminum ion concentration was 1.0 mg / ml. In the Freund's adjuvant immunization protocol, equal volumes of antigen and Freund's adjuvant were emulsified, resulting in a working antigen concentration of 0.2 mg / ml. Example 6. Mouse immunization program and serum collection

[0104] Female BALB / c mice around six weeks old were randomly divided into groups of five. Each mouse was immunized three times on days 0, 21, and 35, with an intraperitoneal injection of 100 μL. The experimental group was injected with antigen / Al. 3+ The negative control group received saline injections. The positive control group received 1 / 15th of the human dose of 4CMenB vaccine or Trumenba vaccine.

[0105] Blood was collected from the eyeballs 14 days after the third immunization to prepare serum. After standing at 37°C for 30 minutes, the serum was collected by centrifugation at 6000 rpm for 10 minutes. Example 7. Determination of specific IgG levels in immune serum

[0106] The specific steps for indirect ELISA detection of serum specific IgG antibody levels are as follows: (1) Antigen coating: Dilute the antigen to a concentration of 0.5 μg / mL with coating buffer (phosphate buffer, pH=9.6), add 100 μL to each well of a 96 plate, seal, and coat overnight at 4°C.

[0107] (2) Washing: Shake off the liquid in the wells thoroughly and pat dry with absorbent paper. Add 200 μL of Wash Buffer (1×PBST) to each well, let stand for 1 min, shake off the liquid, pat dry the ELISA plate, wash five times, and pat dry the ELISA plate.

[0108] (3) Blocking: Block the ELISA plate with 200 μL of PBST solution containing 1% BSA at 37°C for 2 h.

[0109] (4) Washing: The method is the same as step (2).

[0110] (5) Primary antibody incubation: Add 100 μL of 1×Assay Buffer to the blank wells, add 100 μL of mouse serum samples immunized with different antigens after two-fold serial dilution to the experimental wells, and set up PBS negative control wells. Incubate at room temperature for 1 h.

[0111] (6) Washing, the method is the same as step (2).

[0112] (7) Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgG antibody diluted 1:10000 to all wells and incubate at room temperature for 1 h.

[0113] (8) Washing: The method is the same as step (2).

[0114] (9) Color development: Add 100 μL of TMB substrate to each well and incubate at 37 °C in the dark for 15 min.

[0115] (10) Termination of reaction: Add 50 μL of H2SO4 to each well to terminate the reaction.

[0116] (11) Measurement: Turn on the microplate reader, wait for the microplate reader to react for several minutes, and read the OD value after zeroing the blank well at a wavelength of 450 nm.

[0117] (12) Experimental results and analysis: In immunization regimens formulated with Freund's adjuvant, the results of serum IgG antibody titers induced by different subtypes of the fHbp-NHBA fusion antigen are shown in [the table below]. Figure 9The geometric mean titer of anti-fHbp IgG induced by VB17 immune serum was 8.67E+4, and the geometric mean titer of anti-NHBA IgG was 9.96E+4; the geometric mean titer of anti-fHbp IgG induced by VB17T immune serum was 1.24E+5, and the geometric mean titer of anti-NHBA IgG was 1.35E+5; the geometric mean titer of anti-fHbp IgG induced by VB82 immune serum was 4.74E+4, and the geometric mean titer of anti-NHBA IgG was 2.08E+4; the geometric mean titer of anti-fHbp IgG induced by VB96 immune serum was 3.72E+5, and the geometric mean titer of anti-NHBA IgG was 2.89E+4; the geometric mean titer of anti-fHbp IgG induced by VB100 immune serum was 1.35E+5, and the geometric mean titer of anti-NHBA IgG was 3.60E+4. The geometric mean titer of anti-fHbp IgG induced by VB84 immune serum was 4.74E+4, and the geometric mean titer of anti-NHBA IgG was 1.49E+4; the geometric mean titer of anti-fHbp IgG induced by VB88 immune serum was 8.67E+4, and the geometric mean titer of anti-NHBA IgG was 8.67E+4; the geometric mean titer of anti-fHbp IgG induced by VB94 immune serum was 1.04E+4, and the geometric mean titer of anti-NHBA IgG was 2.14E+4; the geometric mean titer of anti-fHbp IgG induced by VB98 immune serum was 2.89E+4, and the geometric mean titer of anti-NHBA IgG was 8.67E+4; the geometric mean titer of anti-fHbp IgG induced by VB102 immune serum was 6.96E+4, and the geometric mean titer of anti-NHBA IgG was 1.00E+4. The geometric mean titer of anti-fHbp IgG induced by VB104 immune serum was 1.14E+4, and the geometric mean titer of anti-NHBA IgG was 1.00E+4.

[0118] In immunization regimens using aluminum phosphate adjuvant, different subtypes of the fHbp-NHBA fusion antigen also induced high levels of serum IgG antibody titers. The results are shown in [see attached table]. Figure 10 .

[0119] In the fHbp-V1.13+NHBA-P688 fusion design, the geometric mean titer of anti-fHbp IgG induced by VB86T immune serum was 2.30E+6, and the geometric mean titer of anti-NHBA IgG was 6.31E+5; the geometric mean titer of anti-fHbp IgG induced by VB135 immune serum was 4.00E+6, and the geometric mean titer of anti-NHBA IgG was 8.33E+5. In the fHbp-V2.16+NHBA-P503 fusion design, the geometric mean titer of anti-fHbp IgG induced by VB178 immune serum was 3.98E+5, and the geometric mean titer of anti-NHBA IgG was 1.00E+5; the geometric mean titer of anti-fHbp IgG induced by VB129 immune serum was 1.00E+6, and the geometric mean titer of anti-NHBA IgG was 3.98E+5; the geometric mean titer of anti-fHbp IgG induced by VB137 immune serum was 1.00E+5, and the geometric mean titer of anti-NHBA IgG was 1.00E+5. In the fHbp-V2.18+NHBA-P20 fusion design, the geometric mean titer of anti-fHbp IgG induced by VB179 immune serum was 1.54E+6, and the geometric mean titer of anti-NHBA IgG was 6.31E+5; the geometric mean titer of anti-fHbp IgG induced by VB172 immune serum was 8.87E+5, and the geometric mean titer of anti-NHBA IgG was 7.58E+5; the geometric mean titer of anti-fHbp IgG induced by VB138 immune serum was 3.98E+5, and the geometric mean titer of anti-NHBA IgG was 8.33E+5. In the fHbp-V2.22+NHBA-P945 fusion design, the geometric mean titer of anti-fHbp IgG induced by VB180 immune serum was 3.31E+5, and the geometric mean titer of anti-NHBA IgG was 6.31E+4; the geometric mean titer of anti-fHbp IgG induced by VB174 immune serum was 1.00E+5, and the geometric mean titer of anti-NHBA IgG was 6.31E+4; the geometric mean titer of anti-fHbp IgG induced by VB139 immune serum was 3.31E+5, and the geometric mean titer of anti-NHBA IgG was 1.58E+5. In the fHbp-V3.31+NHBA-P669 fusion design, the geometric mean titer of anti-fHbp IgG induced by VB96T immune serum was 5.25E+5, and the geometric mean titer of anti-NHBA IgG was 6.31E+4; the geometric mean titer of anti-fHbp IgG induced by VB136 immune serum was 3.16E+5, and the geometric mean titer of anti-NHBA IgG was 1.00E+5. Example 8. Serum bactericidal test (SBA)

[0120] 8.1 Preparation of bacterial culture One day in advance, spread an appropriate amount of frozen MC58 bacterial suspension onto blood agar plates and incubate overnight at 37°C and 5% CO2. The next day, scrape an appropriate amount of bacterial colony from the blood agar plate and transfer it to PBS, shake thoroughly, then spread it onto a new blood agar plate and incubate at 37°C and 5% CO2 for 4-6 hours. Scrape an appropriate amount of colonies and transfer them to DPBS (containing 1% glucose) bacterial suspension, diluting the bacterial suspension to a concentration of approximately 2.0 × 10⁻⁶. 4 CFU / mL, the specific procedure is as follows: First, dilute the bacterial suspension to OD500. 600 =Approximately 0.36, then dilute the bacterial solution 20,000 times and set aside.

[0121] 8.2 Preparation of complement Complement was selected from adult serum that had IgM and IgG removed. The experimental group used active complement, while the control group used inactivated complement (inactivated by incubation in a 56°C water bath for 30 min). 8.3 Dilution of mouse serum samples to be tested The serum to be tested was first incubated in a 56°C water bath for 30 min to inactivate endogenous complement. Then, it was diluted twofold with DPBS (containing 1% glucose) in a 96-well plate, resulting in nine dilution gradients from 1:4 to 1:1024. Specifically, 20 μL of DPBS was added to wells 1-9 of the 96-well plate. Then, 20 μL of the serum to be tested was added to wells 1-4 in the first column. After mixing by pipetting, 20 μL of the liquid was transferred to wells 2 in the second column, and so on until well 9. Finally, 20 μL of the liquid was discarded. The first nine columns were the experimental group. A control group was set up in column 12, containing 10 μL of DPBS and 10 μL of the serum to be tested. Columns 10 and 11 were the mouse serum-free control group; column 10 was the complement control group; and column 11 was the colony control group, containing 20 μL of DPBS.

[0122] 8.4 Sample Mixing Add complement to the 96-well plate containing diluted serum. Add 10 μL of human complement to columns 1-10 and 10 μL of inactivated human complement to columns 11-12. Then add 10 μL of diluted bacterial culture to all sample wells, mix well, and incubate at 37°C and 65 rpm for one hour.

[0123] 8.5 Coated Plate After incubation, remove the 96-well plate, spread the sample mixture onto a blood agar plate, and invert the plate in a 5% CO2 incubator overnight. Observe and count the samples the next day.

[0124] When interpreting the results, non-specific killing rates (NSK) were calculated based on the colony counts of the control group and the complement control group. The experiment was considered valid when the NSK was less than 25%. Subsequently, the bactericidal titer of the serum was calculated. The serum bactericidal titer is the reciprocal of the maximum serum dilution that inhibits the growth of 50% of Neisseria meningitidis.

[0125] 8.6 Experimental Results and Analysis Serum bactericidal assay (SBA) is the gold standard for evaluating the protective efficacy of meningococcal vaccines. Studies have confirmed that when using human complement, an hSBA ≥ 1:4 indicates protective efficacy of the meningococcal vaccine; when using rabbit complement, an rSBA ≥ 1:8 indicates protective efficacy of the meningococcal vaccine.

[0126] 1) Vaccine effectiveness analysis To assess vaccine efficacy, serum bactericidal experiments were performed using MenB strains expressing the homologous fHbp subtype. Results are shown below. Figure 11 .

[0127] The target bacteria used in this experiment include the following: target bacteria ST01-420 (fHbp-V1.5 / NHBA-p9), target bacteria ST01-354 (fHbp-V2.16 / NHBA-p910), ST01-324 (fHbp-V2.16 / NHBA-p688), target bacteria ST01-044 (fHbp-V2.18 / NHBA-p945), target bacteria ST01-119 (fHbp-V2.22 / NHBA-p1101), and target bacteria ST01-401 (fHbp-V3.1239 / NHBA-p669).

[0128] When using the target bacterium ST01-420 (fHbp-V1.5 / NHBA-p9) for detection, the SBA titer of 4CMenB vaccine immune serum was 668.03; the SBA titer of Trumenba vaccine immune serum was 15.15; and the SBA titers of immune serum containing the fusion antigen of fHbp V1.13 were VB82 (97.56), VB86T (546.5), and VB135 (478.01), respectively.

[0129] When using the target bacterium ST01-354 (fHbp-V2.16 / NHBA-p910) for detection, the SBA titer of 4CMenB vaccine immune serum was 4; the SBA titer of Trumenba vaccine immune serum was 3; and the SBA titers of immune serum containing the fusion antigen of fHbpV2.16 were VB102 (27.33), VB104 (4.36), VB178 (4.67), VB129 (24.81) and VB137 (6.42), respectively.

[0130] When using the target bacterium ST01-324 (fHbp-V2.16 / NHBA-p688) for detection, the SBA titer of 4CMenB vaccine immune serum was 14.30; the SBA titer of Trumenba vaccine immune serum was 42.31; and the SBA titers of immune serum containing the fusion antigen of fHbpV2.16 were VB102 (9.54), VB104 (5.03), VB178 (73.62), VB129 (130.25) and VB137 (64.70), respectively.

[0131] When using the target bacteria ST01-044 (fHbp-V2.18 / NHBA-p945) for detection, the SBA titer of the 4CMenB vaccine immune serum was 3; the SBA titer of the Trumenba vaccine immune serum was 3; and the SBA titers of the fusion antigen immune serum containing fHbpV2.18 were VB94 (3), VB98 (3), VB179 (6.92), VB172 (4.25), and VB138 (96.08), respectively.

[0132] When using the target bacterium ST01-119 (fHbp-V2.22 / NHBA-p1101) for detection, the SBA titer of 4CMenB vaccine immune serum was 514.45; the SBA titer of Trumenba vaccine immune serum was 34.68; and the SBA titers of immune serum containing the fusion antigen of fHbpV2.22 were VB84 (14.17), VB88 (61.93), VB180 (9.20), VB174 (100.23), and VB139 (155.56).

[0133] When using the target bacterium ST01-401 (fHbp-V3.1239 / NHBA-p669) for detection, the SBA titer of 4CMenB vaccine immune serum was 560.35; the SBA titer of Trumenba vaccine immune serum was 62.63; and the SBA titers of immune serum containing the fusion antigen fHbpV3.31 were VB96 (121.98), VB100 (149.68), VB96-T (343.7) and VB136 (364.87), respectively.

[0134] The above experimental results show that the fHbp-NHBA fusion antigen designs based on multiple subtypes can induce serum bactericidal activity comparable to and significantly higher than that of the 4CMenB vaccine and the Trumenba vaccine. Among them, the fusion antigen designs including the fHbp-V2.16 and fHbp-V2.18 subtypes (such as VB102, VB178, VB129, VB137, VB179, VB172, and VB138) induced bactericidal activity significantly better than the two control vaccines, 4CMenB and Trumenba. The results indicate that, compared to MenB vaccines marketed in Europe and the United States, the fHbp-NHBA fusion antigen design in this patent exhibits superior immunoprotective potential against prevalent fHbp Variant 2 strains.

[0135] 2) Analysis of the broad spectrum of vaccines Different subtypes of fHbp-NHBA fusion antigen-immunized sera were mixed, and the bactericidal effect of the mixed serum against various target bacteria was detected. The results are as follows: Figure 12 As shown.

[0136] The bactericidal titers of the mixed serum containing VB135, VB136, VB137, VB138, and VB139 against target bacteria ST01-420 (fHbp-V1.5 / NHBA-p9) were 814.47; against target bacteria ST01-324 (fHbp-V2.16 / NHBA-p688) were 1044.73; against target bacteria ST01-044 (fHbp-V2.18 / NHBA-p945) were 151.40; against target bacteria ST01-119 (fHbp-V2.22 / NHBA-p1101) were 1130.70; and against target bacteria ST01-401 (fHbp-V3.1239 / NHBA-p669) were 928.45. The mixed serum showed significantly higher immunoprotective effects against all tested strains than the 4CMenB vaccine and the Trumenba vaccine.

[0137] It is noteworthy that when immune sera containing different subtypes of fHbp-NHBA fusion antigen were mixed, their bactericidal titers were significantly higher than those of immune sera containing any single subtype of fusion antigen. This indicates that combined immunization with different subtypes of fHbp-NHBA fusion antigen can induce synergistic bactericidal effects, thereby further enhancing the immunogenicity and broad-spectrum protective ability of the vaccine. Example 9. Evaluation of the in vivo protective effect of the fusion antigen against MenB strain-infected mice

[0138] 9.1 Mouse Immunization Protocol Six-week-old female CD-1 mice were randomly divided into groups of five. The negative control group was injected with PBS / Al(OH)3; experimental group 1 was injected with a mixture of VB135+VB136+VB137+VB138+VB139 antigens adsorbed with Al(OH)3 adjuvant; experimental group 2 was injected with a mixture of VB86T+VB96T+VB129+VB172+VB174 antigens adsorbed with Al(OH)3 adjuvant. The immunization dose of each antigen was 40ug, administered subcutaneously, with three injections given at two-week intervals.

[0139] 9.2 Pre-infection treatment MenB strain was inoculated onto blood agar plates and incubated overnight at 37°C with 5% CO2. Single colonies were picked and inoculated into liquid BHI medium containing 10 μg / ml deferoxamine (DFOM) and cultured until the logarithmic growth phase (OD200). 650 ≈ 0.4). Collect bacterial cells by centrifugation, wash and resuspend with sterile PBS, and adjust to the predetermined challenge concentration, approximately 10. 6 -10 8 CFU / each.

[0140] 9.3 Mouse challenge Twenty-four hours before challenge, mice were intraperitoneally injected with human transferrin (dosage: 10 mg / mouse) to provide exogenous bound iron for the bacteria and promote their proliferation in the host.

[0141] On the day of challenge, mice were intraperitoneally injected with 100 μL of freshly prepared bacterial solution (approximately 10 μL of ... 6 -10 8 (CFU / mouse). Closely monitor the mice's condition daily after injection.

[0142] 9.4 Analysis of Experimental Results The in vivo protective effects of different subtypes of fHbp-NHBA fusion antigen immunization mixtures against MenB bacteria-infected mice were as follows: Figure 13 As shown, the immunization mixture containing five antigens—VB86T, VB96T, VB129, VB172, and VB174—exhibited protective effects against multiple MenB target bacteria: 100% protection against mice infected with target bacteria ST01-448 (fHbp-V1.248 / NHBA-P688); 80% protection against mice infected with target bacteria ST01-324 (fHbp-V2.16 / NHBA-P688); 80% protection against mice infected with target bacteria ST01-105 (fHbp-V2.16 / NHBA-P73); 100% protection against mice infected with target bacteria ST01-119 (fHbp-V2.22 / NHBA-P1101); and 100% protection against mice infected with target bacteria ST01-401 (fHbp-V3.1239 / NHBA-P669).

[0143] In addition, the immune mixture containing five antigens, namely VB135, VB136, VB137, VB138 and VB139, also showed 100% protection against mice infected with the target bacterium ST01-401 (fHbp-V3.1239 / NHBA-P669).

[0144] In summary, this embodiment designed a fusion protein of group B meningococcal fHbp-NHBA, which is applicable to antigenic gene subtypes of different prevalent strains. Experimental results demonstrate that this fusion protein exhibits good drug-likeness and immunogenicity. Furthermore, immune compositions containing different subtypes of fHbp-NHBA fusion proteins showed broad-spectrum immunoprotective effects against a variety of prevalent strains both in vivo and in vitro.

[0145] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0146] The embodiments described above are merely illustrative of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the protection scope of this disclosure. Therefore, the protection scope of this patent should be determined by the appended claims. Table 6. Amino acid sequences

[0147] Table 7. Nucleotide Sequences

[0148]

Claims

1. An fHbp-NHBA fusion antigen, said fusion antigen comprising one or more fHbp antigens and one or more NHBA antigens.

2. The fusion antigen according to claim 1, wherein the fHbp antigen comprises fHbp-V1.1 or its mutant, fHbp-V1.13 or its mutant, fHbp-V2.22 or its mutant, fHbp-V2.18 or its mutant, fHbp-V2.16 or its mutant, and fHbp-V3.31 or its mutant; Preferably, the amino acid sequence of fHbp-V1.1 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 1; more preferably, the amino acid sequence of fHbp-V1.1 is as shown in SEQ ID NO:

1. Preferably, the mutant of fHbp-V1.1 has E218A and / or E239A mutations; more preferably, it has both E218A and E239A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 7; Preferably, the amino acid sequence of fHbp-V1.13 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 2; more preferably, the amino acid sequence of fHbp-V1.13 is as shown in SEQ ID NO:

2. Preferably, the mutant of fHbp-V1.13 has E218A and / or E239A mutations; more preferably, it has both E218A and E239A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 8; Preferably, the amino acid sequence of fHbp-V2.22 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 3; more preferably, the amino acid sequence of fHbp-V2.22 is as shown in SEQ ID NO: 3; Preferably, the mutant of fHbp-V2.22 has one or more mutations selected from L130R, G133D, E217A, and T238A; more preferably, it has both L130R and G133D mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:13; or, it has both L130R, G133D, E217A, and T238A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:

14. Preferably, the amino acid sequence of fHbp-V2.18 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 4; more preferably, the amino acid sequence of fHbp-V2.18 is as shown in SEQ ID NO:

4. Preferably, the mutant of fHbp-V2.18 has one or more mutations selected from L130R, G133D, E217A, and T238A; more preferably, it has both L130R and G133D mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:11; or, it has both L130R, G133D, E217A, and T238A mutations; more preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO:

12. Preferably, the amino acid sequence of fHbp-V2.16 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 5; more preferably, the amino acid sequence of fHbp-V2.16 is as shown in SEQ ID NO:

5. Preferably, the mutant of fHbp-V2.16 has one or more mutations selected from L130R, G133D, E217A, and T238A; more preferably, it has both L130R and G133D mutations; more preferably, the amino acid sequence of the mutant is shown in SEQ ID NO: 9; or, it has both L130R, G133D, E217A, and T238A mutations; more preferably, the amino acid sequence of the mutant is shown in SEQ ID NO:

10. Preferably, the amino acid sequence of fHbp-V3.31 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 6; more preferably, the amino acid sequence of fHbp-V3.31 is as shown in SEQ ID NO:

6. Preferably, the mutant of fHbp-V3.31 has E225A and / or T246A mutations; more preferably, it has both E225A and T246A mutations; more preferably, the amino acid sequence of the mutant is shown in SEQ ID NO:

15.

3. The fusion antigen according to claim 1 or 2, wherein the NHBA antigen comprises NHBA-P3 or a truncated form thereof, NHBA-P669 or a truncated form thereof, NHBA-P688 or a truncated form thereof, NHBA-P503 or a truncated form thereof, NHBA-P20 or a truncated form thereof, and NHBA-P945 or a truncated form thereof; Preferably, the amino acid sequence of NHBA-P3 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 16; preferably, the amino acid sequence of NHBA-P3 is as shown in SEQ ID NO:

16. Preferably, the amino acid sequence of NHBA-P669 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 21; preferably, the amino acid sequence of NHBA-P669 is as shown in SEQ ID NO:

21. Preferably, the amino acid sequence of NHBA-P688 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 26; preferably, the amino acid sequence of NHBA-P688 is as shown in SEQ ID NO:

26. Preferably, the amino acid sequence of NHBA-P503 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 31; preferably, the amino acid sequence of NHBA-P503 is as shown in SEQ ID NO:

31. Preferably, the amino acid sequence of NHBA-P20 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 36; preferably, the amino acid sequence of NHBA-P20 is as shown in SEQ ID NO:

36. Preferably, the amino acid sequence of NHBA-P945 has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 41; preferably, the amino acid sequence of NHBA-P945 is as shown in SEQ ID NO:

41. Preferably, the amino acid sequence of the NHBA-P3 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 17; more preferably, the amino acid sequence of the NHBA-P3 truncated form is as shown in SEQ ID NO:

17. Alternatively, the NHBA-P3 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 18-19, either directly or via peptide linkers; preferably, the NHBA-P3 truncated form is composed of SEQ ID NO: 18-19 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P3 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 20; even more preferably, the amino acid sequence of the NHBA-P3 truncated form is as shown in SEQ ID NO:

20. Preferably, the amino acid sequence of the NHBA-P669 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 22; more preferably, the amino acid sequence of the NHBA-P669 truncated form is as shown in SEQ ID NO:

22. Alternatively, the NHBA-P669 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 23-24, either directly or via peptide linkers; preferably, the NHBA-P669 truncated form is composed of SEQ ID NO: 23-24 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P669 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 25; even more preferably, the amino acid sequence of the NHBA-P669 truncated form is as shown in SEQ ID NO: 25; Preferably, the amino acid sequence of the NHBA-P688 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 27; more preferably, the amino acid sequence of the NHBA-P688 truncated form is as shown in SEQ ID NO:

27. Alternatively, the NHBA-P688 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 28-29, either directly or via peptide linkers; preferably, the NHBA-P688 truncated form is composed of SEQ ID NO: 28-29 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P688 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 30; even more preferably, the amino acid sequence of the NHBA-P688 truncated form is as shown in SEQ ID NO: 30; Preferably, the amino acid sequence of the NHBA-P503 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 32; more preferably, the amino acid sequence of the NHBA-P503 truncated form is as shown in SEQ ID NO:

32. Alternatively, the NHBA-P503 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 33-34, either directly or via peptide linkers; preferably, the NHBA-P503 truncated form is composed of SEQ ID NO: 33-34 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P503 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 35; even more preferably, the amino acid sequence of the NHBA-P503 truncated form is as shown in SEQ ID NO: 35; Preferably, the amino acid sequence of the NHBA-P20 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 37; more preferably, the amino acid sequence of the NHBA-P20 truncated form is as shown in SEQ ID NO:

37. Alternatively, the NHBA-P20 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 38-39, either directly or via peptide linkers; preferably, the NHBA-P20 truncated form is composed of SEQ ID NO: 38-39 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P20 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 40; even more preferably, the amino acid sequence of the NHBA-P20 truncated form is as shown in SEQ ID NO:

40. Preferably, the amino acid sequence of the NHBA-P945 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 42; more preferably, the amino acid sequence of the NHBA-P945 truncated form is as shown in SEQ ID NO:

42. Alternatively, the NHBA-P945 truncated form is composed of amino acid sequences having at least 80% homology with SEQ ID NO: 43-44, either directly or via peptide linkers; preferably, the NHBA-P945 truncated form is composed of SEQ ID NO: 43-44 linked via a first peptide linker; more preferably, the amino acid sequence of the NHBA-P945 truncated form has at least 80% homology with the amino acid sequence shown in SEQ ID NO: 45; even more preferably, the amino acid sequence of the NHBA-P945 truncated form is as shown in SEQ ID NO:

45.

4. The fusion antigen according to claim 3, wherein the first peptide linker comprises (G)n, (GS)n, (GSG)n, (GGGS)n, (GSGGS)n, (GGGGS)n, and (GGGGS)n, wherein n comprises 1, 2, 3, 4, 5, 6, 7, or 8; more preferably, the first peptide linker comprises GSGGSGGSGGSG.

5. The fusion antigen according to any one of claims 1-4, wherein the fHbp antigen and the NHBA antigen are directly linked or linked through a peptide linker in the fusion antigen; More preferably, the connection method between the fHbp antigen and the NHBA antigen includes: fHbp antigen or its fragment-second peptide linker-NHBA antigen or its fragment, NHBA antigen or its fragment-second peptide linker-fHbp antigen or its fragment, fHbp antigen or its fragment-NHBA antigen or its fragment, NHBA antigen or its fragment-fHbp antigen or its fragment; Preferably, the connection is made via a second peptide linker; Preferably, the second peptide linker includes (G)n, (GS)n, (GSG)n, (GGGS)n, (GSGGS)n, (GGGGS)n, and (GGGGS)n, wherein n includes 1, 2, 3, 4, 5, 6, 7, or 8; more preferably, the second peptide linker includes GGGSGGGSGGGS.

6. The fusion antigen according to any one of claims 1-5, wherein the amino acid sequence of the fusion antigen has at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 46-90; more preferably, the amino acid sequence of the fusion antigen is as shown in any one of SEQ ID NO: 46-90.

7. An immunogenic composition comprising one or more of the fusion antigens described in any one of claims 1-6.

8. The immunogenic composition according to claim 7, wherein the immunogenic composition further comprises an antigen fragment having at least 80% homology with the amino acid sequence shown in any one of SEQ ID NO: 46-90; Preferably, the amino acid sequence of the immunogenic composition is as shown in any one of SEQ ID NO: 46-90; Preferably, the immunogenic composition contains a fusion antigen with amino acid sequences as shown in SEQ ID NO: 63, 66, 83, 84, 85, or the immunogenic composition contains a fusion antigen with amino acid sequences as shown in SEQ ID NO: 86-90.

9. The immunogenic composition according to claim 7 or 8, wherein the immunogenic composition further comprises one or more of an adjuvant, a buffer solution, a stabilizer, and a surfactant; Preferably, the adjuvant is at least one selected from aluminum adjuvant, Freund's adjuvant, liposome, MF59, monophospholipid A, flagellin, CpG-ODN, and Poly(I:C). Preferably, the final concentration of the fusion antigen or antigen fragment in the immunogenic composition is 0.1-1 mg / mL; preferably, the final concentration of the fusion antigen or antigen fragment in the immunogenic composition is 0.2 or 0.4 mg / mL. Preferably, the immunogenic composition is an injectable agent; preferably, the injectable agent includes a liquid solution or an emulsion.

10. Use of the fusion antigen according to any one of claims 1-6 and / or the immunogenic composition according to any one of claims 7-9 in the preparation of vaccines and / or medicaments for the prevention and / or treatment of group B meningitis.

11. The use of the fusion antigen of any one of claims 1-6 and / or the immunogenic composition of any one of claims 7-9 in combination with other serotype meningitis vaccines in the preparation of vaccines and / or medicaments for the prevention and / or treatment of meningitis; Preferably, the other serotype meningitis vaccines include one or more of group A meningitis, group C meningitis, group W135 meningitis, and group Y meningitis.

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