Group b neisseria meningitides antigen fusion protein combination and use thereof

Abstract

The present invention relates to the field of protein engineering modification. Provided are a group B neisseria meningitides antigen fusion protein combination and the use thereof. The antigen fusion protein combination comprises an antigen fusion protein, the antigen fusion protein comprising two or more antigens or fragments thereof that are optionally selected from the following: (i) an fHbp antigen; (ii) an NHBA antigen; (iii) an NadA antigen; or (iv) a PorA loop. The designed antigen fusion protein can combine one or more different subtypes for administration, thereby producing more broad-spectrum immune effects, improving the stability of the antigens, simplifying the production process and reducing costs.

Description

Antigen fusion protein combinations of group B meningococci and their uses

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. 2024114518927, filed on October 16, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of protein engineering, specifically to an antigen fusion protein combination of group B meningococcus and its uses. Background Technology

[0004] Epidemic cerebrospinal meningitis (meningococcal meningitis) is a respiratory infectious disease caused by infection with Gram-negative Neisseria meningitidis (Nm), characterized primarily by cerebrospinal meningitis and bacteremia. It typically occurs endemicly worldwide, with approximately 500,000 cases and 50,000 deaths annually globally. In my country, meningococcal meningitis is classified as a Class B infectious disease, primarily affecting infants and adolescents. Neisseria meningitidis can colonize the nasopharyngeal mucosa, causing local infection, or cross the mucosal barrier, leading to invasive sepsis or meningococcal meningitis. Despite antibiotic treatment and vaccination, the global mortality rate for meningococcal meningitis remains as high as 10%-15%, accompanied by a high rate of disability, posing a significant disease burden worldwide.

[0005] 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. The main component of the capsular polysaccharide of Neisseria meningitidis (MenB) is structurally similar to the human central nervous system antigen N-acetylneuraminic acid polymer, which means that capsular polysaccharide vaccines against serogroup B meningococcal meningitis cannot produce an effective immune response and may cause cross-reactivity. It is noteworthy that there are currently no serogroup B meningococcal vaccines available in my country.

[0006] Vaccine development against serogroup B meningococci primarily focuses on outer membrane vesicle (OMV) vaccines and recombinant protein vaccines. OMV vaccines are internationally used serogroup B meningococcal vaccines targeting a specific circulating strain, with their main component being meningeal porin A (PorA). However, due to the highly complex protein typing of PorA and the low cross-protective properties between serotypes and subtypes, it is difficult to obtain an internationally universally applicable vaccine.

[0007] Recombinant protein vaccines based on reverse vaccinology have greatly accelerated the development of meningococcal B vaccines. Factor H binding protein (fHbp), Neisseria heparin binding antigen (NHBA), and Neisseria adhesin A (NadA) are widely used antigens. Among them, fHbp is the most important antigen in meningococcal B, evoking the strongest immune response. Currently, there are two marketed meningococcal B vaccines: Bexsero developed by GSK and Trumenba developed by Pfizer. Neither of these vaccines is available in China (excluding Hong Kong, Macau, and Taiwan). Therefore, the development of a meningococcal B vaccine is crucial for the control of meningococcal B in my country.

[0008] GSK's Bexsero, a group B meningitis vaccine, was approved in 2013. Its main components are a mixture of three proteins: fHbp, NHBA, and NadA, and OMV. fHbp and NHBA are fused with GNA2091 and GNA1030, respectively, to form the GNA2091-fHbp and NHBA-GNA1030 fusion proteins. The vaccine's complex composition requires separate expression and purification of the antigens, and the complex OMV production process makes quality control difficult, increasing the complexity of vaccine preparation. Furthermore, OMV has significant side effects, posing a safety risk.

[0009] Pfizer's Trumenba meningitis vaccine, approved by the FDA in 2014, contains two subgroups of fHbp (V1.55 / B01 and V3.45 / A05) as its primary antigens. The fHbp is lipid-modified to enhance immunogenicity. The expression level of fHbp varies considerably among different strains, with lower expression levels observed in some Variant 2 and Variant 3 strains. Therefore, vaccines containing only one type of fHbp antigen offer limited coverage against different prevalent strains.

[0010] The genotype of serogroup B meningitis antigen exhibits polymorphism, with significant differences between the prevalent strains in China and those abroad. This is particularly true compared to the antigen components in recombinant protein vaccines marketed overseas. Therefore, the protective efficacy of the Bexsero and Trumenba vaccines against the prevalent serogroup B Nm strain in my country is not ideal. Furthermore, existing serogroup B meningitis vaccines are complex, requiring lipid modification or containing OMV components, resulting in complex manufacturing 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 increasing the content and immunogenicity of effective antigen components. Summary of the Invention

[0011] In order to address the problems existing in the prior art, the purpose of this application is to provide an antigen fusion protein combination of group B meningococcus and its use.

[0012] To solve the above technical problems, this application adopts the following technical solution:

[0013] In one aspect, this application provides an antigen fusion protein combination comprising antigen fusion proteins, said antigen fusion proteins comprising two or more antigens or fragments thereof selected from the following:

[0014] (i)fHbp antigen;

[0015] (ii) NHBA antigen;

[0016] (iii) NadA antigen; or

[0017] (iv) PorA loop.

[0018] On the other hand, this application provides an immunogenic composition comprising the aforementioned antigen fusion protein.

[0019] On the other hand, this application provides an immunogenic composition comprising at least one antigen fusion protein selected from the following free combinations of antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA.

[0020] 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 antigen fusion protein combination and / or the aforementioned immunogenic composition.

[0021] On the other hand, this application provides a method for inducing an individual to neutralize an antigen-specific immune response, the method comprising administering to a subject an effective amount of the aforementioned antigen fusion protein combination and / or the aforementioned immunogenic composition.

[0022] On the other hand, this application provides the use of the aforementioned antigen fusion protein combination and / or the aforementioned immunogenic composition in the preparation of a medicament for the prevention and / or treatment of group B meningitis.

[0023] The beneficial effects of this application are at least as follows:

[0024] (1) The immunogenic composition of group B meningococcus designed in this application covers four antigens: fHbp, NHBA, NadA, and PorA. The antigen coverage is high, and the bactericidal titer induced by the immunogenic composition is significantly higher than that of a single antigen. In addition, the stability of the recombinant antigen is significantly better than that of a single antigen.

[0025] (2) The immunogenic composition prepared in this application has simple components, consisting of a first fusion protein composed of a first antigen and a second antigen, and a second fusion protein composed of a third antigen and a fourth antigen. Only the first and second fusion proteins need to be expressed and purified separately; the antigens do not require lipid modification and are free of OMV, simplifying the production process and reducing costs, while improving the stability and safety of the antigens.

[0026] (3) The fusion method of the recombinant antigen designed in this application can be applied to different gene subtype antigens contained in different prevalent strains. One or more can be used in combination to produce a broader spectrum of immune protection against prevalent strains in different regions. Attached Figure Description

[0027] Figure 1 shows a schematic diagram of the three-dimensional structure analysis of antigens and truncated fragments based on structural biology. In Figures a and b, respectively, the three-dimensional protein structures of fHbp, NHBA, PorA, and NadA are analyzed.

[0028] Figure 2 shows the insertion site of the VR2 loop region of PorA in the head domain of NadA.

[0029] Figure 3 shows the insertion site of the VR2 loop region of PorA in NHBA.

[0030] Figure 4 shows the purity and uniformity characterization results of some recombinant proteins.

[0031] Figure 5 shows the long-term stability characterization results of the VB16-T13 recombinant protein.

[0032] Figure 6 shows the Tagg value detection results of different subtypes of fHbp-NHBA fusion antigen.

[0033] Figure 7 shows the results of detecting the total antibody level in serum immunized with high-dose / Freudian adjuvant designed for the antigen subtype design of MC58 standard strain.

[0034] Figure 8 shows the results of detecting the total antibody level in serum immunized with low-dose / Freudian adjuvant designed for the antigen subtype design of MC58 standard strain.

[0035] Figure 9 shows the results of detecting the total antibody level in serum immunized with the partially fusion antigen / aluminum adjuvant designed for the MC58 standard strain antigen subtype.

[0036] Figure 10 shows the results of total antibody titer detection in serum immunized with fHbp-NHBA fusion antigen / Freuder adjuvant in the design of antigen subtypes of the Chinese epidemic strain.

[0037] Figure 11 shows the results of total antibody titer detection in serum immunized with fHbp-NHBA fusion antigen / aluminum adjuvant in the design of antigen subtypes of the Chinese epidemic strain.

[0038] Figure 12 shows the results of total antibody titer detection in serum immunized with NadA:PorAVR2loop chimeric antigen / Freudian adjuvant in the design of antigen subtypes of the Chinese epidemic strain.

[0039] Figure 13 shows the results of the geometric mean titer of bactericidal serum obtained from high-dose / Freuder adjuvant immunotherapy designed for the antigen subtype of MC58 standard strain.

[0040] Figure 14 shows the results of the geometric mean titer of bactericidal serum obtained from low-dose / Freudian adjuvant immunotherapy designed for the antigen subtype of MC58 standard strain.

[0041] Figure 15 shows the results of the detection of the geometric mean titer of bactericidal serum of the partially fused antigen / aluminum adjuvant immune serum of the MC58 standard strain antigen subtype design.

[0042] Figure 16 shows the results of the geometric mean titer of bactericidal serum against the fHbp-NHBA fusion antigen in the design of antigen subtypes of the Chinese prevalent strain. Detailed Implementation

[0043] I. Definition

[0044] 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.

[0045] 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.

[0046] 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.

[0047] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the terms listed preceding the stated term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB if the order is significant in the particular context. Continuing this example, combinations containing repetitions of one or more items or terms are explicitly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that there is generally no limit to the number of items or terms in any combination unless otherwise apparent from the context.

[0048] Regarding polypeptide sequences, the phrase "substantially identical" can be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference polypeptide sequence. Regarding nucleic acid sequences, the term can be understood as a nucleotide sequence exhibiting at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference nucleic acid sequence.

[0049] 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.

[0050] 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. The most prevalent fHbp genotypes V2.22, V2.18, V2.16, V3.31, and V1.13 among the circulating strains of serogroup B meningococcus in China were selected for vaccine antigen design to improve vaccine coverage. MC58 is the standard strain for serogroup B meningococcus, with fHbp genotype V1.1, containing 320 amino acids, of which 1-65 are the signal peptide and transmembrane region, anchoring 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).

[0051] 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 Neisseria meningitidis 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. The N-terminus of NHBA is highly flexible and lacks a stable three-dimensional domain. AlphaFold predicts that the amino acids at positions 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.

[0052] The term "NadA" refers to Neisseria adhesin A (NadA), which forms a stable trimer structure on the bacterial surface, mediating epithelial cell adhesion and invasion. Based on amino acid homology, NadA contains six variants: NadA1, NadA2, NadA3, NadA4, NadA5, and NadA6. NadA1, NadA2, and NadA3 are the most common variants, found in highly pathogenic bacteria. The NadA genotype V1 from strain MC58 contains 364 amino acids and is a trimer, with amino acids 1-25 forming the signal peptide and 311-364 forming the transmembrane region. The three-dimensional domain is mainly divided into two parts: the N-terminal head domain (26-84) and the C-terminal rod domain (85-310). The head domain is mainly composed of α-helices and β-sheets, while the C-terminal rod domain is composed of α-helices.

[0053] The term "PorA" refers to an outer membrane porin, which comprises a transmembrane domain and a surface loop domain. A β-barrel with a three-dimensional domain is embedded in the cell membrane, and eight flexible loops are exposed on the cell surface. Loop 1 and Loop 4 are variable regions 1 and 2 (VR1 and VR2). The VR2 loop dominates PorA-specific immunity and is a highly variable region, exhibiting significant amino acid sequence differences across different strains, thus limiting cross-protective effects between variants. For example, the VR2 loop amino acid region derived from strain MC58 consists of amino acids 197-210. This application selects the highly immunogenic VR2 loop region for chimeric antigen design. Genotypes P1.23, P1.23-7, P1.13-1, and P1.14, which have the highest VR2 prevalence among prevalent strains of serogroup B meningococcal meningitis in China, were selected to improve the vaccine product's coverage against different prevalent strains.

[0054] 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.

[0055] 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.

[0056] 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, for example, 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, secretion 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.

[0057] The term "β-turn" refers to a flexible region connecting two β-sheets or between an α-helix and a β-sheet, ranging from one to a dozen amino acids. Each antigen has multiple β-turns, and the insertion site can be any one of these flexible β-turn regions.

[0058] The term "inter-domain" refers to the inter-domain structure, also known as the inter-domain structure.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] The term "host cell" preferably refers to a bacterial or yeast cell expressing the protein or polypeptide of this application. The bacteria may be, for example, meningococcus or Escherichia coli. The bacteria may express the protein or polypeptide, but in some embodiments, expression may be controlled by an inducible promoter.

[0063] 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.

[0064] 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.

[0065] II. Detailed Description of Implementation Methods

[0066] In one aspect, this application provides an antigen fusion protein combination comprising antigen fusion proteins, said antigen fusion proteins comprising two or more antigens or fragments thereof selected from the following:

[0067] (i)fHbp antigen;

[0068] (ii) NHBA antigen;

[0069] (iii) NadA antigen; or

[0070] (iv) PorA loop.

[0071] In some implementations, the PorA ring region is the PorA VR2 ring region.

[0072] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein, wherein the antigen fusion protein is linked in the following manner: (i) the N-terminal amino acid of one antigen or a fragment thereof is directly fused to the C-terminal amino acid of another antigen or a fragment thereof or fused through a peptide linker; (ii) a loop region of one antigen or a fragment thereof is inserted into another antigen or a fragment thereof to form a chimera.

[0073] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein selected from the following antigens or free combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA.

[0074] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein, which comprises, from the N-terminus to the C-terminus, the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA.

[0075] In some embodiments, the antigen fusion protein combination comprises at least two antigen fusion proteins selected from at least two of the following antigens or fragments thereof in free linkage combinations: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0076] In some embodiments, the antigen fusion protein combination comprises at least two antigen fusion proteins, wherein the antigen fusion proteins comprise, from the N-terminus to the C-terminus, at least two of the following antigens or combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0077] In some embodiments, the antigen fusion protein combination comprises at least three antigen fusion proteins selected from at least three of the following antigens or fragments thereof in free linkage combinations: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0078] In some embodiments, the antigen fusion protein combination comprises at least three antigen fusion proteins, wherein the antigen fusion proteins comprise, from the N-terminus to the C-terminus, at least three of the following antigens or combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0079] In some embodiments, the antigen fusion protein combination comprises four antigen fusion proteins selected from four of the following free linkage combinations of antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0080] In some embodiments, the antigen fusion protein combination comprises four antigen fusion proteins, which, from the N-terminus to the C-terminus, comprise four of the following antigens or combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0081] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein selected from the following antigens or free combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, and NadA-PorA.

[0082] In some embodiments, the antigen fusion protein combination comprises at least two antigen fusion proteins selected from the following: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and fHbp-PorA, fHbp-NHBA and NHBA-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and fHbp-PorA, fHbp-NadA and NHBA-NadA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, fHbp-PorA and NHBA-NadA. dA, fHbp-PorA and NHBA-PorA, fHbp-PorA and NadA-PorA, NHBA-NadA and NHBA-PorA, NHBA-NadA and NadA-PorA, fHbp-NHBA and fHbp-NadA and fHbp-PorA, fHbp-NHBA and fHbp-NadA and NHBA-NadA, fHbp-NHBA and fHbp-NadA and NHBA-PorA, fHbp-NHBA and fHbp-NadA and NadA-PorA, fHbp-NHBA and fHbp-PorA and NHBA-NadA, fHbp-NHBA and fHbp-PorA and NHBA-NadA, fHbp-NHBA and fHbp-PorA and fHBA-NadA Hbp-PorA and NHBA-PorA, fHbp-NHBA and fHbp-PorA and NadA-PorA, fHbp-NHBA and NHBA-NadA and NHBA-PorA, fHbp-NHBA and NHBA-NadA and NadA-PorA, fHbp-NHBA and NHBA-PorA and NadA-PorA, fHbp-NadA and fHbp-PorA and NHBA-NadA, fHbp-NadA and fHbp-PorA and fHbp-PorA and NHBA-PorA, fHbp-NadA and fHbp-PorA and NadA-PorA, fHbp-NadA and NHBA- NadA and NHBA-PorA, fHbp-NadA and NHBA-NadA and NadA-PorA, fHbp-NadA and NHBA-PorA and NadA-PorA, fHbp-PorA and NHBA-NadA and NHBA-PorA, fHbp-PorA and NHBA-Na dA and NadA-PorA, NHBA-NadA and NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-NadA and NHBA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-NadA and NadA-PorA,fHbp-NHBA and fHbp-PorA and NHBA-NadA and NHBA-PorA, fHbp-NHBA and fHbp-PorA and NHBA-NadA and NadA-PorA, fHbp-NHBA and NHBA-NadA and NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and fHbp-PorA and NHBA-NadA, fHbp-NHBA and fHbp-NadA and fHbp-PorA and NHBA-PorA fHbp-NHBA and fHbp-NadA and fHbp-PorA and NadA-PorA, fHbp-NadA and fHbp-PorA and NHBA-NadA and NHBA-PorA, fHbp-NadA and fHbp-PorA and NHBA-NadA and NadA-PorA, fHbp-PorA and NHBA-NadA and NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA and NadA-PorA.

[0083] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein, which comprises, from the N-terminus to the C-terminus, the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, and NadA-PorA.

[0084] In some embodiments, the antigen fusion protein combination comprises at least two antigen fusion proteins selected from the following antigens or combinations of linked fragments thereof: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and fHbp-PorA, fHbp-NHBA and NHBA-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and fHbp-PorA, fHbp-NadA and NHBA-NadA. BA-NadA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, fHbp-PorA and NHBA-NadA, fHbp-PorA and NHBA-PorA, fHbp-P orA and NadA-PorA, NHBA-NadA and NHBA-PorA, NHBA-NadA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA and NadA-PorA.

[0085] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein selected from the following antigens or free combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, NHBA-PorA, NadA-PorA.

[0086] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein selected from the following antigens or fragments thereof, freely linked combinations: fHbp-NHBA, fHbp-NadA, NHBA-PorA, NadA-PorA; wherein, a portion of the antigen fusion protein comprises, from the N-terminus to the C-terminus, the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA; and a portion of the antigen or fusion fragment thereof forms a chimera by inserting a loop region of one antigen or fragment thereof into another antigen or fragment thereof: NHBA-PorA, NadA-PorA.

[0087] In some embodiments, the antigen fusion protein combination comprises at least two antigen fusion proteins selected from the following antigens or combinations of linked fragments thereof: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA, fHbp-NHBA and fHbp-NadA and NadA-PorA, fHbp-NadA and NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA and NadA-PorA.

[0088] In some embodiments, the antigen fusion protein combination comprises at least two antigen fusion proteins, which comprise the following antigens or fragments thereof: fHbp-NHBA and NadA-PorA VR2, fHbp-NadA and NHBA-PorA VR2.

[0089] In some embodiments, the antigen fusion protein includes different three-dimensional domains. Specifically, fHbp includes an N-terminal β-sheet and a C-terminal β-barrel. In some embodiments, NHBA includes an N-terminal flexible region, an intermediate domain, a C-terminal β-sheet, and a β-barrel. In some embodiments, NadA includes an N-terminal head domain and a C-terminal rod domain. In some embodiments, PorA includes a transmembrane domain and a surface loop domain. In some embodiments, fHbp includes an N-terminal β-sheet and a C-terminal β-barrel; NHBA includes an N-terminal flexible region, an intermediate domain, a C-terminal β-sheet, and a β-barrel; NadA includes an N-terminal head domain and a C-terminal rod domain; and PorA includes a transmembrane domain and a surface loop domain.

[0090] In some embodiments, the fHbp antigen or fragment thereof has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 1-6, preferably having an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of identity, and more preferably having an amino acid sequence with 98% or 99% or more of identity. In some embodiments, the amino acid sequence of the fHbp antigen or fragment thereof is as shown in any one of SEQ ID NO. 1-6.

[0091] In some embodiments, the NHBA antigen or fragment thereof has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 7-25, preferably having an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and more preferably having an amino acid sequence with at least 98% or 99% identity. In some embodiments, the amino acid sequence of the NHBA antigen or fragment thereof is as shown in any one of SEQ ID NO. 7-25.

[0092] In some embodiments, the NadA antigen or fragment thereof has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 26-31, preferably having an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and more preferably having an amino acid sequence with at least 98% or 99% identity. In some embodiments, the amino acid sequence of the NadA antigen or fragment thereof is as shown in any one of SEQ ID NO. 26-31.

[0093] In some embodiments, the PorA ring region has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 32-36, preferably having an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of identity, and more preferably having an amino acid sequence with 98% or 99% or more of identity. In some embodiments, the amino acid sequence of the PorA ring region is as shown in any one of SEQ ID NO. 32-36.

[0094] In some embodiments, the N-terminal amino acid of one antigen or a fragment thereof in the antigen fusion protein is directly fused to the C-terminal amino acid of another antigen or a fragment thereof.

[0095] In some embodiments, the N-terminal amino acid of one antigen or a fragment thereof in the antigen fusion protein is fused to the C-terminal amino acid of another antigen or a fragment thereof via a peptide linker.

[0096] In some embodiments, the peptide linker is selected from (G). n (GS) n (GSG) n (GGGS) n (GSGGS) n Or (GGGGS) n One or more of the following, where 1 <= n <= 8. For example, the peptide linker can be (GSG)4, (GGGS)3, etc.

[0097] In some embodiments, the amino acid sequence of the peptide linker is shown in any one of SEQ ID NO.249-256.

[0098] In some embodiments, the amino acid sequence of the peptide linker is as shown in SEQ ID NO.255 ​​or SEQ ID NO.256.

[0099] In some embodiments, the amino acid sequence of the antigen fusion protein is selected from any one of SEQ ID NO.37-124.

[0100] In some embodiments, the connection of the antigen fusion protein further includes: inserting a loop region of one antigen or a fragment therein into another antigen or a fragment therein to form a chimera.

[0101] In some embodiments, the antigen fusion protein comprises a PorA loop region antigen or a fragment thereof.

[0102] In some embodiments, the antigen fusion protein is a chimera formed by inserting the PorA loop region into the NHBA or NadA antigen or a fragment thereof.

[0103] In some embodiments, the insertion site of the PorA loop region is a loop region between the β-turn of the corresponding antigen or its fragments, or between the α-helix and the β-sheet.

[0104] In some embodiments, the antigen fusion protein is a chimera obtained by inserting the porin PorA loop region as shown in any one of SEQ ID NO. 32-36 into the NHBA antigen or a fragment thereof as shown in any one of SEQ ID NO. 7-25. In some embodiments, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO. 55-61.

[0105] In some embodiments, the antigen fusion protein is a chimera obtained by inserting the porin PorA loop region, as shown in any one of the amino acid sequences of SEQ ID NO. 32-36, into the NadA antigen or a fragment thereof, as shown in any one of the amino acid sequences of SEQ ID NO. 26-31. In some embodiments, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO. 48-51 or SEQ ID NO. 113-124.

[0106] In some embodiments, the antigen fusion protein comprises:

[0107] (i) the fHbp antigen or a fragment thereof as shown in any of the amino acid sequences in SEQ ID NO. 1-6; and

[0108] (ii) NHBA antigen or fragment thereof as shown in any of the amino acid sequences in SEQ ID NO.7-25.

[0109] In some embodiments, the C-terminal amino acid of the fHbp antigen or a fragment thereof in the antigen fusion protein is fused to the N-terminal amino acid of the NHBA antigen or a fragment thereof via a peptide linker. In some embodiments, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO. 37-40 and SEQ ID NO. 62-74. In some embodiments, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO. 40 and SEQ ID NO. 62-74. In some embodiments, the antigen fusion protein is further mutated and / or truncated to form a mutant and / or truncated variant. In some embodiments, the fHbp antigen or a fragment thereof in the antigen fusion protein contains a mutation, the mutation being selected from any combination of the following:

[0110] (1) For fHbp Variant 1, the mutation sites are E218A / E239A;

[0111] (2) For fHbp Variant 2, the mutation sites are E217A / T238A and / or L130R / G133D;

[0112] (3) For fHbp Variant 3, the mutation site is E225A / T246A.

[0113] In some embodiments, the amino acid sequence of the mutant is selected from any one of SEQ ID NO. 45 and SEQ ID NO. 75-87. In some embodiments, the amino acid sequence of the truncated mutant is selected from any one of SEQ ID NO. 46 and SEQ ID NO. 88-97. In some embodiments, the amino acid sequences of both the mutant and the truncated mutant are selected from any one of SEQ ID NO. 47 and SEQ ID NO. 98-112.

[0114] In some embodiments, the N-terminal amino acid of the fHbp antigen or a fragment thereof in the antigen fusion protein is fused to the C-terminal amino acid of the NHBA antigen or a fragment thereof via a peptide linker. In some embodiments, the amino acid sequence of the antigen fusion protein is selected from any one of SEQ ID NO. 41-44.

[0115] In some embodiments, the antigen fusion protein comprises:

[0116] (i) the fHbp antigen or a fragment thereof as shown in any of the amino acid sequences in SEQ ID NO. 1-6; and

[0117] (ii) NadA antigen or fragment thereof as shown in any of the amino acid sequences in SEQ ID NO.26-31.

[0118] In some embodiments, the C-terminal amino acid of the fHbp antigen or a fragment thereof in the antigen fusion protein is fused to the N-terminal amino acid of the NadA antigen or a fragment thereof via a peptide linker. In some embodiments, the amino acid sequence of the antigen fusion protein is selected from any one of SEQ ID NO. 52-54.

[0119] In some embodiments, the amino acid sequence of the antigen fusion protein is selected from any one of SEQ ID NO. 40, 45-47, 62-112.

[0120] In some embodiments, the dosage of the antigen fusion protein is 10-100 μg; in some embodiments, the dosage of the antigen fusion protein is 20 μg; and in some embodiments, the dosage of the antigen fusion protein is 50 μg.

[0121] In some embodiments, the concentration of the antigen fusion protein is 0.1 mg / mL to 1 mg / mL; in some embodiments, the concentration of the antigen fusion protein is 0.2 mg / mL; in some embodiments, the concentration of the antigen fusion protein is 0.5 mg / mL.

[0122] On the other hand, this application provides an immunogenic composition comprising the aforementioned antigen fusion protein combination.

[0123] On the other hand, this application provides an immunogenic composition comprising at least one antigen fusion protein selected from the following free combinations of antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA.

[0124] In some implementations, the PorA ring region is the PorA VR2 ring region.

[0125] In some embodiments, the antigen fusion protein combination comprises at least one antigen fusion protein, wherein the antigen fusion protein is linked in the following manner: (i) the N-terminal amino acid of one antigen or a fragment thereof is directly fused to the C-terminal amino acid of another antigen or a fragment thereof or fused through a peptide linker; (ii) a loop region of one antigen or a fragment thereof is inserted into another antigen or a fragment thereof to form a chimera.

[0126] In some embodiments, the immunogenic composition comprises at least one antigen fusion protein, the antigen fusion protein comprising, from the N-terminus to the C-terminus, the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA.

[0127] In some embodiments, the immunogenic composition further comprises any combination of at least two antigen fusion proteins.

[0128] In some embodiments, the immunogenic composition comprises an antigen fusion protein selected from at least two of the following antigens or freely linked combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0129] In some embodiments, the immunogenic composition comprises an antigen fusion protein comprising, from the N-terminus to the C-terminus, at least two of the following antigens or combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

[0130] In some embodiments, the immunogenic composition comprises at least one antigen fusion protein selected from the following antigens or free combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, and NadA-PorA.

[0131] In some embodiments, the immunogenic composition comprises at least one antigen fusion protein, which comprises, from the N-terminus to the C-terminus, the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, and NadA-PorA.

[0132] In some embodiments, the immunogenic composition comprises at least two antigen fusion proteins selected from the following antigens or combinations of linked fragments thereof: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and fHbp-PorA, fHbp-NHBA and NHBA-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and fHbp-PorA, fHbp-NadA and NHB A-NadA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, fHbp-PorA and NHBA-NadA, fHbp-PorA and NHBA-PorA, fHbp-P orA and NadA-PorA, NHBA-NadA and NHBA-PorA, NHBA-NadA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA and NadA-PorA.

[0133] In some embodiments, the immunogenic composition comprises at least one antigen fusion protein selected from the following: fHbp-NHBA, fHbp-NadA, NHBA-PorA, and NadA-PorA.

[0134] In some embodiments, the immunogenic composition comprises at least one antigen fusion protein selected from free combinations of the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, NHBA-PorA, NadA-PorA; wherein, a portion of the antigen fusion protein comprises, from the N-terminus to the C-terminus, the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA; and a portion of the antigen or fusion fragment thereof forms a chimera by inserting a loop region of one antigen or fragment thereof into another antigen or fragment thereof: NHBA-PorA, NadA-PorA.

[0135] In some embodiments, the immunogenic composition comprises at least two antigen fusion proteins selected from the following antigens or combinations of linked fragments thereof: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA, fHbp-NHBA and fHbp-NadA and NadA-PorA, fHbp-NadA and NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA and NadA-PorA.

[0136] In some embodiments, the immunogenic composition comprises at least two antigen fusion proteins, the antigen fusion proteins comprising the following antigens or fragments thereof: fHbp-NHBA and NadA-PorA VR2, fHbp-NadA and NHBA-PorA VR2.

[0137] In some embodiments, the antigen fusion protein includes different three-dimensional domains. Specifically, fHbp includes an N-terminal β-sheet and a C-terminal β-barrel. In some embodiments, NHBA includes an N-terminal flexible region, an intermediate domain, a C-terminal β-sheet, and a β-barrel. In some embodiments, NadA includes an N-terminal head domain and a C-terminal rod domain. In some embodiments, PorA includes a transmembrane domain and a surface loop domain. In some embodiments, fHbp includes an N-terminal β-sheet and a C-terminal β-barrel; NHBA includes an N-terminal flexible region, an intermediate domain, a C-terminal β-sheet, and a β-barrel; NadA includes an N-terminal head domain and a C-terminal rod domain; and PorA includes a transmembrane domain and a surface loop domain.

[0138] In some embodiments, the N-terminal amino acid of one antigen or a fragment thereof in the antigen fusion protein is directly fused to the C-terminal amino acid of another antigen or a fragment thereof.

[0139] In some embodiments, the N-terminal amino acid of one antigen or a fragment thereof in the antigen fusion protein is fused to the C-terminal amino acid of another antigen or a fragment thereof via a peptide linker.

[0140] In some embodiments, the immunogenic composition comprises a first fusion protein and / or a second fusion protein, the first fusion protein further comprising a first antigen and a second antigen, and the second fusion protein comprising a third antigen and a fourth antigen.

[0141] In some implementations, the C-terminal amino acid of the first antigen in the first fusion protein is directly fused to the N-terminal amino acid of the second antigen.

[0142] In some embodiments, the C-terminal amino acid of the first antigen in the first fusion protein is fused to the N-terminal amino acid of the second antigen via a peptide linker.

[0143] In some embodiments, the peptide linker is selected from (G). n (GS) n (GSG) n (GGGS) n (GSGGS) n Or (GGGGS) n One or more of the following, where 1 <= n <= 8. For example, the peptide linker can be (GSG)4, (GGGS)3, etc.

[0144] In some embodiments, the amino acid sequence of the peptide linker is shown in any one of SEQ ID NO.249-256.

[0145] In some embodiments, the amino acid sequence of the peptide linker is as shown in SEQ ID NO.255 ​​or SEQ ID NO.256.

[0146] In some embodiments, the second fusion protein is a chimera obtained by inserting a third antigen into a fourth antigen.

[0147] In some implementations, the first, second, third, and fourth antigens are selected from any one of the following antigens or fragments thereof:

[0148] (i)fHbp antigen;

[0149] (ii) NHBA antigen;

[0150] (iii) NadA antigen; or

[0151] (iv)PorA ring area.

[0152] In some embodiments, the fHbp antigen or fragment thereof has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 1-6, preferably having an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more of identity, and more preferably having an amino acid sequence with 98% or 99% or more of identity. In some embodiments, the amino acid sequence of the fHbp antigen or fragment thereof is as shown in any one of SEQ ID NO. 1-6.

[0153] In some embodiments, the NHBA antigen or fragment thereof has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 7-25, preferably having an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and more preferably having an amino acid sequence with at least 98% or 99% identity. In some embodiments, the amino acid sequence of the NHBA antigen or fragment thereof is as shown in any one of SEQ ID NO. 7-25.

[0154] In some embodiments, the NadA antigen or fragment thereof has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 26-31, preferably having an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, and more preferably having an amino acid sequence with at least 98% or 99% identity. In some embodiments, the amino acid sequence of the NadA antigen or fragment thereof is as shown in any one of SEQ ID NO. 26-31.

[0155] In some embodiments, the PorA ring region has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 32-36, preferably having an amino acid sequence with 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably having an amino acid sequence with 98% or 99% or more identical. In some embodiments, the amino acid sequence of the PorA ring region is as shown in any one of SEQ ID NO. 32-36.

[0156] In some embodiments, the fHbp antigen or a fragment thereof in the immune composition may or may not contain a mutation. In some embodiments, the fHbp antigen or a fragment thereof contains a mutation selected from any combination of the following:

[0157] (1) For fHbp Variant 1, the mutation sites are E218A / E239A;

[0158] (2) For fHbp Variant 2, the mutation sites are E217A / T238A and / or L130R / G133D;

[0159] (3) For fHbp Variant 3, the mutation site is E225A / T246A.

[0160] In some embodiments, the immunogenic composition:

[0161] (1) Contains a first fusion protein, wherein the first fusion protein is fHbp-NHBA or fHbp-NadA protein; or

[0162] (2) Contains a second fusion protein, wherein the second fusion protein is a NadA-PorA or NHBA-PorA protein; or

[0163] (3) Contains a first fusion protein and a second fusion protein, wherein the first fusion protein is fHbp-NHBA protein and the second fusion protein is NadA-PorA protein; or

[0164] (4) It contains a first fusion protein and a second fusion protein, wherein the first fusion protein is fHbp-NadA protein and the second fusion protein is NHBA-PorA protein.

[0165] In some embodiments, the first fusion protein and / or the second fusion protein are further mutated and / or truncated to form mutants and / or truncated forms.

[0166] In some embodiments, the amino acid sequence of the first fusion protein mutant is selected from any one of SEQ ID NO. 45 and SEQ ID NO. 75-87. In some embodiments, the amino acid sequence of the first fusion protein truncated variant is selected from any one of SEQ ID NO. 46 and SEQ ID NO. 88-97. In some embodiments, the amino acid sequences of the first fusion protein mutant and the truncated variant are selected from any one of SEQ ID NO. 47 and SEQ ID NO. 98-112.

[0167] In some embodiments, the immunogenic composition comprises a first fusion protein and a second fusion protein, wherein the amino acid sequences of the first fusion protein and / or the second fusion protein are each independently selected from any one of SEQ ID NO.37-124.

[0168] In some embodiments, the immunogenic composition comprises a first fusion protein and a second fusion protein, wherein the C-terminal amino acid of the fHbp antigen or a fragment thereof in the first fusion protein is fused to the N-terminal amino acid of the NHBA antigen or a fragment thereof via a peptide linker, and the amino acid sequence of the first fusion protein is shown in any one of SEQ ID NO. 40, SEQ ID NO. 45-47, and SEQ ID NO. 62-112; the second fusion protein is a chimera obtained by inserting the PorA loop region of porin into a NadA antigen fragment, and the amino acid sequence of the second fusion protein is shown in any one of SEQ ID NO. 48-51 and SEQ ID NO. 113-124.

[0169] In some embodiments, the immunogenic composition comprises a first fusion protein and a second fusion protein, wherein the first fusion protein is fused with the N-terminal amino acid of the fHbp antigen or a fragment thereof to the N-terminal amino acid of the NadA antigen or a fragment thereof via a peptide linker, the amino acid sequence of the first fusion protein is shown in any one of SEQ ID NO. 52-54, and the second fusion protein is a chimera obtained by embedding the porin PorA loop region into the NHBA antigen fragment, the amino acid sequence of the second fusion protein is shown in any one of SEQ ID NO. 55-61.

[0170] In some embodiments, the immunogenic composition further comprises one or more of an adjuvant, a buffer solution, a stabilizer, and a surfactant.

[0171] In some embodiments, the adjuvant is at least one of aluminum salt adjuvant, Freund's adjuvant, liposome, MF59, monophospholipid A, flagellin, CpG-ODN, and Poly(I:C).

[0172] In some embodiments, the final concentration of the immunogenic composition is 0.1-1 mg / mL; in some embodiments, the final concentration of the immunogenic composition is 0.2 mg / mL; and in some embodiments, the final concentration of the immunogenic composition is 0.5 mg / mL.

[0173] In another aspect, this application provides a method for preventing and / or treating serogroup B meningitis, the method comprising administering to a subject an effective amount of the aforementioned antigen fusion protein combination and / or the aforementioned immunogenic composition. In some embodiments, the dose of the antigen fusion protein or immunogenic composition is 10-100 μg. In some embodiments, the method further comprises administering one or more vaccines in combination. In some embodiments, the vaccines include, but are not limited to, other serogroup meningitis vaccines, such as the ACYW tetravalent polysaccharide conjugate meningitis vaccine.

[0174] In some embodiments, the fusion protein or immunogenic composition can 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 virus 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.

[0175] On the other hand, this application provides a method for inducing a neutralizing antigen-specific immune response in an individual, the method comprising administering to a subject an effective amount of the aforementioned antigen fusion protein combination and / or the aforementioned immunogenic composition. In some embodiments, the dose of the antigen fusion protein or immunogenic composition is 10-100 μg.

[0176] On the other hand, this application provides the use of the aforementioned antigen fusion protein combination and / or the aforementioned immunogenic composition in the preparation of a medicament for the prevention and / or treatment of group B meningitis.

[0177] In some embodiments, the vaccine composition or immunogenic composition may be formulated as an injectable preparation, such as a liquid solution or emulsion; it may 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 may be administered as a single-dose or multi-dose regimen. The vaccine may be administered in combination with other immunomodulators.

[0178] Example

[0179] 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.

[0180] The raw materials and equipment used in the specific embodiments of this application are all known products, obtained by purchasing commercially available products.

[0181] Example 1. Design of a fusion protein of group B meningococcal antigen

[0182] The three-dimensional structures of fHbp, NHBA, NadA, and PorA proteins were analyzed. While ensuring antigen immunogenicity and three-dimensional structural stability, different domains of the different antigens were truncated. The schematic diagrams of the antigen three-dimensional structures and truncated fragments are shown in Figure 1. The amino acid and nucleotide sequences of the different truncated fragments are shown in Table 1 below. The genotypes of the four antigens selected in the MC58 standard strain were: fHbp Variant 1.1NHBA peptide 3, NadA Variant 1, and PorA VR2 P1.16-2. The genotypes of the four antigens in the CC4821 clonal group of the Chinese prevalent strain are as follows: five subtypes of fHbp (Variant 1.13, Variant 2.22, Variant 2.18, Variant 2.16, and Variant 3.31); five subtypes of NHBA (Peptide 669, Peptide 688, Peptide 503, Peptide 20, and Peptide 945); NadA-2 / 3 subtype; and four subtypes of PorA VR2 loop region (P1.23, P1.23-7, P1.13-1, and P1.14).

[0183] Table 1. Truncated antigen fragments

[0184] Different truncated variants of antigens are fused in pairs to form fusion proteins covering multiple antigens. 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. The amino acid sequence of the peptide linker is shown in any one of SEQ ID NO. 249-256. Simultaneously, the PorA loop region can be inserted into NHBA or NadA antigens or their fragments to form antigen fusion proteins. The specific insertion sites are the loop region between the β-turns of NHBA or NadA antigens or their fragments, or between the α-helix and the β-sheet. The NadA head domain VR2 loop insertion site is shown in Figure 2. The NHBA VR2 loop insertion site is shown in Figure 3.

[0185] This embodiment provides fusion proteins with different antigen combinations as shown in Table 2, including but not limited to: fHbp-NHBA fusion protein, fHbp-NadA fusion protein, NadA-PorA VR2 loop chimera, and NHBA-PorA VR2 loop chimera.

[0186] Table 2. Different fusion proteins and their amino acid sequences Note: All chimeric proteins constructed by insertion are indicated by ":", which is different from the "+" notation for fusion proteins linked by peptide linkers. The superscript in the upper right corner of the chimeric protein constructed by insertion indicates the insertion site, for example, NadA-2 / 3(24-163) shown in VB113. T65 In VR2 P1.23, "T65" indicates that the insertion site of NadA into the PorA loop region is threonine at position 65 of the NadA antigen.

[0187] The specific sequence information for the aforementioned sequences is shown in Tables 5 and 6.

[0188] Any two of the aforementioned antigen fusion proteins can also be combined to form an immunogenic composition, such as an immunogenic composition formed by combining fHbp-NHBA fusion protein and NadA-PorA VR2 loop region, or an immunogenic composition formed by combining fHbp-NadA fusion protein and NHBA-PorA VR2 loop region.

[0189] Furthermore, the aforementioned sequences can be combined to form immunogenic compositions of different subtypes of fHbp-NHBA fusion antigens. Examples include fHbp V1.13-NHBA P688, fHbpV3.31-NHBA P669, fHbpV2.16-NHBA P503, fHbpV2.18-NHBA P20, and fHbpV2.22-NHBA P945.

[0190] Example 2. Construction of prokaryotic expression plasmids

[0191] 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. This full-length DNA sequence was ligated into the expression vector pET28a or pET30a, a process performed by Genscript Biotech. The constructed expression plasmid was transformed into *E. coli* BL21(DE3) and cultured overnight at 37°C in a culture dish containing 50 μg / ml kanamycin to obtain the expression strain. Single clones were picked and inoculated into liquid LB medium, cultured at 37°C with shaking at 200 rpm until the bacterial density reached OD0.05. 600 At approximately 0.6–0.8, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM was added, and the mixture was cultured at 37°C with shaking for 4 hours. SDS-PAGE analysis revealed a clear expression band after induction.

[0192] Example 3. Expression and purification of recombinant proteins

[0193] 3.1 Mass cultivation

[0194] The selected bacterial strains were inoculated into LB liquid medium and incubated 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.

[0195] 3.2 Cell disruption

[0196] Add PBS to the collected bacterial cells, homogenize using high pressure, centrifuge at high speed (15000 rpm, 30 min, 4℃), and collect the supernatant.

[0197] 3.3 Protein Extraction

[0198] 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).

[0199] 3.4 Protein purification

[0200] The protein purified by the nickel column was concentrated to 1 mL and then subjected to gel filtration chromatography. A suitable molecular sieve type (Superdex 200 or Superdex 75) was selected based on the protein's molecular weight. The molecular sieve was then inoculated. The high-performance liquid chromatography system was pre-equilibrated with a molecular sieve containing 50 mM Tris, 150 mM NaCl, and pH 8.0. The equilibration volume was one column volume, approximately 24 mL. The concentrated protein sample was then fed into the molecular sieve through a loading loop, and the protein was eluted at a flow rate of 0.5 mL / min. The sample was then collected, with an elution volume of one column volume.

[0201] 3.5 SDS-PAGE Identification

[0202] The collected samples were validated by SDS-PAGE to ensure that the purity of the samples reached more than 95%.

[0203] 3.6 Protein endotoxin removal

[0204] Endotoxin removal was performed using a Thermo Fisher endotoxin removal column (catalog number 88276), followed by testing for residual endotoxin using a horseshoe crab reagent. Once the endotoxin content was deemed acceptable (less than 100 EU per dose), the next step of vaccine preparation could proceed.

[0205] Example 4: Drug-likeness analysis of recombinant protein

[0206] 4.1 Analysis of the purity, uniformity, and yield of recombinant proteins

[0207] The purity and homogeneity of the recombinant protein were identified by molecular gel filtration chromatography and SDS-PAGE, and the yield of the recombinant protein was calculated. For specific procedures, please refer to Example 3, Protein Purification.

[0208] The results of purity and homogeneity analysis of some recombinant proteins are shown in Figure 4, and the yield statistics are shown in Table 3.

[0209] Figure 4 shows the molecular gel filtration chromatography results. Multiple peaks appeared during the purification of the full-length fHbp-V1.1 (VB4), NHBA-P3 (VB6), and NadA-1 (VB11) antigens, indicating poor homogeneity, which is detrimental to antigen purification in vaccine production. In the fHbp-NHBA fusion antigen design, the homogeneity of VB14, VB15, and VB16 was significantly increased. In the NadA:PorAVR2loop chimeric antigen design, compared with the full-length NadA antigen (VB11), the homogeneity of all recombinant antigens was improved, with VB80-T5 showing a significant increase in homogeneity.

[0210] Furthermore, as shown in Table 3, the expression levels of both fusion antigens and chimeric antigens were significantly increased compared to single antigens.

[0211] 4.2 Thermal stability analysis of recombinant proteins

[0212] The thermostability of recombinant proteins was determined using the UncleAnalysis instrument. The purified test protein was uniformly diluted to a concentration of 1 mg / mL, centrifuged at 14000 rpm for 5 minutes before loading, and then 9 μL of the supernatant was added to the sample well. The Tm and Tagg values ​​of the recombinant proteins were analyzed using the instrument, and the results are shown in Table 3.

[0213] Table 3. Results of stability and yield tests for some recombinant proteins

[0214] The stability of antigens determines the conditions for vaccine production, storage, and transportation. Melting temperature (Tm) and aggregation temperature (Tagg) reflect antigen stability. Table 3 shows that the stability analysis results for full-length fHbp, NHBA, and NadA individual antigens all exhibited poor stability. Specifically, the full-length NHBA (VB6) had a Tm value of 56℃ and a Tagg value of 48.5℃; the full-length NadA (VB11) had a Tm value of 48.9℃ and a Tagg value of 52.3℃. In the fHbp-NHBA fusion design, the Tm values ​​of VB13, VB14, VB15, and VB16 were significantly increased. In the NadA:PorAVR2loop chimeric antigen design, compared with the full-length NadA (VB11), the Tm values ​​of VB80-T5, VB80-T7, and VB80-T8 antigens were significantly increased.

[0215] To further improve the long-term stability of VB16, VB16 was modified by deleting a potential protease cleavage site (GVLI) to obtain VB16-T13. Its Tm value remained at 83℃, while its antigen Tagg value increased to 57.8℃. Simultaneously, the long-term stability of the VB16-T13 recombinant protein was tested, and the results showed that it could remain stable at room temperature for 14 days, as shown in Figure 5.

[0216] Tagg values ​​reflect the aggregation properties of antigens. When Tm values ​​are similar, higher Tagg values ​​indicate that antigens are less likely to aggregate, which is more conducive to antigen production and storage. Experiments have shown that, compared with fHbp alone, different subtypes of fHbp-NHBA fusion antigens all exhibited elevated Tagg values, as shown in Figure 6.

[0217] 4.3 Optimization of recombinant protein stability and immunogenicity

[0218] To further improve the stability and immunogenicity of the antigen, we performed a series of truncated variant constructions and point mutations on the target antigen. Details are as follows:

[0219] To improve the stability of different subtype fusion antigens, potential protease cleavage sites (GVLI) were deleted.

[0220] To reduce the interaction between fHbp and human factor H (FH) and improve the immunogenicity of the vaccine, we introduced point mutations E218A / E239A against the fHbp Variant 1 antigen, E217A / T238A against the fHbp Variant 2 antigen, and E225A / T246A against the fHbp Variant 3 antigen, based on our designed fusion protein.

[0221] To address the inherent instability of the fHbp Variant 2 antigen, we introduced a point mutation L130R / G133D targeting the fHbp Variant 2 antigen into our designed fusion protein.

[0222] The corresponding truncation and mutation sites of the fusion proteins are shown in Table 2, and the stability analysis of some modified antigens is shown in Table 4.

[0223] Table 4. Stability analysis of mutant antigens

[0224] Example 5. Vaccine Preparation

[0225] First, the protein sample concentration was determined using the BCA method, followed by the preparation of antigens and adjuvants. The purified antigens and buffer solutions were filtered for sterilization. The antigens and Freund's adjuvant were then emulsified in a 1:1 ratio using an emulsifier, and mice were subsequently immunized.

[0226] Alternatively, the purified, filtered, and sterilized antigen can be incubated with aluminum hydroxide adjuvant or aluminum phosphate adjuvant at room temperature for 1 hour before being used to immunize mice, where the working concentration of Al ions is 1 mg / ml.

[0227] Example 6. Mouse immunization program

[0228] Female BALB / c mice, approximately six weeks old, were randomly divided into groups of five. Each group received an immunization three times, on days 0, 21, and 35, with an antigen dose of 20 μg or 50 μg per mouse. The control group received saline. Fourteen days after the third immunization, blood was collected from the mice's eyes to prepare serum. The serum was collected after standing at 37°C for 30 min, followed by centrifugation at 6000 rpm for 10 min.

[0229] Example 7. Determination of specific IgG levels in immune serum

[0230] The specific steps for indirect ELISA detection of serum specific IgG antibody levels are as follows:

[0231] (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.

[0232] (2) Washing: Shake off all liquid from the wells and blot dry with absorbent paper. Add 200 μL of Wash Buffer (1×PBST) to each well, let stand for 1 min, shake off, blot dry the ELISA plate, wash five times, and blot dry the ELISA plate.

[0233] (3) Blocking: Block the ELISA plate with 200 μL of PBST solution containing 1% BSA at 37°C for 2 h.

[0234] (4) Washing: The method is the same as step (2).

[0235] (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 diluted twice to the experimental wells, and set up PBS negative control wells. Incubate at room temperature for 1 h.

[0236] (6) Washing, the method is the same as step (2).

[0237] (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.

[0238] (8) Washing: The method is the same as step (2).

[0239] (9) Color development: Add 100 μL of TMB substrate to each well and incubate at 37°C in the dark for 15 min.

[0240] (10) Termination of reaction: Add 50 μL of H2SO4 to each well to terminate the reaction.

[0241] (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 450nm.

[0242] (12) Experimental results and analysis:

[0243] 12.1) MC58 standard strain antigen subtype design / Freudian adjuvant immunization experiment

[0244] 12.1.1) Immunoassay with 50 μg antigen / Freud's adjuvant and strong adjuvant

[0245] The results of the 50 μg antigen / Freudian adjuvant strong adjuvant immunization experiment are shown in Figure 7. The serum antibody titer induced by a single fHbp antigen was 2.89E+6, and the serum antibody titer induced by a single NHBA or NadA antigen was 3.62E+5.

[0246] In the fHbp-NHBA fusion antigen design, the serum antibody titer generated by VB13, VB15, and VB16 was 4.10E+6, and the serum antibody titer generated by VB23-VB26 was 3.57E+6.

[0247] In the fHbp-NadA fusion design, the serum antibody titer generated by VB33T was 1.24E+7, and the serum antibody titer generated by VB40 was 9.41E+6.

[0248] In the NHBA:PorAVR2loop chimeric antigen design, the antibody titers induced by VB74-VB78 were 2.93E+6, 1.17E+7, 3.87E+6, 2.41E+6, and 5.34E+6, respectively.

[0249] 12.1.2) Immunoassay with 20 μg antigen / Freud's adjuvant and strong adjuvant

[0250] The results of the immunization assay using 20 μg antigen / Freudian adjuvant with strong adjuvant are shown in Figure 8. The serum antibody titer induced by a single fHbp antigen was 8.06E+5, and the serum antibody titer induced by a single NHBA antigen was 4.91E+5. In the fHbp-NHBA fusion antigen design, the serum antibody titers induced by VB13-VB16 were 1.04E+6, 1.05E+6, 9.95E+5, and 1.11E+6, respectively. The serum antibody titer induced by VB16-T13 was 3.97E+6, and the serum antibody titer induced by VB16MT was 2.51E+6.

[0251] The serum antibody titer induced by a single NadA antigen was 1.25E+6. In the NadA:PorAVR2loop chimeric antigen design, the antibody titer induced by VB80-T5 was 1.02E+6, the antibody titer induced by VB80-T7 was 1.92E+6, and the antibody titer induced by VB80-T8 was 2.05E+6.

[0252] Immunization with a combination of VB16T-13 and VB80-T5 induced a serum antibody titer of 3.57E+6. This demonstrates that combined immunization with a fusion antigen and a chimeric antigen induces a stronger immune response.

[0253] 12.2) MC58 standard strain antigen subtype design / immunoassay with weak aluminum phosphate adjuvant

[0254] The results of the antigen / aluminum phosphate weak adjuvant immunization experiment are shown in Figure 9. In the 20 μg antigen immunization group, the serum antibody titer induced by fHbp was 2.03E+5, the serum antibody titer induced by NHBA was 5.24E+5, the serum antibody titer induced by VB16-T13 was 2.39E+6, and the serum antibody titer induced by VB16MT was 7.95E+5.

[0255] In the 40μg antigen immunization group, the serum antibody titer induced by fHbp was 6.89E+5, the serum antibody titer induced by NHBA was 1.03E+6, and the serum antibody titer induced by VB16-T13 was 3.99E+6.

[0256] 12.3) Antigen subtype design of the Chinese prevalent strain fHbp-NHBA / adjuvant immunization experiment

[0257] 12.3.1) Freund's adjuvant immunoassay

[0258] The results of the Chinese epidemic strain antigen subtype fHbp-NHBA fusion design / Freudian adjuvant immunization experiment are shown in Figure 10. Compared with fHbp alone, different subtypes of fHbp-NHBA fusion antigens can induce stronger immune responses and produce higher serum antibody titers.

[0259] In the fHbpV1.13 subtype fusion design experimental group, the serum antibody titer produced by mice immunized with the fHbpV1.13 (VB105) antigen alone was 1.99E+5. The serum antibody titer produced by mice immunized with the VB81 fusion antigen was 1.72E+6. The serum antibody titer produced by mice immunized with the VB85 fusion antigen was 1.14E+6.

[0260] In the fHbpV2.22 subtype fusion design experimental group, the serum antibody titer produced by mice immunized with the fHbpV2.22 (VB106) antigen alone was 4.31E+5. The serum antibody titer produced by mice immunized with the VB83 fusion antigen was 4.31E+5. The serum antibody titer produced by mice immunized with the VB87 fusion antigen was 2.70E+6.

[0261] In the fHbpV2.18 subtype fusion design experimental group, the serum antibody titer produced by mice immunized with the fHbpV2.18 (VB107) antigen alone was 3.66E+5. The serum antibody titer produced by mice immunized with the VB93 fusion antigen was 2.44E+6. The serum antibody titer produced by mice immunized with the VB97 fusion antigen was 5.88E+5.

[0262] In the fHbpV3.31 subtype fusion design experimental group, the serum antibody titer produced by mice immunized with the fHbpV3.31 (VB109) antigen alone was 7.46E+5. The serum antibody titer produced by mice immunized with the VB95 fusion antigen was 2.55E+6. The serum antibody titer produced by mice immunized with the VB99 fusion antigen was 3.57E+6.

[0263] In the fHbpV2.16 subtype fusion design experimental group, the serum antibody titer produced by mice immunized with the fHbpV2.16 (VB108) antigen alone was 7.08E+4. The serum antibody titer produced by mice immunized with the VB101 fusion antigen was 1.81E+5. The serum antibody titer produced by mice immunized with the VB103 fusion antigen was 2.83E+5.

[0264] 12.3.2) Immunoassay with weak adjuvant aluminum phosphate / aluminum hydroxide

[0265] When using weak adjuvants such as aluminum hydroxide or aluminum phosphate, the serum antibody titer induced by the fHbp-NHBA fusion design was significantly higher than that induced by a single fHbp antigen, as shown in Figure 11.

[0266] In the aluminum hydroxide experimental group, the antibody titers generated by different subtypes of fHbp antigen were as follows: VB107 (4.87E+6), VB109 (2.90E+6), VB108 (3.44E+6). Correspondingly, the antibody titers generated by the fHbp-NHBA fusion design were VB93 (1.16E+7), VB97 (1.16E+7), VB95 (9.74E+6), VB99 (1.64E+7), VB103 (6.89E+6), VB103MT (5.79E+6).

[0267] In the aluminum phosphate experimental group, the antibody titers generated by different subtypes of fHbp antigen were as follows: VB107 (2.44E+6), VB109 (2.44E+6), VB108 (2.44E+6), and the corresponding antibody titers generated by the fHbp-NHBA fusion design were VB93 (4.87E+6), VB97 (8.52E+6), VB95 (1.42E+7), VB99 (1.64E+7), VB103 (4.87E+6), and VB103MT (4.87E+6).

[0268] 12.4) Immunoassay using NadA:PorA VR2loop chimeric antigen / Freuder adjuvant designed for prevalent Chinese strains.

[0269] The results of the NadA:PorA VR2loop chimeric design / Freudian adjuvant immunoassay for the Chinese prevalent strain antigen subtypes are shown in Figure 12. The serum antibody titer induced by a single NadA antigen (VB112) was 2.38E+5. After inserting VR2loops at different sites, the immunogenicity of most antigen constructions was improved. Specifically, the antibody titers generated by VB113 and VB114 were 7.76E+5, those generated by VB116 were 4.21E+5, those generated by VB118 and VB119 were 3.52E+5, those generated by VB122 were 1.18E+6, those generated by VB123 were 4.46E+5, and those generated by VB124 were 2.56E+5.

[0270] In summary, compared with single antigens, all fusion antigen designs and chimeric antigen designs were able to induce high levels of serum antibody titers in mice.

[0271] Example 8. Serum bactericidal test (SBA)

[0272] 8.1 Preparation of bacterial culture

[0273] 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 colony and transfer it 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.

[0274] 8.2 Preparation of complement

[0275] Complement was selected from adult serum that had IgM and IgG removed. The experimental group received active complement, while the control group received inactivated complement (inactivated by incubation in a 56°C water bath for 30 min).

[0276] 8.3 Dilution of mouse serum samples to be tested

[0277] 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 aspirated and 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.

[0278] 8.4 Sample Mixing

[0279] 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 solution to all sample wells, mix well, and incubate at 37°C and 65 rpm for one hour.

[0280] 8.5 Coated Board

[0281] 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.

[0282] 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.

[0283] 8.6 Experimental Results and Analysis

[0284] 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.

[0285] First, the feasibility of fusion antigen design and chimeric antigen design was verified using the MC58 standard strain antigen subtype. Experimental results confirmed that, compared to single antigens, most fusion antigen designs and chimeric antigen designs induced higher immunogenicity.

[0286] 1) In the 50 μg antigen / Freudian adjuvant strong adjuvant immunization experiment, the results are shown in Figure 13. The SBA bactericidal titer generated by fHbp immunization was 525, the SBA bactericidal titer generated by NHBA immunization was 4, and the SBA bactericidal titer generated by NadA immunization was 36. The SBA bactericidal titers induced by the fusion antigen design were VB24 (1066), VB25 (1546), VB26 (1373), and VB40 (902), which were significantly higher than the bactericidal titers generated by fHbp, NHBA, and NadA alone. This demonstrates the effectiveness of the fHbp-NHBA and fHbp-NadA fusion antigen design.

[0287] The NHBA:PorAVR2loop chimeric antigen design, such as the VB77 immune serum with an SBA bactericidal titer of 32, significantly higher than the immunogenicity produced by a single NHBA, demonstrates the effectiveness of the NHBA:PorAVR2loop chimeric antigen design.

[0288] 2) In the 20 μg antigen / Freudian adjuvant strong adjuvant immunization assay, the results are shown in Figure 14. The SBA bactericidal titer produced by fHbp immunization was 136, and the SBA bactericidal titer produced by NHBA immunization was 12. Most fHbp-NHBA fusion antigen designs produced stronger immunogenicity, among which the SBA bactericidal titers of fHbp-NHBA intermediate domain fusion designs such as VB16, VB16MT, and VB16-T13 were increased to 310, 296, and 1062, respectively. This confirms the effectiveness of fHbp-NHBA fusion antigen design.

[0289] The SBA bactericidal titer generated by NadA immunization was 4, while in the NadA:PorAVR2loop chimeric antigen design, the SBA bactericidal titer induced by VB80-T5 increased to 142. Although the total serum antibody titer generated by VB80-T5 did not increase, the bactericidal antibody titer increased significantly. This confirms the effectiveness of the NadA:PorAVR2loop chimeric antigen design.

[0290] 3) When using aluminum phosphate as a weak adjuvant for immunization experiments, the results are shown in Figure 15. In the 20 μg immunization group, the SBA bactericidal titer produced by the fHbp antigen immune serum was less than 4, while the bactericidal titer produced by the NHBA antigen was 8. The fusion antigen design, such as VB16-T13, showed more prominent immunogenicity, with an SBA bactericidal titer of 104. In the 40 μg immunization group, the SBA bactericidal titer produced by the fHbp antigen immune serum was 8, while the bactericidal titer produced by the NHBA antigen was 16. The SBA bactericidal titer produced by the fusion antigen design, such as VB16-T13 antigen, was 400, showing more prominent immunogenicity.

[0291] The above experimental results demonstrate that the immunogenicity of fusion antigen designs and chimeric antigen designs is superior to that of single antigen designs. Furthermore, fusion antigens and chimeric antigens exhibit higher stability and yield, which is beneficial for vaccine production. Among these, the fHbp-NHBA intermediate domain fusion design showed the best performance.

[0292] Subsequently, the fHbp-NHBA intermediate domain fusion design was adopted, and the immunogenicity of the fusion antigen was further verified using antigen subtypes of the Chinese prevalent strain. As shown in Figure 16, compared with fHbp alone, different subtypes of the fHbp-NHBA fusion antigen could induce the production of higher levels of bactericidal antibodies.

[0293] In the fHbpV1.13 subtype fusion design experimental group, mice immunized with the fHbpV1.13 (VB105) antigen alone produced SBA bactericidal titers of 54 (target bacteria 527) and 102 (target bacteria 420). Mice immunized with the VB81 fusion antigen produced SBA bactericidal titers of 167 (target bacteria 527) and 250 (target bacteria 420). Mice immunized with the VB85 fusion antigen produced SBA bactericidal titers of 382 (target bacteria 527) and 143 (target bacteria 420).

[0294] In the fHbpV2.22 subtype fusion design experimental group, the SBA bactericidal titer produced by mice immunized with the fHbpV2.22 (VB106) antigen alone was 12 (target bacteria MC58), while the SBA bactericidal titer produced by mice immunized with the VB83 fusion antigen was 791 (target bacteria MC58).

[0295] In the fHbpV2.18 subtype fusion design experimental group, mice immunized with the fHbpV2.18 (VB107) antigen alone produced an SBA bactericidal titer of 28 (target bacteria O44). Mice immunized with the VB93 fusion antigen produced an SBA bactericidal titer of 192 (target bacteria O44). Mice immunized with the VB97 fusion antigen produced an SBA bactericidal titer of 98 (target bacteria O44).

[0296] In the fHbpV2.16 subtype fusion design experimental group, mice immunized with the fHbpV2.16 (VB108) antigen alone produced an SBA bactericidal titer of 32 (target bacteria 324). Mice immunized with the VB103 fusion antigen produced an SBA bactericidal titer of 192 (target bacteria 324). Mice immunized with the VB103MT fusion antigen produced an SBA bactericidal titer of 256 (target bacteria 324).

[0297] In the fHbpV3.31 subtype fusion design experimental group, mice immunized with the fHbpV3.31 (VB109) antigen alone produced an SBA bactericidal titer of 21 (401 target bacteria). Mice immunized with the VB95 fusion antigen produced an SBA bactericidal titer of 148 (401 target bacteria). Mice immunized with the VB99 fusion antigen produced an SBA bactericidal titer of 293 (401 target bacteria).

[0298] Furthermore, the aforementioned different subtypes of fHbp-NHBA fusion antigens, such as fHbp V1.13-NHBA P688, fHbpV3.31-NHBA P669, fHbpV2.16-NHBA P503, fHbpV2.18-NHBA P20, and fHbpV2.22-NHBA P945, can be combined arbitrarily to produce a more broad-spectrum immunogenic composition.

[0299] In addition, any two of the aforementioned antigen fusion proteins can be combined to form an immunogenic composition, such as an immunogenic composition formed by combining fHbp-NHBA fusion protein and NadA-PorA VR2 loop region, or an immunogenic composition formed by combining fHbp-NadA fusion protein and NHBA-PorA VR2 loop region.

[0300] In summary, this embodiment designed a novel group B meningococcal antigen fusion protein. This fusion form can be applied to different genotypes of antigens from different prevalent strains. Experiments have shown that this form of antigen fusion protein or immune composition has good immunogenicity and drug-like properties.

[0301] 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.

[0302] 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.

[0303] Table 5. Amino acid sequences

[0304] Table 6. Nucleotide Sequences

Claims

1. An antigen fusion protein assembly comprising an antigen fusion protein, said antigen fusion protein comprising two or more antigens selected from the following: (i)fHbp antigen; (ii) NHBA antigen; (iii) NadA antigen; or (iv) PorA loop.

2. The antigen fusion protein combination according to claim 1, comprising at least one antigen fusion protein, said antigen fusion protein being selected from the following antigens or free combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA; The free linking combination is achieved through the following methods: (i) the N-terminal amino acid of one antigen or its fragment is directly fused with the C-terminal amino acid of another antigen or its fragment, or fused through a peptide linker; (ii) the loop region of one antigen or its fragment is inserted into another antigen or its fragment to form a chimera. Preferably, it comprises at least one antigen fusion protein, which comprises the following antigens or fragments thereof from the N-terminus to the C-terminus: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA.

3. The antigen fusion protein combination according to claim 1 or 2, comprising at least two antigen fusion proteins, wherein the antigen fusion proteins are selected from at least two of the following antigens or fragments thereof in free linkage combinations: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA; Preferably, it comprises at least two antigen fusion proteins, wherein the antigen fusion proteins comprise, from the N-terminus to the C-terminus, at least two of the following antigens or combinations thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

4. The antigen fusion protein combination according to claim 2, comprising at least one antigen fusion protein, said antigen fusion protein being selected from the following antigens or free linkage combinations of their fragments: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA; Preferably, it comprises at least one antigen fusion protein, which comprises the following antigens or fragments thereof from the N-terminus to the C-terminus: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, and NadA-PorA.

5. The antigen fusion protein combination according to claim 4, comprising at least two antigen fusion proteins, wherein the antigen fusion protein combination is selected from the following: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and fHbp-PorA, fHbp-NHBA and NHBA-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and fHbp-PorA, fHbp-NadA and NHBA-NadA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, fHbp-PorA and NHBA-NadA, fHbp-PorA and NHBA-PorA, fHbp-PorA and NadA-PorA, NHBA-NadA and NHBA-PorA, NHBA-NadA and NadA-PorA.

6. The antigen fusion protein combination according to claim 2, comprising at least one antigen fusion protein selected from the following antigens or free linkages of fragments thereof: fHbp-NHBA, fHbp-NadA, NHBA-PorA, NadA-PorA.

7. The antigen fusion protein combination according to claim 6, comprising at least two antigen fusion proteins selected from the following antigens or combinations of their fragments: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA, fHbp-NHBA and fHbp-NadA and NadA-PorA, fHbp-NadA and NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA and NadA-PorA; Preferably, it comprises at least two antigen fusion proteins, wherein the antigen fusion proteins comprise the following antigens or fragments thereof: fHbp-NHBA and NadA-PorA, fHbp-NadA and NHBA-PorA.

8. The combination of antigen fusion proteins according to any one of claims 1 to 7, said antigens having a three-dimensional domain, wherein, fHbp consists of an N-terminal β-sheet and a C-terminal β-bucket; Preferably, the NHBA comprises an N-terminal flexible region, an intermediate structural domain, a C-terminal β-sheet, and a β-barrel; Preferably, NadA comprises an N-terminal head domain composed of α-helices and β-sheets and a C-terminal rod-like domain composed of α-helices; Preferably, PorA includes a transmembrane domain and a surface loop domain.

9. The combination of antigen fusion proteins according to any one of claims 1 to 8, wherein, The fHbp antigen or its fragment has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 1-6, preferably having an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably having an amino acid sequence that is 98% or 99% or more identical.

10. The combination of antigen fusion proteins according to any one of claims 1 to 8, wherein, The NHBA antigen or its fragment has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 7-25, preferably having an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably having an amino acid sequence that is 98% or 99% or more identical.

11. The combination of antigen fusion proteins according to any one of claims 1 to 8, wherein, The NadA antigen or its fragment has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO.26-31, preferably having an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably having an amino acid sequence that is 98% or 99% or more identical.

12. The combination of antigen fusion proteins according to any one of claims 1-8, wherein, The PorA ring region has an amino acid sequence that is at least 80% or more identical to any one of SEQ ID NO. 32-36, preferably having an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical, and more preferably having an amino acid sequence that is 98% or 99% or more identical.

13. The combination of antigen fusion proteins according to any one of claims 1 to 12, wherein, In the antigen fusion protein, 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.

14. The combination of antigen fusion proteins according to any one of claims 1 to 13, wherein, In the antigen fusion protein, the N-terminal amino acid of one antigen or its fragment is fused to the C-terminal amino acid of another antigen or its fragment via a peptide linker.

15. The combination of antigen fusion proteins according to claim 14, wherein, the peptide linker is selected from one or more of (G) n , (GS) n , (GSG) n , (GGGS) n , (GSGGS) n , or (GGGGS) n , 1<=n<=8; Preferably, the amino acid sequence of the peptide linker is as shown in any one of SEQ ID NO.249-256; Preferably, the amino acid sequence of the peptide linker is as shown in SEQ ID NO.255 ​​or SEQ ID NO.

256.

16. The combination of antigen fusion proteins according to any one of claims 1 to 12, wherein, The connection method of the antigen fusion protein also includes: inserting a loop region of one antigen or its fragment into another antigen or its fragment to form a chimera.

17. The combination of antigen fusion proteins according to any one of claims 1 to 16, wherein, The antigen fusion protein contains the PorA loop region antigen or a fragment thereof.

18. The combination of antigen fusion proteins according to claim 17, wherein, The antigen fusion protein is a chimera formed by inserting the PorA loop region into the NHBA or NadA antigen or a fragment thereof.

19. The combination of antigen fusion proteins according to claim 18, wherein, The insertion site of the PorA loop region is the loop region between the β-turn of the corresponding antigen or its fragment, or between the α-helix and the β-sheet.

20. The combination of antigen fusion proteins according to any one of claims 17-19, wherein, The antigen fusion protein is a chimera obtained by inserting the porin PorA loop region as shown in any one of the amino acid sequences of SEQ ID NO. 32-36 into the NHBA antigen or a fragment thereof as shown in any one of the amino acid sequences of SEQ ID NO. 7-25; Preferably, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO.55-61.

21. The combination of antigen fusion proteins according to any one of claims 17-19, wherein, The antigen fusion protein is a chimera obtained by inserting the porin PorA loop region shown in any one of the amino acid sequences of SEQ ID NO. 32-36 into the NadA antigen or a fragment thereof shown in any one of the amino acid sequences of SEQ ID NO. 26-31; Preferably, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO.48-51 and SEQ ID NO.113-124.

22. The combination of antigen fusion proteins according to any one of claims 1-15, wherein, The antigen fusion protein comprises: (i) the fHbp antigen or a fragment thereof as shown in any of the amino acid sequences in SEQ ID NO. 1-6; and (ii) NHBA antigen or fragment thereof as shown in any of the amino acid sequences in SEQ ID NO.7-25.

23. The combination of antigen fusion proteins according to claim 22, wherein, The C-terminal amino acid of the fHbp antigen or its fragment is fused to the N-terminal amino acid of the NHBA antigen or its fragment via a peptide linker in the antigen fusion protein. Preferably, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO.37-40 and SEQ ID NO.62-74; Preferably, the amino acid sequence of the fusion protein is selected from any one of SEQ ID NO.40 and SEQ ID NO.62-74; Preferably, the antigen fusion protein is further mutated and / or truncated to form a mutant and / or a truncated form; Preferably, the fHbp antigen or a fragment thereof in the antigen fusion protein contains a mutation, the mutation being selected from any of the following combinations: (1) For fHbp Variant 1, the mutation sites are E218A / E239A; (2) For fHbp Variant 2, the mutation sites are E217A / T238A and / or L130R / G133D; (3) For fHbp Variant 3, the mutation site is E225A / T246A; Preferably, the amino acid sequence of the mutant is selected from any one of SEQ ID NO.45, SEQ ID NO.75-87; Preferably, the amino acid sequence of the truncated form is selected from any one of SEQ ID NO.46, SEQ ID NO.88-97; Preferably, the amino acid sequences of the mutant and the truncated mutant are selected from any one of SEQ ID NO.47, SEQ ID NO.98-112.

24. The combination of antigen fusion proteins according to claim 22, wherein, The N-terminal amino acid of the fHbp antigen or its fragment is fused to the C-terminal amino acid of the NHBA antigen or its fragment via a peptide linker in the antigen fusion protein. Preferably, the amino acid sequence of the antigen fusion protein is selected from any one of SEQ ID NO.41-44.

25. The combination of antigen fusion proteins according to any one of claims 1-15, wherein, The antigen fusion protein comprises: (i) the fHbp antigen or a fragment thereof as shown in any of the amino acid sequences in SEQ ID NO. 1-6; and (ii) NadA antigen or fragment thereof as shown in any of the amino acid sequences in SEQ ID NO.26-31.

26. The combination of antigen fusion proteins according to claim 25, wherein, In the antigen fusion protein, the C-terminal amino acid of the fHbp antigen or its fragment is fused with the N-terminal amino acid of the NadA antigen or its fragment via a peptide linker. Preferably, the amino acid sequence of the antigen fusion protein is selected from any one of SEQ ID NO.52-54.

27. The combination of antigen fusion proteins according to any one of claims 1 to 26, wherein, The amino acid sequence of the antigen fusion protein is selected from any one of SEQ ID NO.40, 45-47, 62-112.

28. An immunogenic composition comprising the antigen fusion protein combination according to any one of claims 1-27.

29. An immunogenic composition comprising at least one antigen fusion protein selected from the following free combinations of antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA; The free linking combination is achieved by: (i) the N-terminal amino acid of one antigen or its fragment being directly fused with the C-terminal amino acid of another antigen or its fragment, or by fusion through a peptide linker; (ii) the loop region of one antigen or its fragment being inserted into another antigen or its fragment to form a chimera.

30. The immunogenic composition according to claim 29, comprising at least one antigen fusion protein, said antigen fusion protein comprising, from the N-terminus to the C-terminus, the following antigens or fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA.

31. The immunogenic composition according to claim 29, further comprising any combination of at least two antigen fusion proteins.

32. The immunogenic composition according to claim 31, wherein the antigen fusion protein is selected from at least two of the following antigens or free combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, fHbp-NHBA-NadA-PorA; Preferably, it comprises at least two antigen fusion proteins, wherein the antigen fusion proteins comprise, from the N-terminus to the C-terminus, at least two of the following antigens or combinations thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA, fHbp-NHBA-NadA, fHbp-NHBA-PorA, fHbp-NadA-PorA, NHBA-NadA-PorA, and fHbp-NHBA-NadA-PorA.

33. The immunogenic composition according to claim 29, comprising at least one antigen fusion protein selected from the following antigens or free combinations of fragments thereof: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, NadA-PorA; Preferably, it comprises at least one antigen fusion protein, which comprises the following antigens or fragments thereof from the N-terminus to the C-terminus: fHbp-NHBA, fHbp-NadA, fHbp-PorA, NHBA-NadA, NHBA-PorA, and NadA-PorA.

34. The immunogenic composition of claim 33, comprising at least two antigen fusion proteins selected from the following antigens or combinations of linked fragments thereof: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and fHbp-PorA, fHbp-NHBA and NHBA-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and fHbp-PorA, fHbp-NadA and NHBA-NadA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, fHbp-PorA and NHBA-NadA, fHbp-PorA and NHBA-PorA, fHbp-PorA and NadA-PorA, NHBA-NadA and NHBA-PorA, NHBA-NadA and NadA-PorA.

35. The immunogenic composition according to claim 29, comprising at least one antigen fusion protein selected from the following: fHbp-NHBA, fHbp-NadA, NHBA-PorA, NadA-PorA.

36. The immunogenic composition of claim 35, comprising at least two antigen fusion proteins selected from the following antigens or combinations of linked fragments thereof: fHbp-NHBA and fHbp-NadA, fHbp-NHBA and NHBA-PorA, fHbp-NHBA and NadA-PorA, fHbp-NadA and NHBA-PorA, fHbp-NadA and NadA-PorA, NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA, fHbp-NHBA and fHbp-NadA and NadA-PorA, fHbp-NadA and NHBA-PorA and NadA-PorA, fHbp-NHBA and fHbp-NadA and NHBA-PorA and NadA-PorA.

37. The immunogenic composition of claim 29, further comprising a first fusion protein and / or a second fusion protein, wherein the first fusion protein further comprises a first antigen and a second antigen, and the second fusion protein comprises a third antigen and a fourth antigen.

38. The immunogenic composition of claim 37, wherein, In the first fusion protein, the C-terminal amino acid of the first antigen is directly fused to the N-terminal amino acid of the second antigen or fused through a peptide linker.

39. The immunogenic composition of claim 37, wherein, The second fusion protein is a chimera obtained by inserting the third antigen into the fourth antigen.

40. The immunogenic composition of any one of claims 37-39, wherein, The first, second, third, and fourth antigens are selected from any one of the following antigens or fragments thereof: (i)fHbp antigen; (ii) NHBA antigen; (iii) NadA antigen; or (iv)PorA ring area.

41. The immunogenic composition of any one of claims 37-40, wherein, The immunogenic composition: (1) Contains a first fusion protein, wherein the first fusion protein is fHbp-NHBA or fHbp-NadA protein; or (2) Contains a second fusion protein, wherein the second fusion protein is a NadA-PorA or NHBA-PorA protein; or (3) Contains a first fusion protein and a second fusion protein, wherein the first fusion protein is fHbp-NHBA protein and the second fusion protein is NadA-PorA protein; or (4) It includes a first fusion protein and a second fusion protein, wherein the first fusion protein is fHbp-NadA protein and the second fusion protein is NHBA-PorA protein; Preferably, the first fusion protein and / or the second fusion protein are further mutated and / or truncated to form mutants and / or truncated forms.

42. The immunogenic composition of any one of claims 37-41, comprising a first fusion protein and a second fusion protein, wherein, In the first fusion protein, the C-terminal amino acid of the fHbp antigen or its fragment is fused with the N-terminal amino acid of the NHBA antigen or its fragment via a peptide linker. The amino acid sequence of the first fusion protein is selected from any one of SEQ ID NO.40, SEQ ID NO.45-47, and SEQ ID NO.62-112. The second fusion protein is a chimera obtained by inserting the PorA loop region of porin into the NadA antigen fragment. The amino acid sequence of the second fusion protein is selected from any one of SEQ ID NO.48-51 and SEQ ID NO.113-124.

43. The immunogenic composition of any one of claims 37-41, comprising a first fusion protein and a second fusion protein, wherein, In the first fusion protein, the C-terminal amino acid of the fHbp antigen or its fragment is fused with the N-terminal amino acid of the NadA antigen or its fragment via a peptide linker. The amino acid sequence of the first fusion protein is shown in any one of SEQ ID NO. 52-54. The second fusion protein is a chimera obtained by embedding the porin PorA loop region into the NHBA antigen fragment. The amino acid sequence of the second fusion protein is shown in any one of SEQ ID NO. 55-61.

44. The immunogenic composition according to any one of claims 28-43, further comprising one or more of an adjuvant, a buffer solution, a stabilizer, and a surfactant.

45. The immunogenic composition of claim 44, wherein, The adjuvant is at least one of aluminum salt adjuvant, Freund's adjuvant, liposome, MF59, monophospholipid A, flagellin, CpG-ODN, and Poly(I:C).

46. ​​A method for preventing and / or treating group B meningitis, the method comprising administering to a subject an effective amount of the antigen fusion protein combination of any one of claims 1-27 and / or the immunogenic composition of any one of claims 28-45; Preferably, the dosage of the antigen fusion protein or immunogenic composition is 10-100 μg.

47. The method of claim 46, wherein, The method also includes the combined administration of one or more vaccines; Preferably, the vaccine is a meningitis vaccine of other serogroups.

48. A method for inducing an individual to neutralize an antigen-specific immune response, the method comprising administering to a subject an effective amount of the antigen fusion protein combination of any one of claims 1-27 and / or the immunogenic composition of any one of claims 28-45; Preferably, the dosage of the antigen fusion protein or immunogenic composition is 10-100 μg.

49. Use of the antigen fusion protein combination according to any one of claims 1-27 and / or the immunogenic composition according to any one of claims 28-45 in the preparation of a medicament for the prevention and / or treatment of group B meningitis.

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