Immune composition against meningococcus and use thereof

Abstract

Provided is an immunogenic composition comprising at least two antigens. Provided is a specific combination of Neisseria meningitidis antigens, which can significantly improve the efficacy of the immunogenic composition against Neisseria infection. The synergistic combination of the specific antigen combination is greater than the additive reaction triggered by the antigens alone. On the basis of epidemiological studies in China, the immunogenic composition targeting a broader coverage of MenB strains is developed.

Description

An anti-meningococcal immune composition and its use Technical Field

[0001] The present invention relates to an immune composition and its use, and in particular to an anti-meningococcal immune composition and its use. Background Art

[0002] Neisseria meningitidis is a Gram-negative, enveloped bacterial pathogen. Although polysaccharide and conjugate vaccines exist for serogroups A, C, W135, and Y, this approach is not applicable to serogroup B.

[0003] Currently, for serogroup B meningococci, a mixture of five defined protein antigens has been found to elicit a good protective immune response: NadA protein; fHbp protein; 936 protein; 953 protein; and 287 protein.

[0004] fHbp is a specific lipoprotein expressed on the surface of nearly all Neisseria meningitidis bacteria, with a relative molecular mass of approximately 29 kDa. It is a key virulence factor and vaccine antigen for Neisseria meningitidis. Based on antigenic cross-reactivity and protein sequence similarity, fHbp is subdivided into three variants: V1, V2, and V3. Within-variant amino acid sequence identity exceeds 90%, while between variants it can be as low as 62.8%. Bactericidal activity assays have shown that antibodies against variant V1 have minimal cross-reactivity against variants V2 and V3 meningococcal isolates.

[0005] According to the polymorphism analysis of fHbp protein of Neisseria meningitidis serogroup B in China, the fHbp of Neisseria meningitidis serogroup B strains isolated in my country is mainly V2 type. There is a need for an immunogenic composition that covers a wider range of MenB strains than BEXSERO or TRUMENBA.

[0006] Summary of the Invention

[0007] The present invention discloses specific combinations of Neisseria meningitidis antigens that can significantly enhance the efficacy of immunogenic compositions against Neisseria meningitidis infections. The synergistic effects of specific antigen combinations are greater than the additive effects elicited by any single antigen. The present invention relates to combinations of two or more different antigens.

[0008] A first object of the present invention is to provide improved immunogenic compositions for providing immunity against meningococci, in particular for providing broad immunity against serogroup B meningococci.

[0009] The second objective of the present invention is to screen anti-meningococcal antigens to obtain antigens or antigen combinations that can be used to elicit a strong immune response.

[0010] The third goal of the present invention is to analyze the spatial structure and immunogenicity of different antigens to screen for antigen combinations that are stable and can induce strong immune responses.

[0011] A fourth objective of the present invention is to develop immunogenic compositions with broader coverage of MenB strains for epidemiological studies in China.

[0012] The present invention provides an immunogenic composition comprising a first antigen and a second antigen, wherein the first antigen is NMB1162; the second antigen is one of NMB1870, NMB2132, NMB0606 and NMB1994.

[0013] The antigens in the composition of the present invention are selected from Neisseria meningitidis serogroups A, B, C, Y and / or W135; preferably Neisseria meningitidis serogroup B; preferably strains are MC58, H44 / 76, BZ83, M02240058, M14530, 4243, CDC1573, CDC983, A4, 2996, M97-252976, M97-251854, CDC1521, M98-250716, 961-5945, CDC1492, L8-M978, M97-252988, M97-252697, 6557, M050240011, M050240138, 17024, M08 240117, M01-240355, M98-250732, M98250771, C2120.

[0014] The antigens in the composition of the present invention may be present in the composition as a single polypeptide; or two or more antigens may be expressed as a single polypeptide chain to form a fusion polypeptide.

[0015] The first antigen and the second antigen in the composition of the present invention may be from the same or different strains.

[0016] The expression of the antigens of the present invention can occur in Neisseria, preferably using a heterologous host. The heterologous host can be a prokaryotic organism (such as a bacterium) or a eukaryotic organism. Preferably, it is Escherichia coli, Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacterium (such as Mycobacterium tuberculosis), yeast, etc.

[0017] The composition of the present invention further comprises a pharmaceutically acceptable buffer, diluent, adjuvant or carrier.

[0018] The carrier of the present invention comprises one or more of a mineral salt adjuvant, an oil-in-water emulsion, a saponin, a virosome and a virus-like particle, an immunostimulatory oligonucleotide, and a human immunomodulator.

[0019] Mineral salt adjuvants such as aluminum salts, calcium salts, phosphates or sulfates or combinations of different mineral salts; the preferred mineral salt adjuvant is aluminum phosphate; oil-in-water emulsions include but are not limited to: squalene-water emulsion, complete Freund's adjuvant or incomplete Freund's adjuvant; saponins can also be used as adjuvants of the present invention. Saponins are a class of heterologous sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of various plants; virosomes and virus-like particles can also be used as adjuvants of the present invention. Virosomes and virus-like particles usually contain one or more Proteins, optionally combined or formulated with phospholipids, suitable viral proteins for virosomes and virus-like particles include those derived from influenza virus (such as HA or NA), hepatitis B virus (such as core or capsid protein), hepatitis E virus, measles virus, Sindbis virus, rotavirus, foot and oral disease virus, retrovirus, Norwalk virus, human papillomavirus, HIV, RNA-phage, Qβ-phage (such as coat protein), GA-phage, fr-phage, AP205 phage. Immunostimulatory oligonucleotides can also be used as adjuvants of the present invention. Immunostimulatory oligonucleotides include nucleotide sequences containing CpG motifs (dinucleotide sequences containing unmethylated cytosine linked to guanosine via phosphate bonds), double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences; human immunomodulators include cytokines, interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, interferons, macrophage colony-stimulating factor, and tumor necrosis factor;

[0020] Adjuvants that can be used in the present invention also include poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrrolidone, cross-linked derivatives of polysaccharides and carboxymethyl cellulose, chitosan, microparticles, polyoxyethylene ether and polyoxyethylene ester formulations, imidazole quinolone compounds, and muramyl peptides.

[0021] The compositions of the present invention can be administered directly to the patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or into the interstitial space of a tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary, or other mucous membrane injection. Intramuscular injections into the thigh or upper arm are preferred. Injection can be performed with a needle (e.g., a hypodermic needle), but needle-free injection can also be used.

[0022] The composition of the present invention can be prepared into mucosal immune preparations, humoral immune preparations, cellular immune preparations, and skin immune preparations.

[0023] The composition of the present invention can be prepared into liquid dosage form, solid dosage form, semisolid dosage form, gas dosage form, and inhalation dosage form.

[0024] The composition of the present invention can be administered by intravenous injection, intramuscular injection, subcutaneous injection, oral administration, buccal administration, sublingual administration, rectal administration, respiratory tract administration, and transdermal administration; the respiratory tract administration is inhalation through the mouth, inhalation through the nose, or inhalation after atomization by an atomizing device.

[0025] In addition to the antigens described above, the compositions of the present invention may also include one or more meningococcal capsular saccharide conjugates. The compositions of the present invention may include one or more of meningococcal serogroups A, C, W135, and Y. The saccharide moiety in the saccharide conjugate in the conjugate may include a full-length saccharide prepared from meningococci, and / or may include a fragment of the full-length saccharide; useful carrier proteins for meningococcal conjugates include bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. When the composition includes conjugates from more than one meningococcal serogroup, the same carrier protein may be used for each individual conjugate, or different carrier proteins may be used. However, in both cases, the mixture of different conjugates is typically formed by preparing each serotype conjugate separately and then mixing them to form a mixture of the individual conjugates.

[0026] The compositions of the present invention will typically be administered directly to the patient. Direct delivery can be achieved by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or into the interstitial space of a tissue), or by rectal, oral, inhalational, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary, or other mucous membrane injection. Intramuscular injection into the thigh or upper arm is preferred. Injection can be performed with a needle (e.g., a hypodermic needle), but needle-free injection can also be used.

[0027] The immunogenic compositions of the present invention typically include a pharmaceutically acceptable carrier, which can be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable carriers can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffering substances, etc., may also be present in these carriers.

[0028] The present invention provides a use of the composition in preparing a vaccine for eliciting an immune response in a mammal, preferably for primary immunization or booster immunization.

[0029] The present invention provides an isolated and purified polypeptide, wherein the polypeptide has an amino acid sequence with a sequence identity of greater than 95% with any one of SEQ ID NO.1 to SEQ ID NO.175.

[0030] In some embodiments, the amino acid sequence of the polypeptide has at least 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequence shown in any one of SEQ ID NO.1-SEQ ID NO.175.

[0031] Preferably, the present invention provides an isolated and purified polypeptide having an amino acid sequence with greater than 95% sequence identity to any one of SEQ ID NO.56 to SEQ ID NO.139.

[0032] In some embodiments, the amino acid sequence of the polypeptide has at least 95%, 96%, 97%, 98%, 99% or 100% homology to the amino acid sequence shown in any one of SEQ ID NO.56 to SEQ ID NO.139.

[0033] The present invention provides a nucleic acid molecule having at least 95% sequence identity with the sequence of a nucleic acid molecule encoding a polypeptide shown in any of SEQ ID NO. 1 to SEQ ID NO. 175.

[0034] In some embodiments, the nucleic acid molecule encoding the polypeptide has at least 95%, 96%, 97%, 98%, 99% or 100% homology with the sequence of the nucleic acid molecule encoding the polypeptide shown in any one of SEQ ID NO.1-SEQ ID NO.175.

[0035] Preferably, the nucleic acid molecule provided by the present invention has at least 95% sequence identity with the sequence of the nucleic acid molecule encoding the polypeptide shown in any of SEQ ID NO. 56 to SEQ ID NO. 139.

[0036] In some embodiments, the nucleic acid molecule encoding the polypeptide has at least 95%, 96%, 97%, 98%, 99% or 100% homology with the sequence of the nucleic acid molecule encoding the polypeptide shown in any one of SEQ ID NO.56-SEQ ID NO.139.

[0037] The antigen NMB1870 described herein is also referred to in other literature as fHbp, GNA1870,741 protein, and f hypothetical protein. A key function of fHbp is its ability to bind human complement factor (fH). Binding of fH to the bacterial surface accelerates the decay of the C3 / C5 convertase, which reduces activation of the alternative pathway and contributes to the organism's vitality, thereby preventing complement-mediated killing by non-immune human serum or blood. As a vaccine antigen, anti-fHbp antibodies can directly activate bacteriolysis via the classical complement pathway and also prevent fH binding to the bacterial surface. fHbp comprises three domains: A, B, and C. Domain A is highly conserved among the three variants. V1 expresses an epitope located in domain B, while the epitopes of V2 and V3 are primarily located in domain C, with only a few amino acid differences in domain C between V2 and V3. In the two currently marketed MenB protein vaccine products, fHbp is expressed as a single variant, either alone or in fusion with a non-fHbp protein. Epidemiological studies have shown that the majority of Neisseria meningitidis strains found in European patients, healthy carriers, and individuals of unknown origin are serogroup B, including 119 V1 strains, 42 V2 strains, and 57 V3 strains. In the marketed GlaxoSmithKline and Pfizer vaccines, fHbp is primarily V1 and V3. Analysis of fHbp protein polymorphisms in serogroup B Neisseria meningitidis isolates in China shows that fHbp is primarily V2.

[0038] In the present invention, the fHbp protein can be in various forms, such as SEQ ID NO: 1; the preferred fHbp form is truncated; the esterification site is preferably removed to further improve safety; the preferred fHbp removes the signal peptide; the β-pleated sleeve is preferably deleted to improve protein solubility; the preferred fHbp form is a protein variant, such as an allelic variant, a homolog, an ortholog, a paralog, a mutant, etc.; the preferred fHbp can contain at least one amino acid change compared to the wild type; the fHbp has 50% or more homology (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) with SEQ ID NO: 1-SEQ ID NO: 30. The above-mentioned fHbp truncation, mutation, and other modifications relative to the wild type can all retain the immunogenicity of fHbp.

[0039] The amino acid sequence of NMB1162 (GenBank Accession: AAF41547) described in the present invention is shown in SEQ ID NO: 50 or SEQ ID NO: 52 or SEQ ID NO: 54. The NMB1162 described in the present invention can be in various forms, and the preferred form of NMB1162 is truncated, and its amino acid sequence is shown in SEQ ID NO: 51 or SEQ ID NO: 53 or SEQ ID NO: 55. Preferably, the signal peptide is removed. The preferred form of NMB1162 is a protein variant: such as an allelic variant, a homolog, a direct homolog, a paralog, a mutant, etc.; the preferred NMB1162 may contain at least one amino acid change compared to the wild type. NMB1162 has 50% or more homology (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) with any one of the amino acid sequences shown in SEQ ID NO: 50-SEQ ID NO: 55. The above-mentioned NMB1162 modification schemes such as truncation and mutation relative to the wild type can all retain the immunogenicity of NMB1162.

[0040] The NMB2132 described herein is a Neisserial heparin binding antigen (NMB2132), also referred to in other literature as protein 287. For example, SEQ ID NO:154 has been identified as a heparin-binding protein and designated NHBA in references. Preferred forms of NMB2132 are truncated, such as SEQ ID NO:155, preferably with the signal peptide removed. Preferred forms of NMB2132 are protein variants, such as allelic variants, homologs, orthologs, paralogs, and mutants. Preferred NMB2132 may contain at least one amino acid change compared to the wild-type. NMB2132 has 50% or greater homology (e.g., 60%, 70%, 80%, 90%, 95%, 99%, or greater) to SEQ ID NO:154 or SEQ ID NO:155. The aforementioned modifications, such as truncations and mutations of NMB2132 relative to the wild-type, can retain the immunogenicity of NMB2132.

[0041] The NMB0606 described in the present invention: Sec translocon accessory complex subunit YajC; such as SEQ ID NO: 156 or SEQ ID NO: 167 or SEQ ID NO: 170, the NMB0606 described in the present invention can take various forms. The preferred form of NMB0606 is truncated; such as SEQ ID NO: 157 or SEQ ID NO: 166 or SEQ ID NO: 168 or SEQ ID NO: 169 or SEQ ID NO: 171 or SEQ ID NO: 172, preferably with the signal peptide removed; the preferred form of NMB0606 is a protein variant: such as an allelic variant, a homolog, a direct homolog, a paralog, a mutant, etc.; the preferred NMB0606 may contain at least one amino acid change compared to the wild type; NMB0606 has 50% or more homology (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more) with any one of SEQ ID NO: 156 or SEQ ID NO: 157 or SEQ ID NO: 166-SEQ ID NO: 172, and the above-mentioned NMB0606 truncation, mutation, etc. modification schemes relative to the wild type can retain the immunogenicity of NMB0606.

[0042] The NMB1030 of the present invention is an ipid / polyisoprenoid-binding YceI-like domain-containing protein, such as SEQ ID NO: 164. The NMB1030 of the present invention can be in various forms. A preferred form of NMB1030 is a truncated form, such as SEQ ID NO: 165, preferably with the signal peptide removed. A preferred form of NMB1030 is a protein variant, such as an allelic variant, homolog, ortholog, paralog, mutant, etc.; a preferred form of NMB1030 can contain at least one amino acid change compared to the wild type. NMB1030 and SEQ ID NO: 164 or SEQ ID NO: 165 have 50% or more homology (e.g., 60%, 70%, 80%, 90%, 95%, 99% or more). The aforementioned modifications of NMB1030, such as truncation and mutation, relative to the wild type, can all retain the immunogenicity of NMB1030.

[0043] The immunogenic composition of the present invention comprises at least two different antigens; preferably, the antigen is a combination of NMB1162 and other antigens; the combination of NMB1162 and other antigens may be present in the composition as a separate protein (or polypeptide); or NMB1162 and one or more other antigens may be expressed as a single polypeptide chain to form a fusion protein (or polypeptide); such as one or more of SEQ ID NO:56-SEQ ID NO:139, or SEQ ID NO:56-SEQ ID NO:139 have 50% or more homology (for example, 60%, 70%, 80%, 90%, 95%, 99% or more); preferably, the amino acid sequence of the fusion protein (or polypeptide) of the present invention has at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:56-SEQ ID NO:139.

[0044] Preferably, the NMB1162 antigen of the present invention is combined or fused with a protein or polypeptide selected from the following sources: one or more of NMB1870, NMB0281, NMB0995, NMB1220, NMB1333, NMB1533, NMB1961, NMB0033, NMB2132, NMB0606, NMB1124, and NMB1030; preferably, the NMB1162 antigen is combined or fused with a protein or polypeptide selected from the following sources: NMB1870, NMB2132, NMB0606, and NMB1994.

[0045] The first antigen and the second antigen in the immunogenic composition of the present invention may be variants or truncated forms of the above antigens, and the variants or truncated forms have the same or similar biological activity as the source antigens.

[0046] The present invention also provides a method for producing the polypeptide of the present invention, comprising the step of culturing a transformed host cell with the inventive nucleic acid of the present invention under conditions that induce expression of the polypeptide.

[0047] The present invention also provides a method for producing the polypeptide of the present invention, comprising the step of synthesizing at least part or all of the polypeptide by chemical methods.

[0048] The present invention provides a method for producing the nucleic acid of the present invention, comprising the step of amplifying the nucleic acid using a primer-based amplification method (such as PCR).

[0049] The present invention provides a method for producing the nucleic acid of the invention, comprising the step of synthesizing at least part or all of the nucleic acid by chemical methods.

[0050] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0051] The polypeptides of the present invention can be prepared in a variety of ways, e.g., by chemical synthesis (at least in part), by digestion of longer polypeptides using proteases, by translation from RNA, by purification from cell culture (e.g., from recombinant expression or from Neisseria meningitidis culture), etc. Heterologous expression in an E. coli host is a preferred expression route.

[0052] The present invention provides nucleic acids encoding polypeptides of the present invention as defined above; nucleic acids of the present invention can be prepared in a variety of ways, for example, by complete or partial chemical synthesis (e.g., phosphoramidite synthesis of DNA), by digestion of longer nucleic acids using nucleases (e.g., restriction enzymes), by ligating shorter nucleic acids or nucleotides from genomic or cDNA libraries (e.g., using ligases or polymerases), and the like.

[0053] The present invention also provides a method of eliciting an antibody response in a mammal, comprising administering to the mammal an immunogenic composition of the present invention.

[0054] The present invention also provides a method for protecting a mammal from Neisseria meningitidis infection, comprising administering the immunogenic composition of the present invention to the mammal.

[0055] It will be appreciated by those of ordinary skill that many different polynucleotides and nucleic acids can encode the same polypeptide due to the degeneracy of the genetic code. It will also be appreciated that one of ordinary skill can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein, thereby reflecting the codon usage of any particular host organism in which the polypeptide is to be expressed. Therefore, unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns. DETAILED DESCRIPTION

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0057] The aforementioned embodiments and methods described in the present invention may vary based on the abilities, experiences and preferences of those skilled in the art. The present invention does not limit the order of the method steps by simply listing the steps in a certain order. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1. Bactericidal activity of 1162 fusion antigens derived from different strains against 70112 in Example 4.

[0059] Figure 2. Bactericidal activity of 1162 fusion antigens derived from different strains against S357 in Example 4.

[0060] Figure 3. Bactericidal activity of 1162 fusion antigens derived from different strains against S1020 in Example 5.

[0061] Figure 4. Bactericidal activity of 1162 fusion antigens derived from different strains against S1229 in Example 5.

[0062] Figure 5. Bactericidal activity of 1162 fusion antigens from different strains against S1563 in Example 6.

[0063] Figure 6. Bactericidal activity of 1162 fusion antigens derived from different strains against S1549 in Example 6.

[0064] Example 1 Preparation of fusion protein

[0065] The following antigens are encoded according to Table 1, where MC58, 331401, and FDAARGOS_214 represent different strain origins.

[0066] Table 1

[0067] After the design of each protein sequence is completed, codon optimization is performed, and plasmid construction is performed using a PET vector to obtain a recombinant plasmid. The plasmid is then chemically transformed into E. coli. After transformation, five transformants are selected from the plate and stored as strains. Induction trials are conducted to screen for dominant transformants. Fermentation is carried out using a high-density fermentation process, and the cells are harvested after fermentation is complete.

[0068] The cells harvested after culture are broken and resuspended in a ratio of 1:5-1:10 between the cells and the resuspension buffer, and after high-pressure homogenization, the cells are clarified and filtered, and the supernatant is collected and subjected to affinity chromatography (Ni column), ion exchange chromatography or hydrophobic chromatography, ultrafiltration and liquid exchange, etc. to obtain the recombinant protein.

[0069] Example 2 Preparation of fusion protein

[0070] The following antigens are encoded according to Table 2, where MC58, 331401, and FDAARGOS_214 represent different strain origins.

[0071] Table 2

[0072] After the design of each protein sequence is completed, codon optimization is performed, and plasmid construction is performed using a PET vector to obtain a recombinant plasmid. The plasmid is then chemically transformed into E. coli. After transformation, five transformants are selected from the plate and stored as strains. Induction trials are conducted to screen for dominant transformants. Fermentation is carried out using a high-density fermentation process, and the cells are harvested after fermentation is complete.

[0073] The cells harvested after culture are disrupted and resuspended in a ratio of 1:5-1:10 between cells and resuspension buffer. After high-pressure homogenization, the supernatant is clarified and filtered, and the supernatant is collected and subjected to affinity chromatography (Ni column), ion exchange chromatography or hydrophobic chromatography, ultrafiltration, etc. to obtain the recombinant protein.

[0074] Example 3 Preparation of fusion protein

[0075] The following antigens are encoded according to Table 3, where MC58, 331401, and FDAARGOS_214 represent different strain origins.

[0076] Table 3

[0077] After the design of each protein sequence is completed, codon optimization is performed, and plasmid construction is performed using a PET vector to obtain a recombinant plasmid. The plasmid is then chemically transformed into E. coli. After transformation, five transformants are selected from the plate and stored as strains. Induction trials are conducted to screen for dominant transformants. Fermentation is carried out using a high-density fermentation process, and the cells are harvested after fermentation is complete.

[0078] The cells harvested after culture are broken and resuspended in a ratio of 1:5-1:10 between the cells and the resuspension buffer, and after high-pressure homogenization, the cells are clarified and filtered, and the supernatant is collected and subjected to affinity chromatography (Ni column), ion exchange chromatography or hydrophobic chromatography, ultrafiltration and liquid exchange, etc. to obtain the recombinant protein.

[0079] Example 4: Study on the immunogenicity of fusion protein

[0080] After the fusion protein obtained in Example 1 was purified, 6-8 week old female CD1 mice were randomly divided into 6 groups and intraperitoneally injected with

[0081] Serum bactericidal assay (SBA)

[0082] Target bacterial suspension preparation: Inoculate the strain into Columbia blood medium and culture overnight in a 37°C, 5% CO2 incubator for 15-20 hours. After the incubation period, select 20-50 representative colonies and transfer them to culture plates for 4-6 hours. Scrape the bacterial lawn into buffer and mix thoroughly. Count the bacteria using turbidimetry, and dilute the suspension to a final concentration of approximately 4000 CFU / ml.

[0083] Sample testing: Incubate the serum to be tested at 56°C for 30 minutes for inactivation. Serially dilute the serum to be tested by 2-fold, and add an equal volume mixture of active complement and working bacterial solution to each sample well. At the same time, set up inactivated complement and active complement controls to observe whether the complement has natural bactericidal activity and whether the target bacteria are growing well. After oscillation and mixing, place the plate in a 37°C incubator and incubate for 1-2 hours. Then remove the culture plate, add 50μl of color development medium to each well, place the culture plate in a 37°C, 5% CO2 incubator, and incubate overnight before observing the results. The maximum dilution factor that can kill more than 50% of the colonies in the complement control well is the bactericidal antibody titer of the serum to be tested.

[0084] Target bacteria are S70112 and S357

[0085] Serum bactericidal assay (SBA) was performed using the serum of each group of mice after three immunizations and rabbit complement to detect the level of neutralizing antibodies.

[0086] As shown in Figures 1 and 2, the results show that 1162 from different strains can produce higher bactericidal activity after fusion with different antigens.

[0087] Example 5: Study on the immunogenicity of fusion protein

[0088] After the fusion protein obtained in Example 2 was purified, 6-8 week old female CD1 mice were randomly divided into 6 groups and intraperitoneally injected with

[0089] Serum bactericidal assay (SBA)

[0090] Target bacterial suspension preparation: Inoculate the strain into Columbia blood medium and culture overnight in a 37°C, 5% CO2 incubator for 15-20 hours. After the incubation period, select 20-50 representative colonies and transfer them to culture plates for 4-6 hours. Scrape the bacterial lawn into buffer and mix thoroughly. Count the bacteria using turbidimetry, and dilute the suspension to a final concentration of approximately 4000 CFU / ml.

[0091] Sample testing: Incubate the serum to be tested at 56°C for 30 minutes for inactivation. Serially dilute the serum to be tested by 2-fold, and add an equal volume mixture of active complement and working bacterial solution to each sample well. At the same time, set up inactivated complement and active complement controls to observe whether the complement has natural bactericidal activity and whether the target bacteria are growing well. After oscillation and mixing, place the plate in a 37°C incubator and incubate for 1-2 hours. Then remove the culture plate, add 50μl of color development medium to each well, place the culture plate in a 37°C, 5% CO2 incubator, and incubate overnight before observing the results. The maximum dilution factor that can kill more than 50% of the colonies in the complement control well is the bactericidal antibody titer of the serum to be tested.

[0092] The target bacteria are S1020 and S1229.

[0093] Serum bactericidal assay (SBA) was performed using the serum of each group of mice after three immunizations and rabbit complement to detect the level of neutralizing antibodies.

[0094] As shown in Figures 3 and 4, the results show that 1162 and V2-Fhbp from different strains can produce high bactericidal activity in type 2 target bacteria after fusion, while the individual fhbp and 2091-fhbp fusion proteins have almost no bactericidal activity. In the single-component verification, 1162 was shown to be able to improve the bactericidal activity of fhbp. There was no significant difference in the effect of 1162 from different strains on the bactericidal activity of the fused type 2 Fhbp.

[0095] Example 6: Study on the immunogenicity of fusion protein

[0096] After the fusion protein obtained in Example 3 was purified, 6-8 week old female CD1 mice were randomly divided into 6 groups and intraperitoneally injected with

[0097] Serum bactericidal assay (SBA)

[0098] Target bacterial suspension preparation: Inoculate the strain into Columbia blood medium and culture overnight in a 37°C, 5% CO2 incubator for 15-20 hours. After the incubation period, select 20-50 representative colonies and transfer them to culture plates for 4-6 hours. Scrape the bacterial lawn into buffer and mix thoroughly. Count the bacteria using turbidimetry, and dilute the suspension to a final concentration of approximately 4000 CFU / ml.

[0099] Sample testing: Incubate the serum to be tested at 56°C for 30 minutes for inactivation. Serially dilute the serum to be tested by 2-fold, and add an equal volume mixture of active complement and working bacterial solution to each sample well. At the same time, set up inactivated complement and active complement controls to observe whether the complement has natural bactericidal activity and whether the target bacteria are growing well. After oscillation and mixing, place the plate in a 37°C incubator and incubate for 1-2 hours. Then remove the culture plate, add 50μl of color development medium to each well, place the culture plate in a 37°C, 5% CO2 incubator, and incubate overnight before observing the results. The maximum dilution factor that can kill more than 50% of the colonies in the complement control well is the bactericidal antibody titer of the serum to be tested.

[0100] The target bacteria are S1563 and S1549.

[0101] Serum bactericidal assay (SBA) was performed using the serum of each group of mice after three immunizations and rabbit complement to detect the level of neutralizing antibodies.

[0102] The results in Figures 5 and 6 show that 1162 and V3-Fhbp from different strains can produce high bactericidal activity in type 3 target bacteria after fusion, while the individual fhbp and 2091-fhbp fusion proteins have almost no bactericidal activity. In the single-component verification, 1162 was shown to be able to improve the bactericidal activity of fhbp. There was no significant difference in the effect of 1162 from different strains on the bactericidal activity of the fused type 3 Fhbp.

Claims

1. An immunogenic composition comprising a first antigen and a second antigen, characterized in that: The first antigen is NMB1162, and the second antigen is one of NMB1870, NMB2132, NMB0606 or NMB1994.

2. The immunogenic composition according to any one of claim 1, characterized in that The first antigen and the second antigen may be present in the composition as separate polypeptides, respectively; or the first antigen and the second antigen may form a fusion polypeptide.

3. The immunogenic composition according to any one of claims 1 to 2, characterized in that: The antigen is derived from Neisseria meningitidis serogroup A, B, C, Y and / or W135; preferably derived from Neisseria meningitidis serogroup B.

4. The immunogenic composition according to any one of claims 1 to 3, characterized in that The expression of the antigen may occur in Neisseria or using a heterologous host, which may be a prokaryotic or eukaryotic organism; preferably, the heterologous host is Escherichia coli, Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteris or yeast.

5. The immunogenic composition according to any one of claims 2 to 4, wherein the first antigen and the second antigen form a fusion polypeptide, and the polypeptide has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.56 to SEQ ID NO.

139.

6. The immunogenic composition according to any one of claims 1 to 5, characterized in that The composition comprises a pharmaceutically acceptable buffer, diluent, adjuvant or carrier.

7. The immunogenic composition according to any one of claims 1 to 6, characterized in that The composition can be prepared as a mucosal immune preparation, a humoral immune preparation, a cellular immune preparation or a skin immune preparation.

8. The immunogenic composition according to any one of claims 1 to 7, characterized in that The composition can be prepared in liquid dosage form, solid dosage form, semisolid dosage form, gas dosage form or inhalation dosage form.

9. The immunogenic composition according to any one of claims 1 to 8, characterized in that The composition can be administered by intravenous injection, intramuscular injection, subcutaneous injection, oral administration, buccal administration, sublingual administration, rectal administration, respiratory tract administration or transdermal administration; the respiratory tract administration is inhalation through the mouth, inhalation through the nose or inhalation after atomization by an atomizing administration device.

10. The immunogenic composition according to any one of claims 1 to 9, characterized in that The composition also includes one or more meningococcal capsular saccharide conjugates from one or more of meningococcal serogroups A, C, W135 and Y.

11. An isolated and purified polypeptide, characterized in that: The polypeptide has at least 95% sequence identity with the amino acid sequence shown in any one of SEQ ID NO.56 to SEQ ID NO.

139.

12. A nucleic acid molecule encoding the polypeptide of claim 11.

13. A method for producing the polypeptide of claim 11, comprising the step of culturing a transformed host cell containing the nucleic acid molecule of claim 12 under conditions that induce expression of the polypeptide.

14. A method for producing the polypeptide of claim 11, comprising the step of synthesizing part or all of the polypeptide by chemical methods.

15. A method for producing the nucleic acid molecule of claim 12, comprising the step of amplifying the nucleic acid using a primer-based amplification method.

16. A method for producing the nucleic acid molecule of claim 12, comprising the step of synthesizing part or all of the nucleic acid by chemical methods.

17. Use of the immunogenic composition according to any one of claims 1 to 10, the polypeptide according to claim 11 or the nucleic acid molecule according to claim 12 in the preparation of a vaccine for eliciting an immune response in a mammal; preferably, the immune response is a primary immunization or a booster immunization.

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