Anti-meningococcus immune composition and application thereof

Abstract

The invention provides an immunogenic composition, the immunogenic composition comprises at least two antigens, a specific combination of Neisseria meningitidis antigens is provided, and the combination enables the effect of the immunogenic composition in resisting neisseria infection to be remarkably improved. The synergistic combination of specific antigen combinations is higher than the addition reaction initiated by the antigen alone. The invention aims at Chinese epidemiological research and develops an immunogenic composition aiming at a wider MenB strain coverage range.

Description

Technical Field

[0001] The present invention relates to an immune composition and its application, and in particular to an immune composition against meningococci and its application. 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 cannot be applied 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 almost all Neisseria meningitidis, with a relative molecular mass of about 29KD. It is an important virulence factor and vaccine antigen of Neisseria meningitidis. fHbp is subdivided into three variants, namely V1, V2 and V3, based on antigen cross-reactivity and protein sequence similarity. The homology of amino acid sequences within the variants is more than 90%, and can be as low as 62.8% between variants. Bactericidal activity experimental analysis shows that anti-variant V1 antibodies have little cross-reactivity to variant V2 and V3 meningitis isolates.

[0005] According to the polymorphism analysis of fHbp protein of Chinese serogroup B meningitidis, the fHbp of Chinese serogroup B Neisseria meningitidis strains isolated from my country is mainly V2 type. An immunogenic composition with a wider coverage of MenB strains than BEXSERO or TRUMENBA is needed. Summary of the invention

[0006] The present invention discloses a specific combination of Neisseria meningitidis antigens, which can significantly improve the efficacy of immunogenic compositions against Neisseria infection. The synergistic combination of the specific antigen combination is higher than the addition reaction induced by the antigen alone. The present invention relates to a combination of two or more different antigens.

[0007] A first object of the present invention is to provide improved immunogenic compositions for providing immunity against meningitidis, in particular for providing broad immunity against serogroup B meningococci.

[0008] The second objective of the present invention is to screen anti-meningococcal antigens to obtain antigens or antigen combinations that can be used to induce a strong immune response.

[0009] The third objective 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.

[0010] A fourth objective of the present invention is to develop immunogenic compositions with broader coverage of MenB strains for Chinese epidemiological studies.

[0011] 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.

[0012] The antigen in the composition of the present invention is selected from Neisseria meningitidis serogroup 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, CD C1521, M98-250716, 961-5945, CDC1492, L8-M978, M97-252988, M97-252697, 6557, M05 0240011, M050240138, 17024, M08240117, M01-240355, M98-250732, M98250771, C2120.

[0013] 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.

[0014] The first antigen and the second antigen in the composition of the present invention may be from the same or different strains.

[0015] The expression of antigens of the present invention can occur in Neisseria, preferably using a heterologous host. The heterologous host can be a prokaryotic (such as bacteria) 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.

[0016] The composition of the present invention further comprises a pharmaceutically acceptable buffer, diluent, adjuvant or carrier.

[0017] The carrier of the present invention comprises one or more of mineral salt adjuvant, oil-in-water emulsion, saponin, virosome and virus-like particle, immunostimulatory oligonucleotide and human immunomodulator.

[0018] 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; water-in-oil emulsions include but are not limited to: squalene-water emulsions, complete Freund's adjuvants or incomplete Freund's adjuvants; saponins can also be used as adjuvants of the present invention, saponins are a class of heterologous sterol glycosides and triterpene glycosides, which exist 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 from viruses 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, including nucleotide sequences containing CpG motifs (dinucleotide sequences containing unmethylated cytosine linked to guanosine by 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;

[0019] 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 cell wall acyl peptides.

[0020] The compositions of the present invention can be administered directly to the patient. Direct delivery can be by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial space of tissue), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucous membranes. Intramuscular injections are preferably given to the thigh or upper arm and can be given by a needle (e.g., a hypodermic needle), but needle-free injections can also be used.

[0021] The composition of the present invention can be prepared into a mucosal immune preparation, a humoral immune preparation, a cellular immune preparation, or a skin immune preparation.

[0022] The composition of the present invention can be prepared into a liquid dosage form, a solid dosage form, a semisolid dosage form, a gas dosage form, or an inhalation dosage form.

[0023] 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 inhaled through the mouth, inhaled through the nose, or inhaled after atomization by an atomizing administration device.

[0024] The composition of the present invention, in addition to the above-mentioned antigens, may also include one or more meningococcal capsular saccharide conjugates. The composition of the present invention may include one or more of meningococcal serogroups A, C, W135 and Y. The sugar moiety in the sugar conjugate in the conjugate may include a full-length sugar prepared from meningococci, and / or may include a fragment of the full-length sugar; 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, a mixture of different conjugates is usually formed by preparing each serotype conjugate separately and then mixing them to form a mixture of individual conjugates.

[0025] The compositions of the present invention will generally be administered directly to the patient. Direct delivery can be by parenteral injection (e.g., subcutaneous, intraperitoneal, intravenous, intramuscular, or interstitial space of tissue), or by rectal, oral, inhalation, vaginal, topical, transdermal, intranasal, ocular, ear, lung or other mucous membranes. Intramuscular injection into the thigh or upper arm is preferred. Injection can be by needle (e.g., hypodermic needle), but needle-free injection can also be used.

[0026] The immunogenic compositions of the present invention generally include a pharmaceutically acceptable carrier, which may be any substance that does not itself induce the production of antibodies harmful to the patient receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable carriers may include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may also be present in these carriers.

[0027] The present invention provides a use of the composition in preparing a vaccine for inducing an immune response in a mammal, preferably, for primary immunization or booster immunization.

[0028] The present invention provides a separated 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-SEQ ID NO.175.

[0029] In some specific embodiments, the amino acid sequence of the polypeptide has at least 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequence shown in any one of SEQ ID NO.1-SEQ ID NO.175.

[0030] Preferably, 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.56 to SEQ ID NO.139.

[0031] In some specific embodiments, the amino acid sequence of the polypeptide has at least 95%, 96%, 97%, 98%, 99% or 100% homology with the amino acid sequence shown in any one of SEQ ID NO.56-SEQ ID NO.139.

[0032] 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.

[0033] In some specific 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.

[0034] 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-SEQ ID NO.139.

[0035] In some specific 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.

[0036] The antigen NMB1870 described in the present invention is also called fHbp, GNA1870, 741 protein, and fhypothetical protein in other documents; an important function of fHbp is that it can bind to human complement factor (fH). The binding of fH to the bacterial surface will accelerate the decline of C3 / C5 convertase, which will reduce the activation of the bypass pathway and contribute to the vitality of the organism, thereby avoiding the killing mediated by non-immune human serum or blood complement. As a vaccine antigen, anti-fHbp antibodies can directly activate the classical complement pathway for bacteriolysis and prevent fH from binding to the bacterial surface. fHbp contains three domains, A, B, and C, and domain A is highly conserved in the three variants. V1 expresses an antigenic epitope located in domain B, and the antigenic epitopes of V2 and V3 are mainly located in domain C, and there are only individual amino acid differences in domain C of V2 and V3. In the two MenB protein vaccine products currently on the market, fHbp is expressed in the form of a single variant alone or fused with non-fHbp proteins. According to epidemiological research, the strains of Neisseria meningitidis from patients, healthy carriers and unknown sources in Europe are mainly group B, including 119 V1, 42 V2 and 57 V3. In the GlaxoSmithKline and Pfizer vaccines that have been launched on the market, the fHbp is selected as V1 and V3. According to the polymorphism analysis of the fHbp protein of group B Neisseria meningitidis in China, the fHbp is mainly V2.

[0037] In the present invention, the fHbp protein can be in a variety of forms; such as SEQ ID NO: 1; the preferred fHbp form is truncated; the preferred esterification site is removed to further improve safety; the preferred fHbp removes the signal peptide; the preferred β-folded sleeve is deleted to improve protein solubility; the preferred fHbp form is a protein variant: such as an allele variant, a homolog, a direct homolog, a paralog, a mutant, etc.; the preferred fHbp may contain at least one amino acid change compared to the wild type; fHbp has 50% or more homology with SEQ ID NO: 1-SEQ ID NO: 30 (for example, 60%, 70%, 80%, 90%, 95%, 99% or more). The above-mentioned fHbp truncation, mutation, etc., relative to the wild type, can retain the immunogenicity of fHbp.

[0038] The amino acid sequence of NMB1162 (GenBank Accession: AAF41547) described in the present invention is as 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. The signal peptide is preferably 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 of the amino acid sequences shown in SEQ ID NO:50-SEQ ID NO:55. The above-mentioned NMB1162 truncation, mutation and other modification schemes relative to the wild type can all retain the immunogenicity of NMB1162.

[0039] The NMB2132 described in the present invention: Neisserial heparin binding antigen, also known as 287 protein in other documents; such as SEQ ID NO: 154, the protein is confirmed to be a heparin binding protein and is named NHBA in the reference; the preferred form of NMB2132 is truncated, such as SEQ ID NO: 155, preferably the signal peptide is removed; the preferred form of NMB2132 is a protein variant: such as an allele variant, a homolog, a direct homolog, a paralog, a mutant, etc.; the preferred NMB2132 can contain at least one amino acid change compared to the wild type. NMB2132 has 50% or more homology with SEQ ID NO: 154 or SEQ ID NO: 155 (for example, 60%, 70%, 80%, 90%, 95%, 99% or more), and the above-mentioned modification schemes of NMB2132 truncation, mutation, etc. relative to the wild type can retain the immunogenicity of NMB2132.

[0040] 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 the signal peptide is removed; the preferred form of NMB0606 is a protein variant: such as an allelic variant, a homolog, a direct homolog, a lateral homolog, 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 (for example, 60%, 70%, 80%, 90%, 95%, 99% or more) with any one of SEQ ID NO:156 or SEQ ID NO:157 or SEQID 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.

[0041] The NMB1030 of the present invention: 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. The preferred form of NMB1030 is truncated, such as SEQ ID NO: 165 preferably removing the signal peptide; the preferred form of NMB1030 is a protein variant: such as an allele variant, a homolog, a direct homolog, a paralog, a mutant, etc.; the preferred 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), and the above-mentioned modification schemes of NMB1030 truncation, mutation, etc. relative to the wild type can retain the immunogenicity of NMB1030.

[0042] 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 single protein (or polypeptide); NMB1162 and one or more other antigens may also 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.

[0043] 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.

[0044] The first antigen and the second antigen in the immunogenic composition of the present invention may be variants or truncated forms derived from the above antigens, and the variants or truncated forms have the same or similar biological activity as the source antigens.

[0045] 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 inducing polypeptide expression.

[0046] 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.

[0047] 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).

[0048] The present invention provides a method for producing the nucleic acid of the present invention, comprising the step of synthesizing at least part or all of the nucleic acid by chemical methods.

[0049] 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.

[0050] The polypeptides of the 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.

[0051] The invention provides nucleic acids encoding polypeptides of the invention as defined above; nucleic acids of the invention can be prepared in a variety of ways, for example by all or part chemical synthesis (e.g., phosphoramidite synthesis of DNA), by digestion of longer nucleic acids using nucleases (e.g., restriction enzymes), by ligation of shorter nucleic acids or nucleotides from genomic or cDNA libraries (e.g., using ligases or polymerases), and the like.

[0052] 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.

[0053] The present invention also provides a method for protecting a mammal from infection by Neisseria meningitidis, comprising administering the immunogenic composition of the present invention to the mammal.

[0054] It will be appreciated by those of ordinary skill that many different polynucleotides and nucleic acids may encode the same polypeptide due to the degeneracy of the genetic code. It will also be appreciated that one of ordinary skill may use conventional techniques to make nucleotide substitutions that will 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. Thus, 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

[0055] 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 principle of the present invention should be included in the protection scope of the present invention.

[0056] The foregoing 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 merely lists the steps of the method in a certain order and does not constitute any limitation on the order of the steps of the method. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1.The bactericidal activity of 1162 fusion antigens from different strains in Example 4 against 70112.

[0058] Figure 2 .The bactericidal activity of 1162 fusion antigens from different strains in Example 4 against S357.

[0059] Figure 3 .The bactericidal activity of 1162 fusion antigens from different strains in Example 5 against S1020.

[0060] Figure 4 .The bactericidal activity of 1162 fusion antigens from different strains in Example 5 against S1229.

[0061] Figure 5 .The bactericidal activity of 1162 fusion antigens from different strains in Example 6 against S1563.

[0062] Figure 6 .The bactericidal activity of 1162 fusion antigens from different strains in Example 6 against S1549.

[0063] Example 1 Preparation of fusion protein

[0064] The following antigens are encoded according to Table 1, where MC58, 331401, and FDAARGOS_214 represent different strain sources.

[0065] Table 1

[0066]

[0067]

[0068] 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, which is then transferred into Escherichia coli using chemical transformation. After the transformation is completed, 5 transformants are picked from the plate for strain preservation, and induction is attempted to screen for superior transformants. A high-density fermentation process is used for fermentation culture, and the bacteria are harvested after the fermentation is completed.

[0069] 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.

[0070] Example 2 Preparation of fusion protein

[0071] The following antigens are encoded according to Table 2, where MC58, 331401, and FDAARGOS_214 represent different strain sources.

[0072] Table 2

[0073]

[0074] 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, which is then transferred into Escherichia coli using chemical transformation. After the transformation is completed, 5 transformants are picked from the plate for strain preservation, and induction is attempted to screen for superior transformants. A high-density fermentation process is used for fermentation culture, and the bacteria are harvested after the fermentation is completed.

[0075] 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.

[0076] Example 3 Preparation of fusion protein

[0077] The following antigens are encoded according to Table 3, where MC58, 331401, and FDAARGOS_214 represent different strain sources.

[0078] Table 3

[0079]

[0080] 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, which is then transferred into Escherichia coli using chemical transformation. After the transformation is completed, 5 transformants are picked from the plate for strain preservation, and induction is attempted to screen for superior transformants. A high-density fermentation process is used for fermentation culture, and the bacteria are harvested after the fermentation is completed.

[0081] 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.

[0082] Example 4: Study on the immunogenicity of fusion protein

[0083] After the fusion protein obtained in Example 1 was purified, 6-8 week old female CD1 mice were randomly divided into 6 mice per group and intraperitoneally injected with

[0084] Serum Bactericidal Assay (SBA)

[0085] Preparation of target bacterial solution: Inoculate the strain into Columbia blood culture medium and culture overnight in a 37°C, 5% CO2 incubator for 15-20 hours. After the culture is completed, pick 20-50 typical colonies and transfer them to the culture medium plate for 4-6 hours. Scrape the bacterial moss into the buffer solution and mix well. Count the bacteria by turbidimetry and dilute the bacterial suspension to a final concentration of about 4000 CFU / ml.

[0086] Sample testing: The serum to be tested is incubated at 56°C for 30 minutes for inactivation. The serum to be tested is diluted 2 times in a row, and an equal volume of a mixture of active complement and working bacterial solution is added to the sample wells. At the same time, inactivated complement and active complement controls are set up to observe whether the complement has natural bactericidal activity and whether the target bacteria grow well. After oscillation and mixing, place it in a 37°C incubator and incubate for 1-2 hours. Then take out the culture plate, add 50μl of color development culture medium to each well, place the culture plate in a 37°C, 5% CO2 incubator and observe the results after incubation overnight. 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.

[0087] Target bacteria are S70112 and S357

[0088] Serum bactericidal assay (SBA) was performed with the mouse sera and rabbit complement of each group after three immunizations to detect the level of neutralizing antibodies.

[0089] like Figure 1 and Figure 2 The results showed that 1162 from different strains and different antigens could produce higher bactericidal activity after fusion.

[0090] Example 5 Study on the immunogenicity of fusion protein

[0091] After the fusion protein obtained in Example 2 was purified, 6-8 week old female CD1 mice were randomly divided into 6 mice per group and intraperitoneally injected with

[0092] Serum Bactericidal Assay (SBA)

[0093] Preparation of target bacterial solution: Inoculate the strain into Columbia blood culture medium and culture overnight in a 37°C, 5% CO2 incubator for 15-20 hours. After the culture is completed, pick 20-50 typical colonies and transfer them to the culture medium plate for 4-6 hours. Scrape the bacterial moss into the buffer solution and mix well. Count the bacteria by turbidimetry and dilute the bacterial suspension to a final concentration of about 4000 CFU / ml.

[0094] Sample testing: The serum to be tested is incubated at 56°C for 30 minutes for inactivation. The serum to be tested is diluted 2 times in a row, and an equal volume of a mixture of active complement and working bacterial solution is added to the sample wells. At the same time, inactivated complement and active complement controls are set up to observe whether the complement has natural bactericidal activity and whether the target bacteria grow well. After oscillation and mixing, place it in a 37°C incubator and incubate for 1-2 hours. Then take out the culture plate, add 50μl of color development culture medium to each well, place the culture plate in a 37°C, 5% CO2 incubator and observe the results after incubation overnight. 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.

[0095] The target bacteria are S1020 and S1229.

[0096] Serum bactericidal assay (SBA) was performed with the mouse sera and rabbit complement of each group after three immunizations to detect the level of neutralizing antibodies.

[0097] like Figure 3 and Figure 4 The results showed that 1162 and V2-Fhbp from different strains could produce high bactericidal activity in type 2 target bacteria after fusion, while the single fhbp and 2091-fhbp fusion proteins had almost no bactericidal activity. In the single-component verification, 1162 was shown to be able to improve the bactericidal activity of fhbp, and there was no significant difference in the effect of 1162 from different strains on the bactericidal activity of the fused type 2 Fhbp.

[0098] Example 6 Study on the immunogenicity of fusion protein

[0099] After the fusion protein obtained in Example 3 was purified, 6-8 week old female CD1 mice were randomly divided into 6 mice per group and intraperitoneally injected with

[0100] Serum Bactericidal Assay (SBA)

[0101] Preparation of target bacterial solution: Inoculate the strain into Columbia blood culture medium and culture overnight in a 37°C, 5% CO2 incubator for 15-20 hours. After the culture is completed, pick 20-50 typical colonies and transfer them to the culture medium plate for 4-6 hours. Scrape the bacterial moss into the buffer solution and mix well. Count the bacteria by turbidimetry and dilute the bacterial suspension to a final concentration of about 4000 CFU / ml.

[0102] Sample testing: The serum to be tested is incubated at 56°C for 30 minutes for inactivation. The serum to be tested is diluted 2 times in a row, and an equal volume of a mixture of active complement and working bacterial solution is added to the sample wells. At the same time, inactivated complement and active complement controls are set up to observe whether the complement has natural bactericidal activity and whether the target bacteria grow well. After oscillation and mixing, place it in a 37°C incubator and incubate for 1-2 hours. Then take out the culture plate, add 50μl of color development culture medium to each well, place the culture plate in a 37°C, 5% CO2 incubator and observe the results after incubation overnight. 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.

[0103] The target bacteria are S1563 and S1549.

[0104] Serum bactericidal assay (SBA) was performed with the mouse sera and rabbit complement of each group after three immunizations to detect the level of neutralizing antibodies.

[0105] Figure 5 and Figure 6 The results showed that 1162 and V3-Fhbp from different strains could produce high bactericidal activity in type 3 target bacteria after fusion, while the single fhbp and 2091-fhbp fusion proteins had almost no bactericidal activity. In the single-component verification, 1162 was shown to be able to improve the bactericidal activity of fhbp, and there was no significant difference in the effect of 1162 from different strains on the bactericidal activity of type 3 Fhbp after fusion.

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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