Broad-spectrum multi-subunit vaccine for preventing group B meningococcus infection and application of broad-spectrum multi-subunit vaccine

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a broad-spectrum multi-subunit vaccine for preventing serogroup B meningococcus infection and application of the broad-spectrum multi-subunit vaccine for preventing serogroup B meningococcus infection. A neisseria heparin binding antigen NHBA; a pile protein; the chimeric antigen is obtained by taking a complement regulatory factor H binding protein fHbp with a signal peptide removed part as a skeleton and carrying out fusion expression with a variable region of an additional antigen Opa; an App protein; an immunoglobulin A (IgA) protein; and an adjuvant CpG. The vaccine is efficient, broad in spectrum and low in price, meanwhile, the vaccine adopts a mucosal immunization way, tissue damage and local side effects are avoided, and the vaccine is easy and convenient to use and easy to popularize.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular relates to a broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection and a use thereof. Background Art

[0002] Neisseria meningitidis is a Gram-negative bacterium that can cause respiratory infectious diseases mainly including cerebrospinal meningitis and bacteremia, also known as epidemic cerebrospinal meningitis (abbreviated as meningococcal meningitis). Meningitis occurs in people of all ages, with the highest incidence in infants under 1 year old. Humans are the only host of Neisseria meningitidis, which exists in the upper respiratory tract of about 10% of adults. It is mainly transmitted through the respiratory tract, crosses the mucosa and blood-brain barrier, and forms invasive meningitis (IMD). Invasive meningitis can cause long-term disability, and the mortality rate in severe cases can reach 10%-20%.

[0003] The capsular polysaccharide of Neisseria meningitidis is its main virulence factor. It can be divided into 12 serogroups according to the different chemical components of its polysaccharide. Most epidemic invasive meningitis is caused by 6 serogroups: A, B, C, W, Y and X. However, the distribution of these serogroups is also affected by the geographical environment and vaccination status. Currently, there are multivalent polysaccharide vaccines for A, C, W, Y and X. However, the chemical composition of group B meningitis capsular polysaccharide is similar to polysialic acid in human glycoproteins, and its immunogenicity and safety are poor, so it cannot be used as a vaccine component.

[0004] The earliest vaccine used to prevent group B meningitis was the detergent-extracted outer membrane vesicle (OMV) vaccine. The main antigen of the OMV vaccine is the porin (PorA). PorA exhibits a high degree of antigenic variability, which limits cross-immune protection against different strains. The non-conservatism of PorA is a challenge for using OMV as a broad-spectrum vaccine. Vaccines with a single recombinant protein component are difficult to achieve good immune effects, and multi-component vaccines have become a development trend, bringing new directions to the research of group B meningococcal vaccines.

[0005] Strain fHbp is one of the main virulence factors of Neisseria meningitidis. It binds to factor H (fH) to allow the pathogen to avoid the alternative complement pathway and survive in human blood. The loss of the fHbp gene results in the failure of fH binding, and the survival ability of the strain in human serum is reduced. Strains carrying different fHbp variants can bind to fH, and the binding ability is related to the fHbp expression level (7,8). At the same time, fHbp is also the main component of two approved type B meningococcal vaccines. Generally speaking, fHbp as an immunogen can induce bactericidal antibodies against strains of the same subfamily and has cross-immune protection.

[0006] Opa (neisserial colony opacity-associated protein adhesins) is expressed during the infection process of pathogens and binds to CEACAMs receptors. It plays an important role in the colonization and infection of pathogens in respiratory epithelial cells and is one of the main virulence factors of Neisseria meningitidis.

[0007] Neisseria heparin binding antigen (NHBA or GNA2132) is a surface-exposed lipoprotein unique to Neisseria. Analysis of the NHBA gene sequence has reported more than 400 different NHBA polypeptide variants (1). The NHBA protein consists of approximately 420-490 amino acids. The N-terminal amino acid sequence of the NHBA protein has approximately 250 residues with large sequence differences, while the C-terminal is composed of approximately 180 amino acids, forming 8 β-folded domains, which are highly conserved. The serum antibodies of mice immunized with recombinant NHBA can bind to the surface of different strains of Neisseria meningitidis and induce complement-mediated bactericidal activity. At the same time, it can passively protect young mice from bacteremia, indicating that it can provide cross-immunity protection against different strains and is one of the candidate antigens for recombinant protein vaccines.

[0008] The Chinese invention CN202011644037.X specification records a Neisseria meningitidis vaccine, which includes bacterial ghosts and factor H binding protein (fHBP). The vaccine ingredients in this invention are simple, and it is difficult to achieve a broad-spectrum immune effect. There is currently a lack of broad-spectrum vaccines for the prevention of group B meningococcal infection, and there are many technical difficulties in developing a broad-spectrum multi-subunit vaccine suitable for group B meningococcal infection. Therefore, it is urgent to develop a broad-spectrum multi-subunit vaccine for the prevention of group B meningococcal infection. Summary of the invention

[0009] The present invention provides a broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection and its use. The vaccine utilizes conserved antigens of multiple group B meningococci to mucosally immunize mice, effectively induces Th17 and antibody responses in mice, effectively prevents the prevalence of group B meningococci, and provides a wide range of protection against group B meningococci. At the same time, the vaccine adopts the mucosal immunization approach, has the characteristics of no tissue damage, no local side effects, and is easy to use, and is easy to promote and use.

[0010] A broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection in the present invention is characterized in that the active ingredients of the vaccine are composed of component A, component B, component C, component D, component E, component F and component G;

[0011] The component A is complement regulatory factor H binding protein fHbp, a fusion protein having the full-length or partial amino acid sequence of fHbp, a protein conjugated with the full-length or partial amino acid sequence of fHbp and an adjuvant protein, a complex connected with the full-length or partial amino acid sequence of fHbp and a polysaccharide, or a DNA expression vector carrying the full-length or partial encoding gene of fHbp;

[0012] The component B is NHBA, a fusion protein having the full or partial amino acid sequence of the NHBA, a protein conjugated with the full or partial amino acid sequence of the NHBA and an adjuvant protein, a complex connected with the full or partial amino acid sequence of the NHBA and a polysaccharide, or a DNA expression vector carrying the full or partial coding gene of the NHBA;

[0013] The component C is fHbp-Opa (HV2), a fusion protein having the full-length or partial amino acid sequence of fHbp-Opa (HV2), a protein conjugated with the full-length or partial amino acid sequence of fHbp-Opa (HV2) and an adjuvant protein, a complex of the full-length or partial amino acid sequence of fHbp-Opa (HV2) and a polysaccharide, or a DNA expression vector carrying the full-length or partial encoding gene of fHbp-Opa (HV2);

[0014] The component D is pile, a fusion protein having the full-length or partial amino acid sequence of pile, a protein conjugated with the full-length or partial amino acid sequence of pile and an adjuvant protein, a complex connected with the full-length or partial amino acid sequence of pile and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of pile;

[0015] The component E is App, a fusion protein having the full or partial amino acid sequence of App, a protein conjugated with the full or partial amino acid sequence of App and an adjuvant protein, a complex connected with the full or partial amino acid sequence of App and a polysaccharide, or a DNA expression vector carrying the full or partial coding gene of App;

[0016] The component is IgA protease, a fusion protein having the full-length or partial amino acid sequence of the IgA protease, a protein conjugated with the full-length or partial amino acid sequence of the IgA protease and an adjuvant protein, a complex connected with the full-length or partial amino acid sequence of the IgA protease and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of the IgA protease;

[0017] The component heptadjuvant is CpG;

[0018] The function of the vaccine is to provide immune protection against group B meningococci;

[0019] The mass ratio of component A, component B, component C, component D, component E, component H and component G is 1:1:1:1:1:1:1:1.

[0020] In a preferred embodiment of the present invention, the component A is fHbp, having the amino acid sequence of the fHbp after removing the signal peptide, specifically as shown in SEQ ID NO: 3;

[0021] The component B is NHBA, having the amino acid sequence of NHBA after removing the signal peptide and the cell wall anchoring domain, specifically as shown in SEQ ID NO:6;

[0022] The component C is fHbp-Opa(HV2)-GST, having the amino acid sequence of the fHbp minus the signal peptide and the Opa(HV2) multivariable region, and a GST tag, as specifically shown in SEQ ID NO:14;

[0023] The component D is pile, having the amino acid sequence of pile after removing the signal peptide, specifically as shown in SEQ ID NO: 17;

[0024] The component E is App, having the amino acid sequence of the binding domain of App, specifically as shown in SEQ ID NO: 20;

[0025] The component is IgA Protease, having the amino acid sequence of IgA Protease after removing the signal peptide and the cell wall anchoring domain, specifically as shown in SEQ ID NO:23.

[0026] The vaccine can be used by inhalation into the lungs, inhalation into the nose, oral administration, subcutaneous injection, intradermal injection, genital tract injection, and anal injection.

[0027] In a preferred embodiment, the vaccine is administered by nasal inhalation.

[0028] In a preferred embodiment, the dosage of component A, component B, component C, component D, component E, component F and component G in the vaccine is 10 μg / time; the number of immunizations is 3 times, with an interval of one week.

[0029] Another aspect of the present invention provides use of any of the above vaccines in the preparation of a medicament for preventing group B meningococcal infection.

[0030] The functions of the vaccine are as follows (I) or (II) or (III): (I) highly effective in inducing IgG in serum and secretory IgA in mucosa; (II) inducing specific T cell responses mainly composed of Th17 in lungs and spleen; (III) mucosal immunity can induce neutralizing antibodies with high bactericidal activity against clinical strains. The clinical strain of group B meningitis is 2015-16#.

[0031] The group B meningococci include different epidemic strains of group B meningococci.

[0032] The prevention of group B meningococcal infection is the prevention of human infection caused by different group B meningococci.

[0033] The human infections are infections of the respiratory system, digestive system, urinary system, reproductive system, skin and blood circulation system.

[0034] Th17 cells are a new type of T cell discovered in recent years. The immune memory Th17 cells produced after mucosal immunization can quickly migrate to the infected mucosal site and play an important role in resisting mucosal bacterial infection. Unlike the immunity provided by B cells, the immunity provided by T cells has the characteristics of tolerance to antigen variation and can provide cross-immune protection against allotypic bacteria. It is the theoretical basis for the construction of new vaccines. The cytokine IL-17 released after Th17 cell activation activates neutrophils and macrophages to phagocytose and effectively kill pathogens that enter the body. The selected vaccine components can induce an immune response dominated by Th17 cells.

[0035] Antigens such as fHbp and NHBA are located on the surface of group B meningococci. Antibodies against these virulence factors can specifically neutralize the pathogenic effects of virulence factors, or play a role in resisting group B meningococcal infection through the antibody-dependent bactericidal activity received by complement.

[0036] After long-term and in-depth research, the inventors found that the combined use of fHbp, NHBA, fHbp-Opa (HV2), pile, App and IgAprotease can not only induce neutralizing antibodies, but also activate the response of Th17 cells, thereby playing a more effective protective role against group B meningococci. Therefore, the combined use of fHbp, NHBA, fHbp-Opa (HV2), pile, App and IgAprotease can have a broad-spectrum bactericidal effect on group B meningococci. Mucosal adjuvants can promote the uptake of vaccine subunits by antigen-processing cells in the mucosal area, significantly enhance their immunogenicity and immune effects, and avoid local tissue reactions caused by adjuvants in muscle or subcutaneous immunization.

[0037] The antigens used in the multi-linked recombinant protein vaccine (hereinafter referred to as Men.B-V6) of the present invention are ubiquitous in Neisseria, with a homology of more than 90%. By making full use of the universality and homology of various antigens in Neisseria, mucosal immunity, and mucosal immune adjuvants to enhance the immunogenicity of antigens, the activation of Th17 cells and the level of antibodies are significantly improved, thereby preventing the colonization of pathogens and rapidly clearing pathogens, and having a protective effect on group B meningococcal Neisseria meningitidis strains, and having the advantages of high efficiency, broad spectrum, and low price. At the same time, the vaccine provided by the present invention adopts the mucosal immunization route, has the characteristics of no tissue damage, no local side effects, and is easy to use, and is easy to promote and use; BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.

[0039] Figure 1 The polyacrylamide gel electrophoresis diagrams during the preparation of fHbp, NHBA, fHbp-Opa (HV2), pile, App, and IgA Protease proteins;

[0040] Figure 2 This is a graph showing the antigen-specific serum IgG response results produced by mice immunized with Men.B-V6 in Example 2;

[0041] Figure 3 This is a graph showing the results of the antigen-specific secretory IgA response produced by mice immunized with Men.B-V6 in Example 2;

[0042] Figure 4 This is a graph showing the results of the Men.B-V6 immunization inducing mice to produce antigen-specific Th17 immune cell responses in Example 3. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with specific embodiments:

[0044] To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0045] CpG: References Iho S, Maeyama J, Suzuki F. CpG oligodeoxynucleotides asmucosal adjuvants. Hum Vaccin Immunother. 2015; 11(3): 755-60.

[0046] Vector pET28a(+): Novagen, Cat.No.69846-3. Vector pGEX6P-1: Miaoling Biotechnology, Cat.P0005. Vector pCold-SUMO: Haiji Biotechnology Co., Ltd., Cat.C1801M. Escherichia coli BL21(DE3): purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number: CD601-02. (ICR) IGS mice: purchased from Charles River; STRAIN CODE: 201.

[0047] Example 1

[0048] Preparation of complement binding protein (fHbp)

[0049] 1. Using the genomic DNA of the clinical strain of group B meningitis 2015-16# as a template, PCR amplification was performed with the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0050] F1: 5'-CATCATATGGTCGCCGCCGACATCGGCGC-3';

[0051] R1: 5'-GATCTCGAGCTACTGTTTGCCGGCGATGC-3'.

[0052] 2. Double digest the PCR amplification product of step 1 with restriction endonucleases NdeI and XhoI, and recover the digestion product.

[0053] 3. Double-digest the vector pET28a(+) with restriction endonucleases NdeI and XhoI to recover the vector backbone of about 5400 bp.

[0054] 4. Connect the digestion product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid pET28a-fHbp. According to the sequencing results, the recombinant plasmid pET28a-fHbp is described as follows: a double-stranded DNA molecule shown in sequence 1 of the sequence table from nucleotides 28-274 at the 5' end is inserted between the NdeI and XhoI restriction sites of the vector pET28a(+). The inserted double-stranded DNA molecule forms a fusion gene shown in sequence 2 of the sequence table with part of the DNA on the vector backbone, and expresses the fusion protein shown in sequence 3 of the sequence table.

[0055] 5. Introduce the recombinant plasmid pET28a-fHbp into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0056] 6. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 50 μg / ml kanamycin, and cultured at 37°C and 220 rpm with shaking until OD560nm = 0.6. IPTG was added for induction at a concentration of 40 μg / ml, and cultured at 37°C and 220 rpm with shaking for 4 hours.

[0057] 7. Take the culture system of step 6, centrifuge at 4°C and 3000 rpm for 20 minutes to collect the bacterial precipitate, suspend the bacterial precipitate with pH 7.4 PBS buffer and perform ultrasonic disruption (power is 200 W, work for 4 seconds and rest for 6 seconds, cycle 99 times), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0058] 8. Load the supernatant obtained in step 7 onto GE Ni Sepharose 6 Fast Flow, first use solution I (pH 7.4, solvent is water, containing 20mM Na2HPO4 and 500mM NaCl) for 10 column volumes to remove impurities, then use solution II (pH 7.4, solvent is water, containing 200mM imidazole, 20mM Na2HPO4 and 500mM NaCl) for 2 column volumes to elute the target protein, collect the post-column solution when eluting with solution II, and name it fHbp solution.

[0059] 10 mg of protein with a purity of more than 90% can be obtained per liter of the culture system in step 6.

[0060] Preparation of NHBA

[0061] 1. The genomic DNA of the clinical strain of group B meningitis 2015-16# was used as a template, and PCR amplification was performed with the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0062] F1: 5'-CCGCCATGGCGAATGGCGGTAGCAATTTT-3';

[0063] R1: 5'-CCGCTCGAGTGCGGCCGCAAGCTTGTCG-3'.

[0064] 2. Double digest the PCR amplification product of step 1 with restriction endonucleases NcoI and XhoI, and recover the digestion product.

[0065] 3. Double-digest the vector pET28a(+) with restriction endonucleases NcoI and XhoI to recover the vector backbone of about 6000 bp.

[0066] 4. Connect the digestion product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid pET28a-NHBA. According to the sequencing results, the recombinant plasmid pET28a-NHBA is described as follows: a double-stranded DNA molecule shown in sequence 4 of the sequence table from nucleotides 397-1284 at the 5' end is inserted between the NcoI and XhoI restriction sites of the vector pET28a(+). The inserted double-stranded DNA molecule forms a fusion gene shown in sequence 5 of the sequence table with part of the DNA on the vector backbone, and expresses the fusion protein shown in sequence 6 of the sequence table.

[0067] 5. Introduce the recombinant plasmid pET28a-NHBA into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0068] 6. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm with shaking until OD560nm = 0.6, then IPTG was added for induction at a concentration of 40 μg / ml, and cultured at 37°C and 220 rpm with shaking for 4 hours.

[0069] 7. Take the culture system of step 6, centrifuge at 4°C and 6000 rpm for 10 minutes to collect the bacterial precipitate, suspend the bacterial precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power is 200 W, work for 4 seconds and rest for 8 seconds, cycle 99 times), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0070] 8. Load the supernatant obtained in step 7 onto GE Ni Sepharose 6 Fast Flow, first use solution I (pH 7.4, solvent is water, containing 20mM Na2HPO4 and 500mM NaCl) for 10 column volumes to remove impurities, then use solution II (pH 7.4, solvent is water, containing 200mM imidazole, 20mM Na2HPO4 and 500mM NaCl) for 2 column volumes to elute the target protein, collect the post-column solution when eluting with solution II, and name it NHBA solution.

[0071] 24 mg of protein with a purity of more than 90% can be obtained per liter of the culture system in step 6.

[0072] Preparation of fHbp-Opa(HV2)

[0073] 1. Opa, as a transmembrane protein, has a more complex structure and 16 transmembrane regions. Soluble proteins are also not available. The virulence factor fHbp is used as the molecular scaffold of the extracellular soluble region of Opa. The extracellular soluble region VR2 loop of Opa and fHbp constitute a chimeric antigen, and the chimeric antigen can produce immune protection. It has an amino acid sequence that deletes the signal peptide of fHbp, deletes its 240th aspartic acid, and inserts the extracellular soluble region VR2 loop of Opa. The genomic DNA of the clinical strain of group B meningococcus 2015-16# was used as a template, and the primer pair composed of F1 and R1 was used for PCR amplification to obtain the PCR amplification product, and the HV2 sequence was obtained by sequencing:

[0074] F1: 5′-TGAAAACCGTAGCCTACGGACACGTTAGGCATCA-3′

[0075] R1: 5′-CAGCATCCGCCGCTTGGGTGGCATACGCCATATCG-3′

[0076] 2. Using the genomic DNA of the standard strain of group B meningococcus MC58 as a template, perform PCR amplification with the primer pair consisting of F1 and R1 to obtain the PCR amplification product and the fHbp skeleton:

[0077] F1: 5′-TGTTCCAGGGGCCCCTGGGATCCGTCGCCGCCGACATCGGT-3′

[0078] R1: 5′-ATATCGGCCTTGCCGCCAAGCAACTCGAGCGGCCGCATCGTG-3′

[0079] 4. Double-digest the vector pGEX6P-1 with restriction endonucleases BamHI and XhoI.

[0080] 5. Homologous recombination of the digestion products of steps 2 and 3 and the vector backbone of step 4 was performed to obtain the recombinant plasmid pGEX6P-1-fHbp-Opa (HV2). According to the sequencing results, the recombinant plasmid pGEX6P-1-fHbp (Opa) was described as follows: the double-stranded DNA molecules shown in sequence 7 and sequence 8 of the sequence table were inserted between the BamHI and XhoI restriction sites of the vector pGEX6P-1. The inserted double-stranded DNA molecules and part of the DNA on the vector backbone formed the fusion gene shown in sequence 9 of the sequence table, and expressed the fusion protein shown in sequence 10 of the sequence table.

[0081] 6. Introduce the recombinant plasmid pGEX6P-1-fHbp-Opa (HV2) into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0082] 7. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37°C and 220 rpm with shaking until OD560nm = 0.6, then IPTG was added for induction, and cultured at 37°C and 220 rpm with shaking for 4 hours.

[0083] 8. Take the culture system of step 7, collect the bacterial precipitate by centrifugation at 4°C and 3000 rpm for 20 minutes, suspend the bacterial precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power is 200 W, work for 4 seconds and rest for 8 seconds, cycle 99 times), then centrifuge at 12000 rpm for 20 minutes and collect the supernatant.

[0084] 9. Load the supernatant obtained in step 8 onto GST rap HP, first elute with equilibration buffer for 10 column volumes to remove impurities, and then elute with eluent to obtain the target protein.

[0085] 10. The eluate from step 9 was further purified by AKTA purifier to obtain a relatively pure target protein.

[0086] Preparation of pile

[0087] 1. Using the genomic DNA of group B meningococcal clinical strain 2015-16# as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0088] F1: 5'-CCGCCATGGCCTGCTTATCAAGACTACACAGC-3';

[0089] R1: 5'-CCGCTCGAGGCTGCCAGATGAAGCGTCGC-3'.

[0090] 2. Double digest the PCR amplification product of step 1 with restriction endonucleases NcoI and XhoI, and recover the digestion product.

[0091] 3. Double-digest the vector pCold-sumo(+) with restriction endonucleases NcoI and XhoI to recover the vector backbone of about 6000 bp.

[0092] 4. Connect the enzyme digestion product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid pCold-sumo-pile. According to the sequencing results, the recombinant plasmid pcold-sumo-pile is described as follows: a double-stranded DNA molecule of nucleotides 85-504 from the 5' end of sequence 11 in the sequence list is inserted between the NcoI and XhoI restriction sites of the vector pCold-sumo(+). The inserted double-stranded DNA molecule and part of the DNA on the vector backbone form a fusion gene shown in sequence 12 in the sequence list, and express the fusion protein shown in sequence 13 in the sequence list.

[0093] 5. Introduce the recombinant plasmid pCold-sumo-pile into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0094] 6. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 50 μg / mL ampicillin, and cultured at 37°C and 220 rpm with shaking until OD560nm = 0.6. IPTG was added for induction at a concentration of 40 μg / ml, and cultured at 37°C and 220 rpm with shaking for 4 hours.

[0095] 7. Take the culture system of step 6, centrifuge at 4°C and 3000 rpm for 20 minutes to collect the bacterial precipitate, suspend the bacterial precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power is 200 W, work for 4 seconds and rest for 8 seconds, cycle 99 times), then centrifuge at 12000 rpm for 20 minutes and collect the supernatant.

[0096] 8. Load the supernatant obtained in step 7 onto GE Ni Sepharose 6 Fast Flow, first use solution I (pH 7.4, solvent is water, containing 20mM Na2HPO4 and 500mM NaCl) for 10 column volumes to remove impurities, then use solution II (pH 7.4, solvent is water, containing 200mM imidazole, 20mM Na2HPO4 and 500mM NaCl) for 2 column volumes to elute the target protein, collect the post-column solution when eluting with solution II, and name it pile solution.

[0097] 12 mg of protein with a purity of more than 90% can be obtained per liter of the culture system in step 6.

[0098] Preparation of App

[0099] 1. Using the genomic DNA of group B meningococcal clinical strain 2015-16# as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0100] F1: 5′-CATGCCATGGGGATCCGAAAAAGACAACG-3′

[0101] R1: 5′-CCGCTCGAGCTCGAGGTCGACGCCTAATT-3′

[0102] 2. Double digest the PCR amplification product of step 1 with restriction endonucleases NcoI and XhoI, and recover the digestion product.

[0103] 3. Double-digest the vector pET28a with restriction endonucleases NcoI and XhoI.

[0104] 4. Connect the digestion product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid pET28a-App. According to the sequencing results, the recombinant plasmid pET28a-App is described as follows: a double-stranded DNA molecule of nucleotides shown in sequence 14 of the sequence list from the 3229-4362th position of the 5' end is inserted between the NcoI and XhoI restriction sites of the vector pET28a-App. The inserted double-stranded DNA molecule and part of the DNA on the vector backbone form a fusion gene shown in sequence 15 of the sequence list, and express the fusion protein shown in sequence 16 of the sequence list.

[0105] 5. Introduce the recombinant plasmid pET28a-App into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0106] 6. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37°C and 220 rpm until OD560nm = 0.6. After the culture medium was cooled to 15°C, IPTG was added for induction, and cultured at 15°C and 220 rpm for 24 hours.

[0107] 7. Take the culture system of step 6, centrifuge at 4°C and 3000 rpm for 20 minutes to collect the bacterial precipitate, suspend the bacterial precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power is 200 W, work for 4 seconds and rest for 8 seconds, cycle 99 times), then centrifuge at 12000 rpm for 20 minutes and collect the supernatant.

[0108] 8. Load the supernatant obtained in step 7 onto Ni Sepharose 6 Fast Flow, first elute with equilibration buffer for 10 column volumes to remove impurities, then elute with 0, 20, 50, 100, and 150 mM imidazole to obtain the target protein, and collect the post-column solution when eluted with 150 mM imidazole.

[0109] 9. The eluate from step 8 was further purified by AKTA purifier to obtain a relatively pure target protein.

[0110] Preparation of IgA Protease

[0111] 1. Using the genomic DNA of group B meningococcal clinical strain 2015-16# as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0112] F1: 5′-CATGCCATGGGCATTGGTCAGAGACGATGTCG-3′

[0113] R1: 5′-ACGCCTCGAG GTTCTCGGCATAAGGATTGTACAAT-3′

[0114] 2. Double digest the PCR amplification product of step 1 with restriction endonucleases NcoI and XhoI, and recover the digestion product.

[0115] 3. Double-digest the vector pET28a with restriction endonucleases NcoI and XhoI.

[0116] 4. Connect the digestion product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid pET28a-IgAProtease. According to the sequencing results, the recombinant plasmid pET28a-IgA Protease is described as follows: a double-stranded DNA molecule of nucleotides 82-2904 from the 5' end of sequence 17 of the sequence list is inserted between the NcoI and XhoI restriction sites of the vector pET28a. The inserted double-stranded DNA molecule and part of the DNA on the vector backbone form a fusion gene shown in sequence 18 of the sequence list, and express the fusion protein shown in sequence 19 of the sequence list.

[0117] 5. Introduce the recombinant plasmid pET28a-IgA Protease into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0118] 6. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 100 μg / mL kanamycin, and cultured at 37°C and 220 rpm until OD560nm = 0.6. After the culture medium was cooled to 15°C, IPTG was added for induction, and cultured at 15°C and 220 rpm for 24 hours.

[0119] 7. Take the culture system of step 6, centrifuge at 4°C and 6000 rpm for 10 minutes to collect the bacterial precipitate, suspend the bacterial precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power is 200 W, work for 4 seconds and rest for 8 seconds, cycle 99 times), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0120] 8. Load the supernatant obtained in step 7 onto Ni Sepharose 6 Fast Flow, first elute with equilibration buffer for 10 column volumes to remove impurities, then elute with 0, 20, 50, 100, and 150 mM imidazole to obtain the target protein, and collect the post-column solution when eluted with 150 mM imidazole.

[0121] 9. The eluate from step 8 was further purified by AKTA purifier to obtain a relatively pure target protein.

[0122] The polyacrylamide gel electrophoresis images of the above preparation process of fHbp, NHBA, fHbp-Opa (HV2), pile, App, and IgAprotease proteins are shown in the attached figure. Figure 1 As shown. Among them, Men.B recombinant proteins are fHbp (Mw≈34kd), NHBA (Mw≈30kd), fHbp-Opa(HV2)-GST (Mw≈59kd), pile (Mw≈27kd), App (Mw≈42kd), and IgA Protease (Mw≈107kd).

[0123] Example 2

[0124] To induce antibody response in mice by Men.B-V6 immunization, 6-8 week old female C57BL / 6 mice were randomly divided into two groups and treated as follows:

[0125] CpG group: CpG solution was instilled into the nasal cavity on the 1st, 7th and 14th days of the experiment;

[0126] Men.B-V6 group: On the first, seventh and 14th day of the experiment, the vaccine solution was dripped into the nasal cavity (the vaccine solution was obtained by mixing the six recombinant proteins prepared in Example 1 with the CpG solution, and each mouse was given 10 μg of each of the six recombinant proteins and 10 μg of CpG each time). On the 21st day of the experiment, blood and oral lavage fluid were collected from the tail of the anesthetized mice, and serum IgG and oral lavage fluid IgA were measured by ELIAS. The results are shown in the attached Figure 2 and attached Figure 3 Compared with the CpG group, the Men.B-V6 group significantly induced antigen-specific serum IgG and secretory IgA.

[0127] Example 3

[0128] Men.B-V6 mucosal immunization induced Th17 cell immune response. Female C57BL / 6 mice aged 6-8 weeks were randomly divided into two groups and treated as follows:

[0129] CpG group: CpG solution was instilled into the nasal cavity on the 1st, 7th and 14th days of the experiment;

[0130] Men.B-V6 group: On the first, seventh and 14th day of the experiment, the vaccine solution was dripped into the nasal cavity (the vaccine solution was obtained by mixing the six recombinant proteins prepared in Example 1 with the CpG solution, and each mouse was given 10 μg of each of the six recombinant proteins and 10 μg of CpG each time). On the 21st day of the experiment, cells were extracted from spleen and lung tissues, and ELISPOT was used to measure Men.B-V6 antigen-specific IL-17+ cells. The results are shown in the attached Figure 4 Compared with the CpG group, the GBSV6 group significantly induced antigen-specific Th17 cells in the spleen and lung tissues, indicating that Men.B-V6 nasal immunization of mice induced Th17 cell responses.

[0131] Example 4

[0132] Complement-mediated bactericidal assay. To prepare mouse antiserum, Men.B-V6 mucosal immunization induced Th17 cell immune response. Female C57BL / 6 mice aged 6-8 weeks were randomly divided into groups and treated as follows:

[0133] Table 1. Formulation grouping of multi-component vaccine ingredients

[0134]

[0135] On the 1st day, 7th day and 14th day of the experiment, the vaccine solution of the control group (CpG adjuvant group) (the vaccine solution is obtained by mixing the 6 recombinant proteins prepared in Example 1 with the CpG solution according to the grouping in Table 1) was dripped into the nasal cavity to immunize the mice. On the 21st day of the experiment, the blood was collected by cutting off the tail and heat-inactivated at 56°C for 30 minutes before the test. The serum from the young rabbit was used as the complement source. The serum bactericidal titer was defined as the serum dilution corresponding to a 50% reduction in the remaining CFU compared with the pre-reaction after the bacteria were incubated with the reaction mixture for 60 minutes. The results are shown in Table 2:

[0136] Table 2 Group B meningitis clinical bacteria bactericidal test BC50 (Titer)

[0137]

[0138] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0139] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.

Claims

1. A broad-spectrum multi-subunit vaccine for preventing serogroup B meningococcal infection, characterized in that: The active ingredients of the vaccine include ingredient A, ingredient B, ingredient C, ingredient D, ingredient E, ingredient F, and ingredient G; The component A is complement regulatory factor H binding protein fHbp, a fusion protein having the full-length or partial amino acid sequence of fHbp, a protein conjugated with the full-length or partial amino acid sequence of fHbp and an adjuvant protein, a complex connected with the full-length or partial amino acid sequence of fHbp and a polysaccharide, or a DNA expression vector carrying the full-length or partial encoding gene of fHbp; The component B is NHBA, a fusion protein having the full or partial amino acid sequence of the NHBA, a protein conjugated with the full or partial amino acid sequence of the NHBA and an adjuvant protein, a complex connected with the full or partial amino acid sequence of the NHBA and a polysaccharide, or a DNA expression vector carrying the full or partial coding gene of the NHBA; The component C is fHbp-Opa (HV2), a fusion protein having the full-length or partial amino acid sequence of fHbp-Opa (HV2), a protein conjugated with the full-length or partial amino acid sequence of fHbp-Opa (HV2) and an adjuvant protein, a complex of the full-length or partial amino acid sequence of fHbp-Opa (HV2) and a polysaccharide, or a DNA expression vector carrying the full-length or partial encoding gene of fHbp-Opa (HV2); The component D is pile, a fusion protein having the full-length or partial amino acid sequence of pile, a protein conjugated with the full-length or partial amino acid sequence of pile and an adjuvant protein, a complex connected with the full-length or partial amino acid sequence of pile and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of pile; The component E is App, a fusion protein having the full or partial amino acid sequence of App, a protein conjugated with the full or partial amino acid sequence of App and an adjuvant protein, a complex connected with the full or partial amino acid sequence of App and a polysaccharide, or a DNA expression vector carrying the full or partial coding gene of App; The component is IgA protease, a fusion protein having the full-length or partial amino acid sequence of the IgA protease, a protein conjugated with the full-length or partial amino acid sequence of the IgA protease and an adjuvant protein, a complex connected with the full-length or partial amino acid sequence of the IgA protease and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of the IgA protease; The component heptadjuvant is CpG; The mass ratio of component A, component B, component C, component D, component E, component H and component G is 1:1:1:1:1:1:1:

1.

2. A broad-spectrum multi-subunit vaccine for preventing serogroup B meningococcal infection according to claim 1, characterized in that: The component A is fHbp, having the amino acid sequence of the fHbp after removing the signal peptide, specifically as shown in SEQ ID NO: 3; The component B is NHBA, having the amino acid sequence of NHBA after removing the signal peptide and the cell wall anchoring domain, specifically as shown in SEQ ID NO:6; The component C is fHbp-Opa(HV2)-GST, having the amino acid sequence of the fHbp minus the signal peptide and the Opa(HV2) multivariable region, and a GST tag, as specifically shown in SEQ ID NO:14; The component D is pile, having the amino acid sequence of pile after removing the signal peptide, specifically as shown in SEQ ID NO: 17; The component E is App, having the amino acid sequence of the binding domain of App, specifically as shown in SEQ ID NO: 20; The component is IgA Protease, having the amino acid sequence of IgA Protease after removing the signal peptide and the cell wall anchoring domain, specifically as shown in SEQ ID NO:

23.

3. A broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection according to claim 1 or 2, characterized in that: The vaccine can be used by inhalation into the lungs, inhalation into the nose, oral administration, subcutaneous injection, intradermal injection, genital tract injection, and anal injection.

4. A broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection according to claim 3, characterized in that: The vaccine is administered by nasal inhalation.

5. A broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection according to claim 4, characterized in that: The dosage of component A, component B, component C, component D, component E, component F and component G in the vaccine is 10 μg / time; the number of immunizations is 3 times, with an interval of one week.

6. Use of the vaccine according to any one of claims 1 to 5 in the preparation of a medicament for preventing group B meningococcal infection.

7. The use according to claim 6, characterized in that: The functions of the vaccine are as follows (I) or (II) or (III): (I) highly efficient inducing IgG in serum and secretory IgA in mucosa; (II) inducing specific T cell responses mainly composed of Th17 in lungs and spleen; and (III) mucosal immunity inducing neutralizing antibodies with high bactericidal activity against clinical strains.

8. The use according to claim 6, characterized in that: The group B meningococci include different epidemic strains of group B meningococci.

9. The use according to claim 6, characterized in that: The prevention of group B meningococcal infection is the prevention of human infection caused by different group B meningococci.

10. The use according to claim 9, characterized in that: The human infections are infections of the respiratory system, digestive system, urinary system, reproductive system, skin and blood circulation system.

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