Broad-spectrum multi-component subunit vaccine for preventing group B meningococcal infection and its uses

Abstract

The present invention belongs to the field of biomedical technologies, and particularly relates to a broad-spectrum multi-subunit vaccine for preventing Neisseria meningitidis serogroup B infection and its uses, including complement factor H-binding protein fHbp; Neisseria heparin-binding antigen NHBA; pile protein; a chimeric antigen obtained by removing the signal peptide part of complement factor H-binding protein fHbp as a backbone and fusing and expressing it with the variable region of an additional antigen Opa; App protein; IgA protease; and adjuvant CpG. The vaccine in the present invention is highly efficient, broad-spectrum and low-cost. At the same time, the vaccine adopts the route of mucosal immunity, has no tissue damage, no local side effects and is easy to use, and is easy to promote.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a broad-spectrum multi-component subunit vaccine for preventing Neisseria meningitidis serogroup B infection and its uses. Background Art

[0002] Neisseria meningitidis is a Gram-negative bacterium that can cause respiratory infectious diseases mainly characterized by cerebrospinal meningitis and bacteremia, also known as epidemic cerebrospinal meningitis (abbreviated as epidemic meningitis). Meningitis occurs in all age groups, 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, crossing the mucosal and blood-brain barriers to form invasive meningitis (IMD). Invasive meningitis can lead to long-term disabilities, and the severe fatality rate can reach 10%-20%.

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

[0004] The earliest vaccine used to prevent Neisseria meningitidis serogroup B is the detergent-extracted outer membrane vesicle (OMV) vaccine. The main antigen of the OMV vaccine is porin (PorA). PorA exhibits a high degree of antigenic variability, which limits cross-immune protection against different strains. The non-conservation 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 the development trend, bringing a new direction to the research of Neisseria meningitidis serogroup B vaccines.

[0005] Strain fHbp is one of the main virulence factors of Neisseria meningitidis. By binding to factor H (fH), the pathogen can avoid the alternative complement pathway and survive in human blood. The deletion of the fHbp gene leads to the failure of fH binding, and the survival ability of the strain in human serum is reduced. Strains carrying different fHbp variants can all bind fH, and the binding ability is related to the expression level of fHbp (7, 8). At the same time, fHbp is also the main component of two approved serogroup 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 pathogenic bacteria. It binds to the CEACAMs receptor and plays an important role in the colonization and infection of pathogenic bacteria in respiratory epithelial cells. It is one of the main virulence factors of Neisseria meningitidis.

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

[0008] Chinese invention CN202011644037.X describes a Neisseria meningitidis vaccine, which includes bacterial ghosts and factor H-binding protein (fHBP). The components of the vaccine in this invention are simple and it is difficult to achieve a broad-spectrum immune effect. Currently, there is still a lack of a broad-spectrum vaccine for preventing 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, there is an urgent need to develop a broad-spectrum multi-subunit vaccine for preventing 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. By using conserved antigens of multiple group B meningococci to immunize mice via mucosal route, it can effectively induce Th17 and antibody responses in mice, effectively prevent the epidemic of group B meningococci, provide broad protection against group B meningococci. At the same time, the vaccine uses the mucosal immunization route, which has the characteristics of no tissue damage, no local side effects, and easy 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 components of the vaccine are composed of component A, component B, component C, component D, component E, component F, and component G;

[0011] 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 in which the full-length or partial amino acid sequence of fHbp is conjugated to an adjuvant protein, a ligation complex of the full-length or partial amino acid sequence of fHbp and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of fHbp;

[0012] Component B is NHBA, a fusion protein having the full-length or partial amino acid sequence of NHBA, a protein in which the full-length or partial amino acid sequence of NHBA is conjugated to an adjuvant protein, a ligation complex of the full-length or partial amino acid sequence of NHBA and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of NHBA;

[0013] Component C is fHbp-Opa(HV2), a fusion protein having the full-length or partial amino acid sequence of fHbp-Opa(HV2), a protein in which the full-length or partial amino acid sequence of fHbp-Opa(HV2) is conjugated to an adjuvant protein, a ligation 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 coding gene of fHbp-Opa(HV2);

[0014] Component D is pile, a fusion protein having the full-length or partial amino acid sequence of pile, a protein in which the full-length or partial amino acid sequence of pile is conjugated to an adjuvant protein, a ligation complex of 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] Component E is App, a fusion protein having the full-length or partial amino acid sequence of App, a protein in which the full-length or partial amino acid sequence of App is conjugated to an adjuvant protein, a ligation complex of the full-length or partial amino acid sequence of App and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of App;

[0016] Component F is IgA protease, a fusion protein having the full-length or partial amino acid sequence of IgA protease, a protein in which the full-length or partial amino acid sequence of IgA protease is conjugated to an adjuvant protein, a ligation complex of the full-length or partial amino acid sequence of IgA protease and a polysaccharide, or a DNA expression vector carrying the full-length or partial coding gene of IgA protease;

[0017] Component G is the immune adjuvant CpG;

[0018] The function of the vaccine is to provide immune protection against Neisseria meningitidis serogroup B;

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

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

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

[0022] Component C is fHbp-Opa(HV2)-GST, which has the amino acid sequence after removing the signal peptide and the variable region of Opa(HV2) of fHbp, as well as a GST tag, specifically as shown in SEQ ID NO:14;

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

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

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

[0026] The usage methods of the vaccine include pulmonary inhalation, nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection, and anal injection.

[0027] In a preferred embodiment, the usage method of the vaccine is nasal inhalation.

[0028] In a preferred embodiment, the dosages of Component A, Component B, Component C, Component D, Component E, Component F, and Component G in the vaccine are 10 μg per administration; the number of immunizations is 3 times, with a one-week interval.

[0029] Another aspect of the present invention provides the use of the vaccine as described above in the preparation of a drug for preventing Neisseria meningitidis serogroup B infection.

[0030] The functions of the vaccine are as follows (I) or (II) or (III): (I) efficiently inducing IgG in serum and secretory IgA in mucosa; (II) inducing a specific T cell response dominated by Th17 in the lung and spleen; (III) mucosal immunity inducing neutralizing antibodies with high bactericidal activity against clinical strains. The clinical strain of Neisseria meningitidis serogroup B is 2015-16#.

[0031] The Neisseria meningitidis serogroup B includes different epidemic strains of Neisseria meningitidis serogroup B.

[0032] The prevention of Neisseria meningitidis serogroup B infection is to prevent human infections caused by different Neisseria meningitidis serogroup B.

[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 newly discovered type of T cells in recent years. The immune memory Th17 cells produced after mucosal immunity can rapidly migrate to the infected mucosal site and play an important role in anti-mucosal bacterial infection. Different from the immunity provided by B cells, the immunity provided by T cells has the characteristic of tolerating antigen variation, can provide cross-immune protection against allogeneic bacteria, and is the theoretical basis for the construction of new vaccines. The cytokine IL-17 released after Th17 cell activation activates neutrophils and macrophages to phagocytize, effectively killing the pathogenic bacteria entering 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 Neisseria meningitidis serogroup B. Antibodies against these virulence factors can specifically neutralize the pathogenic effects of virulence factors or play a role in anti-Neisseria meningitidis serogroup B infection through complement-mediated antibody-dependent bactericidal activity.

[0036] After long-term and in-depth research, the inventors found that the combined use of fHbp, NHBA, fHbp-Opa (HV2), pile, App, and IgA protease can not only induce neutralizing antibodies but also activate the response of Th17 cells, thus providing more effective protection against Neisseria meningitidis serogroup B. Therefore, the combined use of fHbp, NHBA, fHbp-Opa (HV2), pile, App, and IgA protease has a broad-spectrum bactericidal effect on Neisseria meningitidis serogroup B. Mucosal adjuvants can promote the uptake of vaccine subunits by antigen-processing cells at mucosal sites, significantly enhance their immunogenicity and immune effects, and at the same time avoid local tissue reactions caused by adjuvants in muscle or subcutaneous immunization.

[0037] The antigens used in the multi-component recombinant protein vaccine of the present invention (hereinafter referred to as Men.B-V6) 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 route immunization, and mucosal immune adjuvants to enhance the immunogenicity of antigens, the activation of Th17 cells and the level of antibodies are significantly improved, achieving the effects of preventing pathogen colonization and rapidly clearing pathogens, and having a protective effect on Neisseria meningitidis serogroup B strains, with the advantages of high efficiency, broad spectrum, and low cost. At the same time, the vaccine provided by the present invention adopts the mucosal immunization route, with the characteristics of no tissue damage, no local side effects, and simple 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 following will briefly introduce the drawings required for description in the embodiments.

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

[0040] Figure 2 Results diagram of antigen-specific serum IgG response induced by Men.B-V6 immunization in mice in Example 2;

[0041] Figure 3 Results diagram of antigen-specific secretory IgA response induced by Men.B-V6 immunization in mice in Example 2;

[0042] Figure 4 Results diagram of antigen-specific Th17 immune cell response induced by Men.B-V6 immunization in mice in Example 3. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0043] The following further elaborates on the present invention in combination with specific embodiments:

[0044] It is convenient to better understand the present invention, but does not limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from regular biochemical reagent stores unless otherwise specified. The quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged.

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

[0046] Vector pET28a(+): Novagen, Cat. No. 69846-3. Vector pGEX6P-1: Miaoling Biology, Cat. P0005. Vector pCold-SUMO: Haike Biotechnology Co., Ltd., Cat. C1801M. Escherichia coli BL21(DE3): Purchased from Beijing TransGen Biotech Co., Ltd., Product No.: 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 clinical strain 2015-16# of group B meningitis as a template, perform PCR amplification with the primer pair composed of F1 and R1 to obtain a 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 digested product.

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

[0054] 4. Ligate the digested product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid pET28a-fHbp. According to the sequencing results, the description of the recombinant plasmid pET28a-fHbp is as follows: A double-stranded DNA molecule shown by nucleotides at positions 28-274 from the 5’ end of Sequence 1 of the sequence list is inserted between the NdeI and XhoI restriction enzyme sites of the vector pET28a(+). The inserted double-stranded DNA molecule and a part of the DNA on the vector backbone form a fusion gene shown by Sequence 2 of the sequence list, and express a fusion protein shown by Sequence 3 of the sequence list.

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

[0056] 6. Inoculate the recombinant bacteria obtained in step 5 into LB liquid medium containing 50 μg / ml kanamycin, culture with shaking at 37 °C and 220 rpm. When OD560nm = 0.6, add IPTG to make its concentration 40 μg / ml, and continue to culture with shaking at 37 °C and 220 rpm 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 cell precipitate, suspend the cell precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power: 200 W, intermittent for 6 seconds every 4 seconds of working, 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's Ni Sepharose 6 Fast Flow. First, use Solution I (pH 7.4, solvent: water, containing 20 mM Na2HPO4 and 500 mM NaCl) for elution of 10 column volumes to remove impurity proteins, and then use Solution II (pH 7.4, solvent: water, containing 200 mM imidazole, 20 mM Na2HPO4 and 500 mM NaCl) for elution of 2 column volumes to obtain the target protein. Collect the solution after passing through the column during elution with Solution II, and name it fHbp solution.

[0059] 10 mg of protein with purity above 90% can be obtained from each liter of the culture system in step 6.

[0060] Preparation of NHBA

[0061] 1. Using the genomic DNA of the clinical strain of group B meningitis 2015-16# as a template, perform PCR amplification with the primer pair composed 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 the restriction endonucleases NcoI and XhoI, and recover the digested product.

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

[0066] 4. Ligate the digested product from Step 2 and the vector backbone from Step 3 to obtain the recombinant plasmid pET28a-NHBA. According to the sequencing results, the description of the recombinant plasmid pET28a-NHBA is as follows: A double-stranded DNA molecule shown by nucleotides at positions 397 - 1284 from the 5'-end of Sequence 4 in the Sequence Listing was inserted between the NcoI and XhoI digestion sites of the vector pET28a(+). The inserted double-stranded DNA molecule and a part of the DNA on the vector backbone form a fusion gene shown by Sequence 5 in the Sequence Listing, and express a fusion protein shown by Sequence 6 in the Sequence Listing.

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

[0068] 6. Inoculate the recombinant bacterium obtained in Step 5 into an LB liquid medium containing 50 μg / mL kanamycin, and culture it with shaking at 37 °C and 220 rpm. When OD560nm = 0.6, add IPTG to make its concentration 40 μg / ml, and continue culturing with shaking at 37 °C and 220 rpm for 4 hours.

[0069] 7. Take the culture system from Step 6, centrifuge at 4 °C and 6000 rpm for 10 minutes to collect the bacterial cell precipitate, suspend the bacterial cell precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power: 200 W, working for 4 seconds and then intermittent for 8 seconds, cycling 99 times), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0070] 8. Load the supernatant obtained in Step 7 onto Ni Sepharose 6 Fast Flow from GE. First, elute with Solution I (pH 7.4, solvent: water, containing 20 mM Na2HPO4 and 500 mM NaCl) for 10 column volumes to remove impurity proteins, then elute with Solution II (pH 7.4, solvent: water, containing 200 mM imidazole, 20 mM Na2HPO4 and 500 mM NaCl) for 2 column volumes to obtain the target protein. Collect the solution after passing through the column during the elution with Solution II, and name it NHBA solution.

[0071] 24 mg of protein with a purity above 90% can be obtained from each liter of the culture system in Step 6.

[0072] Preparation of fHbp-Opa(HV2)

[0073] 1. As a transmembrane protein, Opa has a more complex structure with 16 transmembrane regions and is also unable to obtain soluble protein. The virulence factor fHbp is used as a molecular scaffold for the extracellular soluble region of Opa. The extracellular soluble region VR2 loop of Opa and fHbp form a chimeric antigen, and the chimeric antigen can generate immune protection. It has the amino acid sequence of deleting the signal peptide of fHbp, while deleting the 240th aspartic acid and inserting the extracellular soluble region VR2 loop of Opa. Among them, using the genomic DNA of Neisseria meningitidis serogroup B clinical strain 2015-16# as a template, PCR amplification is carried out with the primer pair composed of F1 and R1 to obtain a PCR amplification product, and the HV2 sequence is obtained by sequencing:

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

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

[0076] 2. Using the genomic DNA of Neisseria meningitidis serogroup B standard strain MC58 as a template, PCR amplification is carried out with the primer pair composed of F1 and R1 to obtain a PCR amplification product and obtain the fHbp backbone:

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

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

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

[0080] 5. Homologous recombination of the enzyme digestion products in steps 2 and 3 and the vector backbone in step 4 to obtain the recombinant plasmid pGEX6P-1-fHbp-Opa(HV2). According to the sequencing results, the description of the recombinant plasmid pGEX6P-1-fHbp(Opa) is as follows: A double-stranded DNA molecule shown in sequences 7 and 8 of the sequence list is inserted between the BamHI and XhoI restriction enzyme sites of the vector pGEX6P-1. The inserted double-stranded DNA molecule and a part of the DNA on the vector backbone form a fusion gene shown in sequence 9 of the sequence list, and express a fusion protein shown in sequence 10 of the sequence list.

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

[0082] 7. Inoculate the recombinant bacteria obtained in step 5 into LB liquid medium containing 50 μg / mL kanamycin, culture with shaking at 37 °C and 220 rpm. When OD560nm = 0.6, add IPTG for induction, and continue culturing with shaking at 37 °C and 220 rpm for 4 hours.

[0083] 8. Take the culture system in step 7, centrifuge at 4 °C and 3000 rpm for 20 minutes to collect the cell precipitate. Suspend the cell precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power: 200 W, working for 4 seconds and intermittent for 8 seconds, cycling 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 the equilibration buffer for 10 column volumes to remove the impurity proteins, and then elute with the elution buffer to obtain the target protein.

[0085] 10. Further purify the eluate obtained in step 9 with AKTA purifier to obtain a relatively pure target protein.

[0086] Preparation of pile

[0087] 1. Using the genomic DNA of Neisseria meningitidis serogroup B clinical strain 2015 - 16# as a template, perform PCR amplification with the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

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

[0089] R1: 5’-CCGCTCGAGGCTGGCAGATGAAGCGTCGC-3’.

[0090] 2. Double digest the PCR amplification product in step 1 with restriction enzymes NcoI and XhoI, and recover the digested product.

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

[0092] 4. Ligate the 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 description of the recombinant plasmid pcold-sumo-pile is as follows: A double-stranded DNA molecule of nucleotides 85 to 504 from the 5' end of Sequence 11 in the Sequence List was inserted between the NcoI and XhoI digestion sites of the vector pCold-sumo(+). The inserted double-stranded DNA molecule and a part of the DNA on the vector backbone form a fusion gene shown in Sequence 12 of the Sequence List, and express a fusion protein shown in Sequence 13 of the Sequence List.

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

[0094] 6. Inoculate the recombinant bacterium obtained in step 5 into an LB liquid medium containing 50 μg / mL ampicillin, and culture it with shaking at 37 °C and 220 rpm. When OD560nm = 0.6, add IPTG to make its concentration 40 μg / ml, and culture it with shaking at 37 °C and 220 rpm 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 cell pellet, suspend the cell pellet with PBS buffer at pH 7.4 and perform ultrasonic disruption (power: 200 W, intermittent for 8 seconds every 4 seconds of working, 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's Ni Sepharose 6 Fast Flow. First, elute with Solution I (pH 7.4, solvent: water, containing 20 mM Na2HPO4 and 500 mM NaCl) for 10 column volumes to remove impurity proteins, then elute with Solution II (pH 7.4, solvent: water, containing 200 mM imidazole, 20 mM Na2HPO4 and 500 mM NaCl) for 2 column volumes to obtain the target protein. Collect the solution after passing through the column during elution with Solution II, and name it pile solution.

[0097] 12 mg of protein with a purity of over 90% can be obtained from each liter of the culture system of step 6.

[0098] Preparation of App

[0099] 1. Use the genomic DNA of Neisseria meningitidis serogroup B clinical strain 2015 - 16# as a template, and perform PCR amplification with the primer pair composed of F1 and R1 to obtain a 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 digested product.

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

[0104] 4. Ligate the digested product of step 2 and the vector backbone of step 3 to obtain the recombinant plasmid pET28a-App. According to the sequencing results, the description of the recombinant plasmid pET28a-App is as follows: A double-stranded DNA molecule of the nucleotides shown at positions 3229-4362 from the 5′ end of sequence 14 in the sequence list is inserted between the NcoI and XhoI restriction sites of the vector pET28a-App. The inserted double-stranded DNA molecule forms a fusion gene shown in sequence 15 of the sequence list with a part of the DNA on the vector backbone, and expresses a 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 a recombinant bacterium.

[0106] 6. Inoculate the recombinant bacterium obtained in step 5 into an LB liquid medium containing 100 μg / mL ampicillin, and culture it with shaking at 37°C and 220 rpm until OD560nm = 0.6. After the medium is cooled to 15°C, add IPTG for induction, and culture it with shaking 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 cell pellet, suspend the cell pellet with PBS buffer at pH 7.4 and perform ultrasonic disruption (power: 200 W, intermittent for 8 seconds every 4 seconds of work, 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 the equilibration buffer for 10 column volumes to remove the impurity proteins, and then elute with 0, 20, 50, 100, 150 mM imidazole respectively to obtain the target protein, and collect the solution after passing through the column when eluting with 150 mM imidazole.

[0109] 9. Further purify the eluate obtained in step 8 with an AKTA purifier to obtain a relatively pure target protein.

[0110] Preparation of IgA Protease

[0111] 1. Using the genomic DNA of Neisseria meningitidis serogroup B clinical strain 2015-16# as a template, perform PCR amplification with the primer pair composed of F1 and R1 to obtain a PCR amplification product.

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

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

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

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

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

[0118] 6. Inoculate the recombinant bacterium obtained in step 5 into an LB liquid medium containing 100 μg / mL kanamycin, and culture it with shaking at 37 °C and 220 rpm until OD560nm = 0.6. After the medium is cooled to 15 °C, add IPTG for induction, and culture it with shaking at 15 °C and 220 rpm for 24 hours.

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

[0120] 8. Load the supernatant obtained in step 7 onto Ni Sepharose 6 Fast Flow. First, perform elution with the equilibration buffer for 10 column volumes to remove contaminating proteins, and then elute with 0, 20, 50, 100, and 150 mM imidazole respectively to obtain the target protein. Collect the post-column solution when eluting with 150 mM imidazole.

[0121] 9. Further purify the eluate obtained in step 8 using an AKTA purifier to obtain a relatively pure target protein.

[0122] The polyacrylamide gel electrophoresis patterns during the preparation of the above fHbp, NHBA, fHbp - Opa(HV2), pile, App, and IgA protease proteins are as shown in the appendix Figure 1 as follows. Among them, the Men.B recombinant proteins are fHbp (Mw≈34 kd), NHBA (Mw≈30 kd), fHbp - Opa(HV2) - GST (Mw≈59 kd), pile (Mw≈27 kd), App (Mw≈42 kd), and IgA Protease (Mw≈107 kd).

[0123] Example 2

[0124] Antibody response induced by Men.B - V6 immunization in mice. Female C57BL / 6 mice at 6 - 8 weeks of age were randomly divided into two groups, and the grouping treatments were as follows:

[0125] CpG group: Intranasally instill CpG solution on the 1st, 7th, and 14th days of the experiment;

[0126] Men.B - V6 group: Intranasally instill the vaccine solution (the vaccine solution is obtained by mixing the 6 recombinant proteins prepared in Example 1 and CpG solution, and each mouse is given 10 μg of each of the 6 recombinant proteins and 10 μg of CpG each time) on the 1st, 7th, and 14th days of the experiment. On the 21st day of the experiment, anesthetize the mice and collect blood from the tail and oral lavage fluid. Measure serum IgG and oral lavage fluid IgA by ELIAS. The results are shown in the appendix Figure 2 and the appendix 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] Immune response of Men.B - V6 mucosal immunization to induce Th17 cells. Female C57BL / 6 mice at 6 - 8 weeks of age were randomly divided into two groups, and the grouping treatments were as follows:

[0129] CpG group: Intranasally instill CpG solution on the 1st, 7th, and 14th days of the experiment;

[0130] Group Men.B-V6: On the 1st, 7th, and 14th days of the experiment, the vaccine solution was instilled into the nasal cavity respectively (the vaccine solution was obtained by mixing the 6 recombinant proteins prepared in Example 1 and the CpG solution, and each mouse was given 10 μg of each of the 6 recombinant proteins and 10 μg of CpG each time). On the 21st day of the experiment, spleen and lung tissues were taken to extract cells, and ELISPOT was used to measure Men.B-V6 antigen-specific IL-17+ cells. The results are shown in the appendix Figure 4 , compared with the CpG group, the GBSV6 group significantly induced antigen-specific Th17 cells in the spleen and lung tissues, indicating that nasal immunization of mice with Men.B-V6 induced a Th17 cell response.

[0131] Example 4

[0132] Complement-mediated bactericidal assay. To prepare mouse antiserum and for the immune response of Men.B-V6 mucosal immunization to induce Th17 cells, female C57BL / 6 mice at 6-8 weeks of age were randomly grouped, and the grouping treatments were as follows:

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

[0134]

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

[0136] Table 2 Bactericidal activity experiment of clinical bacteria of group B meningitis BC50 (Titer)

[0137]

[0138] The above examples are merely illustrations given for clear explanation and are not limitations on the implementation methods. For those of ordinary skill 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 implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of this invention.

[0139] Although some specific embodiments of the present invention have been described in detail by way of examples, those skilled in the art should understand that the above examples are for illustration purposes only and not for limiting the scope of the present invention. Those skilled in the art should also understand that various modifications can 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 group B meningococcal infection, characterized in that: The active ingredients of the vaccine include component A, component B, component C, component D, component E, component F, and component G; The component A is fHbp, having the amino acid sequence of the fHbp after removing the signal peptide, and the amino acid sequence is shown in SEQ ID NO: 3; The component B is NHBA, having the amino acid sequence of NHBA after removing the signal peptide and cell wall anchoring domain, and the amino acid sequence is shown in SEQ ID NO: 6; The component C is fHbp-Opa (HV2)-GST, which has the amino acid sequence of the fHbp minus the signal peptide and the Opa (HV2) multivariable region, and a GST tag, and the amino acid sequence thereof is shown in SEQ ID NO: 14; The component D is pile, which has the amino acid sequence of pile after removing the signal peptide, and the amino acid sequence is shown in SEQ ID NO: 17; The component E is App, having the amino acid sequence of the binding domain of App, the amino acid sequence of which is 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 cell wall anchoring domain, and the amino acid sequence is shown in SEQ ID NO: 23; The component heptadjuvant is CpG; The mass ratio of component A, component B, component C, component D, component E, component H and component H is 1:1:1:1:1:1:1:

1.

2. A broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection according to claim 1, characterized in that: The vaccine is administered by nasal inhalation.

3. A broad-spectrum multi-subunit vaccine for preventing group B meningococcal infection according to claim 2, 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.

4. Use of the vaccine according to any one of claims 1 to 3 in the preparation of a medicament for preventing group B meningococcal infection.

5. The use according to claim 4, characterized in that: The functions of the vaccine are as follows (I) or (II) or (III): (I) highly efficient induction of IgG in serum and secretory IgA in mucosa; (II) induction of specific T cell responses dominated by Th17 in the lungs and spleen; and (III) mucosal immunity capable of inducing neutralizing antibodies with high bactericidal activity against clinical strains.

6. The use according to claim 4, characterized in that: The group B meningococci include different epidemic strains of group B meningococci.

7. The use according to claim 4, characterized in that: The prevention of group B meningococcal infection is the prevention of human infection caused by different group B meningococci.

8. The use according to claim 7, 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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