A broad-spectrum multi-subunit vaccine for preventing group B streptococcal infection and its use

Abstract

The present invention belongs to the field of biomedical technology, and particularly relates to a broad-spectrum multi-subunit vaccine for preventing group B streptococcal infection and its uses. Component A is sortase A, having the protein sequence of sortase A after removing the signal peptide; component B is CspA, having the protein sequence of CspA after removing the signal peptide and cell wall anchoring domain; component C is C5a peptidase, having the protein sequence of C5a peptidase after removing the signal peptide and cell wall anchoring domain; component D is FbsB, having the protein sequence of FbsB after removing the signal peptide; component E is Srr2, having the protein sequence of the Srr2 binding domain; component F is BibA, having the protein sequence of BibA after removing the signal peptide and cell wall anchoring domain; and component H is the adjuvant CpG. The vaccine of the present invention is highly effective, broad-spectrum, and low-cost. Furthermore, the vaccine utilizes a mucosal immunization pathway, resulting in no tissue damage, no local side effects, and is simple to use and easily disseminated.

Description

Technical Field

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

[0002] Sortase A (SrtA) is a highly conserved membrane surface protein found in a variety of Gram-positive pathogens and is essential for the anchoring of numerous virulence factors. Group B Streptococcus (GBS), also known as Streptococcus agalactiae, lacking sortase A, is a conditionally pathogenic bacterium that asymptomatically colonizes the female vagina or gastrointestinal tract, becoming a source of infection for both neonates and pregnant women. Maternal colonization predisposes pregnant women and neonates to various adverse outcomes. Infection in pregnant women can lead to miscarriage, premature birth, stillbirth, intrauterine growth restriction, premature rupture of membranes, urinary tract infection, upper genital tract infection, and postpartum endometritis. More importantly, maternal colonization increases the risk of neonatal infection. Transmission of GBS to neonates via the placenta or the birth canal during delivery can cause pneumonia, meningitis, sepsis, and even death. Therefore, GBS has long been a concern for the World Health Organization and scientists. According to the World Health Organization, GBS infection causes approximately 150,000 infant deaths, over 500,000 premature births, and severe long-term disability annually.

[0003] The main constraints on vaccine development are that there are 10 strains of Streptococcus B, and the lack of cross-serotype interaction between them makes it difficult to develop a vaccine that protects against all serotypes. Secondly, the vaccine needs to provide immune protection for both pregnant women and fetuses / newborns. Finally, because Streptococcus B colonization in pregnant women can lead to neonatal infection from birth to three months of age, the World Health Organization and experts recommend maternal immunization to protect newborns. However, maternal immunization may pose certain safety concerns, which have seriously hindered the development of a Streptococcus B vaccine. Therefore, there is an urgent need to develop a safe, effective, broad-spectrum vaccine that is easily disseminated and applicable.

[0004] Streptococcus spp. lacking the bacterial sortase A (SrtA) gene results in defects in multiple membrane protein functions, significantly reducing its adhesion to host epithelial cells. CspA (cell-surface-associated protein) is a secreted virulence protein of Streptococcus spp. present in various serotypes of Streptococcus spp. It not only cleaves the α-chain of human fibrinogen but also inhibits neutrophil migration to the site of infection by degrading CXC chemokines, thereby inhibiting host clearance of Streptococcus spp. It has been demonstrated that Streptococcus spp. lacking CspA exhibits significantly reduced virulence and neutrophil phagocytosis. C5a peptidase (ScpB) is a virulence protein located on the surface of Streptococcus spp. and is ubiquitous in all serotypes of Streptococcus spp. C5a peptidase inactivates the normal complement protein C5a in humans and binds to a motif generated by the juxtaposition of multiple adjacent fibronectin molecules, contributing to the invasion of epithelial cells. Studies have demonstrated that respiratory immunization of mother mice with C5a peptidase not only reduces vaginal colonization of Streptococcus Bacillus but also provides immune protection to newborn pups. FbsB (Fibrinogen-binding protein) is a ubiquitous adhesin present in all serotypes of Streptococcus Bacillus that binds to human fibrinogen. FbsB knockout strains exhibit reduced invasive epithelial cell infection, a finding associated with the ability of Streptococcus Bacillus to enter cells. Srr2 (Serine-rich repeat 2) is a cell wall-anchored protein of the highly virulent Streptococcus Bacillus ST-17 strain and an adhesin that binds to fibrinogen and plasminogen. The Srr2 knockout strain reduces brain colonization in mice, and immunization with recombinant Srr2 has been shown to induce the production of specific antibodies against Streptococcus Bacillus, providing immune protection against lethal infection with Streptococcus Bacillus. BibA (GBS immunogenic bacterial adhesin) is a multifunctional protein anchored on the surface of GBS. BibA-deficient strains are impaired in their ability to adhere to human cervical and lung epithelial cells. Furthermore, BibA specifically binds to complement C4 binding protein, protecting against neutrophil cytotoxicity. The BibA gene sequence is highly conserved among clinical isolates, with the N-terminal domain sharing 98-100% identity. Immunization of female mice with BibA has been shown to induce antibody production, which is then transferred across the placenta to pups, providing protection against GBS infection in newborn mice.

[0005] However, there is a technical problem that existing research results confirm that a mixture of single B-streptavidin subunit components cannot provide comprehensive and effective protection against multiple serotypes of B-streptavidin.

[0006] The specification of Chinese invention CN201710792641.9 describes a broad-spectrum, multi-subunit vaccine for preventing group A Streptococcus infection. Its active ingredients consist of component A, component B, component C, component D, component E, and component F. Component A is a sortase or a fusion protein containing the sortase; component B is C5a protease or a fusion protein containing the C5a protease; component C is Spy0269 or a fusion protein containing Spy0269; component D is SCPC or a fusion protein containing SCPC; component E is SLO or a fusion protein containing SLO; and component F contains CpG or other mucosal immune adjuvants. This vaccine boasts high efficacy, broad spectrum, and low cost.

[0007] Group A Streptococcus and Group B Streptococcus belong to the same genus, Streptococcus, and share similarities. The protective efficacy of broad-spectrum, multi-subunit vaccines against Group A Streptococcus demonstrates the superiority of combining multiple virulence factors as vaccine antigens. Researchers are also studying the pathogenicity of Group B Streptococcus and screening for a series of key specific virulence factors involved in its pathogenesis, aiming to develop a broad-spectrum, highly effective, multi-subunit vaccine against Group B Streptococcus. However, due to differences in the populations infected, the modes of infection, the pathological mechanisms of infection, and the diseases caused by Group A Streptococcus and Group B Streptococcus, the development of a broad-spectrum, multi-subunit vaccine suitable for Group B Streptococcus infections presents numerous technical challenges. Consequently, no vaccine for Group B Streptococcus is currently available, either domestically or internationally. Summary of the Invention

[0008] In order to solve the above technical problems, the present invention provides a broad-spectrum multi-subunit vaccine for preventing B-streptococcus infection and its use. It uses the conserved antigens of multiple B-streptococci to mucosally immunize mice, effectively induce Th17 and antibody responses in mice, and provide broad-spectrum and effective protection across serotypes against lethal doses of B-streptococci, B-streptococci infections of different serotypes, and newborn mice. The vaccine is highly effective, broad-spectrum, and low-cost. At the same time, the vaccine 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.

[0009] The present invention solves the above technical problems and provides a broad-spectrum multi-subunit vaccine for preventing Streptococcus B infection, characterized in that the active ingredients of the vaccine are composed of component A, component B, component C, component D, component E, component H, and component H;

[0010] The component A is sortase A, a fusion protein having the full or partial amino acid sequence of sortase A, a protein comprising the full or partial amino acid sequence of sortase A conjugated to an adjuvant protein, a complex comprising the full or partial amino acid sequence of sortase A and a polysaccharide, or a DNA expression vector carrying the full or partial encoding gene of sortase A;

[0011] Component B is CspA, a fusion protein having the full or partial amino acid sequence of CspA, a protein in which the full or partial amino acid sequence of CspA is conjugated to an adjuvant protein, a complex of the full or partial amino acid sequence of CspA and a polysaccharide, or a DNA expression vector carrying the full or partial coding gene of CspA;

[0012] The component C is C5a peptidase, a fusion protein having the full or partial amino acid sequence of the C5a peptidase, a protein conjugated with the full or partial amino acid sequence of the C5a peptidase and an adjuvant protein, a complex connected with the full or partial amino acid sequence of the C5a peptidase and a polysaccharide, or a DNA expression vector carrying the full or partial encoding gene of the C5a peptidase;

[0013] Component D is FbsB, a fusion protein having the full or partial amino acid sequence of FbsB, a protein conjugated with the full or partial amino acid sequence of FbsB and an adjuvant protein, a complex connected with the full or partial amino acid sequence of FbsB and a polysaccharide, or a DNA expression vector carrying the full or partial encoding gene of FbsB;

[0014] Component E is Srr2, a fusion protein having the full or partial amino acid sequence of Srr2, a protein conjugated with the full or partial amino acid sequence of Srr2 and an adjuvant protein, a complex connected with the full or partial amino acid sequence of Srr2 and a polysaccharide, or a DNA expression vector carrying the full or partial coding gene of Srr2;

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

[0016] It also includes component heptadjuvant CpG;

[0017] The function of the vaccine is to prevent infection with Streptococcus B or to provide immune protection to newborn babies through maternal immunity;

[0018] 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.

[0019] In the optimized solution of the present invention, the component A is sortase A, having the protein sequence of sortase A after removing the signal peptide; specifically, a protein consisting of the amino acid sequence shown in sequence 3 in the sequence listing;

[0020] Component B is CspA, having the protein sequence of CspA after removing the signal peptide and cell wall anchoring domain; specifically, a protein consisting of the amino acid sequence shown in Sequence 6 in the sequence listing. Component C is C5a peptidase, having the protein sequence of C5a peptidase after removing the signal peptide and cell wall anchoring domain; specifically, a protein consisting of the amino acid sequence shown in Sequence 9 in the sequence listing. Component D is FbsB, having the protein sequence of FbsB after removing the signal peptide; specifically, a protein consisting of the amino acid sequence shown in Sequence 12 in the sequence listing. Component E is Srr2, having the protein sequence of the Srr2 binding domain; specifically, a protein consisting of the amino acid sequence shown in Sequence 15 in the sequence listing. Component F is BibA, having the protein sequence of BibA after removing the signal peptide and cell wall anchoring domain; specifically, a protein consisting of the amino acid sequence shown in Sequence 18 in the sequence listing.

[0021] The functions of the vaccine of the present invention are as follows (I) or (II) or (III) or (IV): (I) inhibiting different serotypes of Streptococcus B; (II) preventing human infection caused by different serotypes of Streptococcus B; (III) reducing or preventing the colonization of different serotypes of Streptococcus B in the human mucosal system, including the reproductive tract of pregnant women; (IV) producing immune protection for newborns after maternal immunization.

[0022] The different serotypes of Streptococcus B may be the ten serotypes of Streptococcus B that have been discovered and reported so far, namely, Streptococcus B serotypes Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX; and may also be subtypes of the above serotypes that may appear and new serotypes not included in the above serotypes.

[0023] In the optimized solution, the Streptococcus B bacteria may specifically be Streptococcus B bacteria serotype Ib or serotype III.

[0024] The prevention of Streptococcus B infection is the prevention of human mucosal system infection caused by Streptococcus B.

[0025] In a further optimized solution, the human mucosal system may be the respiratory system, digestive system, urinary system, reproductive system or skin.

[0026] The vaccine can be used by nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection or anal injection.

[0027] The present invention is applied to the use of any of the above vaccines in the preparation of drugs for preventing B-streptococcal infection.

[0028] Recommended usage of the vaccine provided by this invention: 10 μg / dose of each of the proteins in Components A, B, C, D, E, and F, and 10 μg / dose of CpG in Component G. Three immunizations are administered, one week apart. Immunization is by nasal inhalation.

[0029] Th17 cells are a newly discovered type of T cell. Memory Th17 cells, generated after mucosal immunization, can rapidly migrate to infected mucosal sites and play a crucial role in combating mucosal bacterial infections. Unlike immunity provided by B cells, T cell immunity tolerates antigenic variation, providing cross-protection against allotypic bacteria and forming the theoretical basis for the development of novel vaccines. The cytokine IL-17 released by activated Th17 cells activates neutrophils and macrophages to phagocytose and effectively kill pathogens that enter the body. Because sortase A is located within the bacterial cell wall, antibodies targeting sortase A are ineffective in providing immune protection. Sortase A can induce an immune response dominated by Th17 cells, targeting Streptococcus spp. in a T cell-dependent manner.

[0030] In addition to SrtA, CspA, C5a peptidase, FbsB, Srr2, and BibA are located on the surface of Streptococcus B or secreted extracellularly. Antibodies against these virulence factors can specifically neutralize the pathogenic effects of virulence factors or play an anti-Streptococcus B infection role through antibody-dependent phagocytic killing.

[0031] After extensive and in-depth research, the inventors discovered that the combined use of sortase A, CspA, C5a peptidase, FbsB, Srr2, and BibA can induce both a Th17 cell response and serotype-independent protective antibodies, thereby providing more effective protection against Streptococcus serotypes. Therefore, the combined use of sortase A, CspA, C5a peptidase, FbsB, Srr2, and BibA can provide broad-spectrum protection against different serotypes of Streptococcus serotypes. Mucosal adjuvants can promote the uptake of vaccine subunits by antigen-processing cells in mucosal areas, significantly enhancing their immunogenicity and immune efficacy, while also avoiding local tissue reactions that can be caused by adjuvants during intramuscular or subcutaneous immunization.

[0032] The vaccine provided by the present invention can be administered by inhalation into the lungs, inhalation into the nasal cavity, oral administration, subcutaneous injection, intradermal injection, genital tract injection, anal injection, etc.

[0033] The vaccine provided by the present invention can be used to prevent and treat mucosal system (respiratory system, digestive system, urinary system, reproductive system or skin) infections caused by various Gram-positive bacteria.

[0034] The antigens used in the multi-linked recombinant protein vaccine of the present invention (hereinafter referred to as GBSV6) are ubiquitous in Streptococci, with a homology of over 90%. By fully utilizing the ubiquity and homology of various antigens in Streptococci, mucosal immunity, and mucosal immune adjuvants to enhance antigen immunogenicity, the vaccine significantly improves Th17 cell activation and antibody levels, preventing the colonization of pathogens and rapidly clearing them. It also has a protective effect against different serotypes of Streptococcus B, and has the advantages of high efficiency, broad spectrum, and low cost. At the same time, the vaccine provided by the present invention adopts the mucosal immunity route, has the characteristics of no tissue damage, no local side effects, and is easy to use, making it easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Polyacrylamide gel electrophoresis diagram of the process of preparing SrtA, ScpB, CspA, Srr2, BibA, and FbsB proteins in the present invention

[0036] Figure 2 This is the result diagram of Example 2 of the present invention (clearance of Streptococcus B in the genital tract infection site after nasal inhalation of GBSV6)

[0037] Figure 3 This is the result of Example 3 of the present invention (immune protection against lethal infection with Streptococcus B bacteria after nasal inhalation of GBSV6)

[0038] Figure 4 This is the result of Example 4 of the present invention (GBSV6 immunization induces mice to produce antigen-specific Th17 immune cell responses)

[0039] Figure 5 This is the result diagram of Example 5 of the present invention (antigen-specific serum IgG response produced by GBSV6 immunization-induced mice)

[0040] Figure 6 This is the result of Example 5 of the present invention (antigen-specific secretory IgA response produced by GBSV6 immunization-induced mice) DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to specific embodiments:

[0042] To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples, unless otherwise specified, are conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0043] Streptococcus serotype Ib and Streptococcus serotype III: obtained from the Microbiology Laboratory of Beijing Children's Hospital, Capital Medical University, Beijing. Reference: Shen AD, Zhang GR, Wang YH, Yang YH. Zhonghua Er Ke Za Zhi. 2005; 43(9): 661-664. CpG: reference: Iho S, Maeyama J, Suzuki F. CpG ligodeoxynucleotides as mucosal adjuvants. Hum Vaccin Immunother. 2015; 11(3): 755-60. Vector pET28a(+): Novagen, Cat. No. 69846-3. 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.

[0044] Example 1

[0045] Preparation of sortase A (SrtA)

[0046] 1. Using the genomic DNA of Streptococcus serotype Ib as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain a PCR amplification product.

[0047] F1:5'-CATG CCATGG GCTCTGCTCAAACGAAATCACA-3';

[0048] R1: 5'-CCG CTCGAG GAGATTAATTTGATTATATT-3'.

[0049] 2. Double-digest the PCR amplification product from step 1 with restriction endonucleases NcoI and XhoI, and recover the digestion product.

[0050] 3. Double-digest the vector pET28a(+) with restriction endonucleases NcoI and XhoI to recover the vector backbone of approximately 5400 bp.

[0051] 4. Ligate the digested product from step 2 with the vector backbone from step 3 to obtain the recombinant plasmid pET28a-SrtA. Based on the sequencing results, the recombinant plasmid pET28a-SrtA was sequenced as follows: A double-stranded DNA molecule represented by nucleotides 244-741 of Sequence 1 in the Sequence Listing, starting from the 5' end, was inserted between the NcoI and XhoI restriction sites of the pET28a(+) vector. This inserted double-stranded DNA molecule, combined with a portion of the DNA from the vector backbone, formed a fusion gene represented by Sequence 2 in the Sequence Listing, expressing the fusion protein represented by Sequence 3 in the Sequence Listing.

[0052] 5. Introduce the recombinant plasmid pET28a-SrtA into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0053] 6. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 50 μg / ml kanamycin, and cultured at 37°C, 220 rpm, and shaken until OD560nm = 0.6. IPTG was added for induction at a concentration of 40 μg / ml, and cultured at 37°C, 220 rpm, and shaken for 4 hours.

[0054] 7. Take the culture system of step 6, centrifuge at 3000 rpm for 20 minutes at 4°C to collect the bacterial precipitate, suspend the bacterial precipitate with PBS buffer (pH 7.4) and perform ultrasonic disruption (power 200 W, 4 seconds of work and 6 seconds of rest, 99 cycles), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0055] 8. The supernatant from step 7 was loaded onto a GE Ni Sepharose 6 Fast Flow. Elution was performed for 10 column volumes with Solution I (pH 7.4, solvent: water, containing 20 mM Na2HPO4 and 500 mM NaCl) to remove contaminants. Elution was then performed for 2 column volumes with Solution II (pH 7.4, solvent: water, containing 200 mM imidazole, 20 mM Na2HPO4, and 500 mM NaCl) to obtain the target protein. The post-column solution from elution with Solution II was collected and designated Sortase A Solution. Per liter of the culture system from step 6, 13 mg of protein with a purity exceeding 90% was obtained.

[0056] Preparation of CspA

[0057] 1. Using the genomic DNA of Streptococcus serotype III as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0058] F1:5'-CATG CCATGG GCGATTCTGTCATAAATAAGCC-3';

[0059] R1: 5'-CCG CTCGAG ATTGCCAATATTGATCAAATCT-3'.

[0060] 2. Use PCR to perform point mutations to mutate Asp172 and Ser567 in the active center to Ala to obtain CspA that retains immunogenicity but loses catalytic activity. Use the following primers for point mutations:

[0061] Asp-Ala(172):

[0062] F1: 5′-GTAGCAATTATTGCTTCAGGACTAGAT-3′

[0063] R1: 5′-ATCTAGTCCTGAAGCAATAATTGCTAC-3′

[0064] Ser-Ala(567):

[0065] F1: 5′-ATGAGTGGGACAGCTATGGCTTCTCCC-3′

[0066] R1: 5′-GGGAGAAGCCATAGCTGTCCCACTCAT-3′

[0067] 3. Double-digest the PCR amplification product from step 1 with restriction endonucleases NcoI and XhoI, and recover the digestion product.

[0068] 4. Double-digest the vector pET28a(+) with restriction endonucleases NcoI and XhoI to recover the vector backbone of approximately 6000 bp.

[0069] 5. Ligate the digested product from step 2 with the vector backbone from step 3 to obtain the recombinant plasmid pET28a-CspA. Based on the sequencing results, the recombinant plasmid pET28a-CspA was sequenced as follows: a double-stranded DNA molecule represented by nucleotides 106-3228 of the 5' terminus of Sequence 7 of the Sequence Listing was inserted between the NcoI and XhoI restriction sites of the pET28a(+) vector. This inserted double-stranded DNA molecule, along with a portion of the DNA from the vector backbone, formed a fusion gene represented by nucleotides 5 of the Sequence Listing, expressing the fusion protein represented by nucleotides 6 of the Sequence Listing.

[0070] 5. Introduce the recombinant plasmid pET28a-CspA into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0071] 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.

[0072] 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 pH 7.4 PBS buffer and perform ultrasonic disruption (power 200 W, 4 seconds of work and 8 seconds of rest, 99 cycles), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0073] 8. The supernatant obtained in step 7 was loaded onto a GE Ni Sepharose 6 Fast Flow. Elution was performed for 10 column volumes with Solution I (pH 7.4, solvent: water, containing 20 mM Na2HPO4 and 500 mM NaCl) to remove contaminants. Elution was then performed for 2 column volumes with Solution II (pH 7.4, solvent: water, containing 200 mM imidazole, 20 mM Na2HPO4, and 500 mM NaCl) to obtain the target protein. The post-column solution from elution with Solution II was collected and designated as the CspA solution. Per liter of the culture system from step 6, 36 mg of protein with a purity exceeding 90% was obtained.

[0074] Preparation of C5a peptidase

[0075] 1. Using the genomic DNA of Streptococcus serotype III as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0076] F1:5′-ATGA CCATGG GCAATACTGTGACAGAAGACACTCC-3′, R1: 5′-CCG CTCGAG AGAGTGGCCCTCCAATAG.

[0077] 2. PCR was used to perform point mutations to mutate Asp130 and Ser512 in the active center to Ala to obtain ScpB that retains immunogenicity but loses catalytic activity. The following primers were used for point mutations:

[0078] Asp-Ala (130): F1: 5′-GTTGCAGTGATTGCTGCTGGTTTTGAT-3′, R1: 5′-ATCAAAACCAGCAGCAATCACTGCAAC-3′.

[0079] Ser-Ala(512): F1: 5′-CTTTCTGGAACTGCTATGTCTGCGCCA-3′, R1: 5′-TGGCGCAGACATAGCAGTTCCAGAAAG-3′.

[0080] 3. Double-digest the PCR amplification product from step 1 with restriction endonucleases NcoI and XhoI, and recover the digestion product.

[0081] 4. Double-digest the vector pET28a(+) with restriction endonucleases NcoI and XhoI.

[0082] 5. The digested product from step 3 was ligated with the vector backbone from step 4 to obtain the recombinant plasmid pET28a-ScpB. Based on the sequencing results, the recombinant plasmid pET28a-ScpB was sequenced as follows: a double-stranded DNA molecule represented by SEQ ID NO: 4 (5' end, nucleotides 94-3096) was inserted between the NcoI and XhoI restriction sites of the pET28a(+) vector. This inserted double-stranded DNA molecule, combined with a portion of the DNA from the vector backbone, formed a fusion gene represented by SEQ ID NO: 8 (5' end, nucleotides 94-3096) and expressed the fusion protein represented by SEQ ID NO: 9 (5' end, nucleotides 94-3096).

[0083] 6. Introduce the recombinant plasmid pET28a-ScpB into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0084] 7. The recombinant bacteria obtained in step 5 were inoculated into LB liquid medium containing 50 μg / mL kanamycin, cultured at 37°C and 220 rpm with shaking until OD560nm = 0.6, and then IPTG was added for induction, and cultured at 37°C and 220 rpm with shaking for 4 hours.

[0085] 8. Take the culture system of step 7, centrifuge at 3000 rpm for 20 minutes at 4°C to collect the bacterial precipitate, suspend the bacterial precipitate with PBS buffer at pH 7.4 and perform ultrasonic disruption (power of 200 W, 4 seconds of work and 8 seconds of rest, 99 cycles), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0086] 9. Load the supernatant obtained in step 8 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.

[0087] 10. The eluate from step 9 was further purified by AKTA purifier to obtain a relatively pure target protein.

[0088] Preparation of Srr2

[0089] 1. Using the genomic DNA of Streptococcus serotype III as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product. F1: 5'-CATG CCATGG GCTCAGAAGCGGCAACGACCGCTAGAG-3';R1:5'-CCG CTCGAG TTGAGCATTTACATCTGAATA-3'.

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

[0091] 3. Double-digest the vector pET28a(+) with restriction endonucleases NdeI and XhoI to recover the vector backbone of approximately 6000 bp.

[0092] 4. Ligate the digested product from step 2 with the vector backbone from step 3 to obtain the recombinant plasmid pET28a-Srr2. Based on the sequencing results, the recombinant plasmid pET28a-Srr2 was characterized as follows: a double-stranded DNA molecule represented by Sequence 10 in the Sequence Listing, starting from nucleotides 571-1629 at the 5' end, was inserted between the NdeI and XhoI restriction sites of the pET28a(+) vector. This inserted double-stranded DNA molecule, combined with a portion of the DNA from the vector backbone, formed the fusion gene represented by Sequence 11 in the Sequence Listing, expressing the fusion protein represented by Sequence 12 in the Sequence Listing.

[0093] 5. Introduce the recombinant plasmid pET28a-Srr2 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 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.

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

[0096] 8. The supernatant from step 7 was loaded onto a GE Ni Sepharose 6 Fast Flow. Elution was performed for 10 column volumes with Solution I (pH 7.4, solvent: water, containing 20 mM Na2HPO4 and 500 mM NaCl) to remove contaminants. Elution was then performed for 2 column volumes with Solution II (pH 7.4, solvent: water, containing 200 mM imidazole, 20 mM Na2HPO4, and 500 mM NaCl) to obtain the target protein. The post-column solution from elution with Solution II was collected and designated as Solution Srr2. 12 mg of protein with a purity exceeding 90% was obtained per liter of the culture system from step 6.

[0097] Preparation of FbsB

[0098] 1. Using the genomic DNA of Streptococcus serotype III as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product.

[0099] F1:5′-C GAGCTC GCCGGGATAACTAAAG-3′,R1:5′-ACGC GTCGAC CTCTTTTATACGCGATGAG-3′.

[0100] 2. Double-digest the PCR amplification product from step 1 with restriction endonucleases SacI and SalI, and recover the digestion product.

[0101] 3. Double-digest the vector pCold-SUMO with restriction endonucleases SacI and SalI.

[0102] 4. Ligate the digested product from step 2 with the vector backbone from step 3 to obtain the recombinant plasmid pCold-SUMO-FbsB. Based on the sequencing results, the recombinant plasmid pCold-SUMO-FbsB was characterized as follows: a double-stranded DNA molecule represented by nucleotides 76-1884 of the 5' end in Sequence 13 of the Sequence Listing was inserted between the SacI and SalI restriction sites of the pCold-SUMO vector. This inserted double-stranded DNA molecule, combined with a portion of the DNA from the vector backbone, formed a fusion gene represented by Sequence 14 of the Sequence Listing, expressing the fusion protein represented by Sequence 15 of the Sequence Listing.

[0103] 5. Introduce the recombinant plasmid pCold-SUMO-FbsB into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0104] 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 with shaking 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 with shaking for 24 hours.

[0105] 7. Take the culture system of step 6, centrifuge at 3000 rpm for 20 minutes at 4°C to collect the bacterial precipitate, suspend the bacterial precipitate with PBS buffer (pH 7.4) and perform ultrasonic disruption (power 200 W, 4 seconds of work and 8 seconds of rest, 99 cycles), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0106] 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.

[0107] 9. The eluate from step 8 was further purified by AKTA purifier to obtain a relatively pure target protein.

[0108] Preparation of BibA

[0109] 1. Using the genomic DNA of Streptococcus serotype Ib as a template, PCR amplification was performed using the primer pair consisting of F1 and R1 to obtain the PCR amplification product. GAGCTC CACGCGGATACTAGTTCAGGA-3′, R1: 5′-ACGC GTCGAC ACCTCTGGTAAGGTCTTGAA-3′.

[0110] 2. Double-digest the PCR amplification product from step 1 with restriction endonucleases SacI and SalI, and recover the digestion product.

[0111] 3. Double-digest the vector pCold-SUMO-BibA with restriction endonucleases SacI and SalI.

[0112] 4. Ligate the digested product from step 2 with the vector backbone from step 3 to obtain the recombinant plasmid pCold-SUMO-BibA. Based on the sequencing results, the recombinant plasmid pCold-SUMO-BibA was characterized as follows: a double-stranded DNA molecule represented by SEQ ID NO: 102-1413 from the 5' end of SEQ ID NO: 16 in the Sequence Listing was inserted between the SacI and SalI restriction sites of the pCold-SUMO vector. This inserted double-stranded DNA molecule, combined with a portion of the DNA from the vector backbone, formed a fusion gene represented by SEQ ID NO: 17 in the Sequence Listing, expressing the fusion protein represented by SEQ ID NO: 18 in the Sequence Listing.

[0113] 5. Introduce the recombinant plasmid pCold-SUMO-BibA into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

[0114] 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 with shaking 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 with shaking for 24 hours.

[0115] 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 pH 7.4 PBS buffer and perform ultrasonic disruption (power 200 W, 4 seconds of work and 8 seconds of rest, 99 cycles), then centrifuge at 12000 rpm for 20 minutes, and collect the supernatant.

[0116] 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.

[0117] 9. The eluate from step 8 was further purified by AKTA purifier to obtain a relatively pure target protein.

[0118] The polyacrylamide gel electrophoresis images of the above preparation process of SrtA, ScpB, CspA, Srr2, BibA, and FbsB proteins are shown in FIG. Figure 1The GBS recombinant proteins are SrtA (amino acid sequence: 82-247, Mw≈19 kd), ScpB (amino acid sequence: 32-1032, Mw≈110 kd), CspA (amino acid sequence: 36-1076, Mw≈114 kd), Srr2 (amino acid sequence: 192-543, Mw≈40 kd), BibA (amino acid sequence: 34-471, Mw≈64 kd), and FbsB (amino acid sequence: 19-628, Mw≈86 kd).

[0119] Example 2

[0120] To investigate the clearance of Streptococcus B in genital tract infection sites after nasal inhalation of GBSV6+CpG, 6-8 week-old female ICR mice were randomly divided into two groups and treated as follows:

[0121] CpG group: PBS buffer was instilled into the nasal cavity on the 1st, 7th, and 14th days of the experiment;

[0122] GBSV6 group: The vaccine solution was dripped into the nasal cavity on the 1st, 7th and 14th days of the experiment (the vaccine solution was obtained by mixing the 6 recombinant proteins prepared in Example 1 with CpG solution, and each mouse was given 10 μg of each of the 5 recombinant proteins and 10 μg of CpG each time).

[0123] On day 21 of the experiment, mice were challenged with live bacteria (Streptococcus serotype Ib or III) via genital tract instillation (10 μl of the bacterial solution at a concentration of 1×107 CFU / 10 μl per mouse). Genital tract lavage fluid was collected 1, 3, 6, 9, 15, and 35 days after challenge, and the number of live Streptococcus serotype B in the genital tract lavage fluid was determined by blood agar plate culture.

[0124] See the results Figure 2 Each black dot represents a mouse (the vertical axis refers to the number of CFUs of Streptococcus B in the entire genital tract lavage fluid of each mouse). After challenge with Streptococcus B serotype Ib or III, the number of viable bacteria in the genital tract lavage fluid of the GBSV6 group of mice was significantly lower than that of the CpG group, indicating that the vaccine provided by the present invention can effectively eliminate genital Streptococcus B serotype Ib or III infection through respiratory mucosal immunization.

[0125] Example 3

[0126] To protect against lethal infection with Streptococcus spp. after intranasal inhalation of GBSV6, 6-8 week-old female ICR mice were randomly divided into two groups and treated as follows:

[0127] CpG group: CpG solution was instilled into the nasal cavity on the 1st, 7th and 14th day of the experiment; GBSV6 group: vaccine solution was instilled into the nasal cavity on the 1st, 7th and 14th day of the experiment (the vaccine solution was obtained by mixing the 6 recombinant proteins prepared in Example 1 with CpG solution, and each mouse was given 10 μg of each of the 6 recombinant proteins and 10 μg of CpG each time).

[0128] On the 21st day of the experiment, mice were challenged with a lethal dose of live bacteria (Streptococcus B serotype Ib or III) via tail vein injection (the concentration of the bacterial solution was 2×108 CFU / 50μl, and 50μl was dripped into each mouse). The survival of the mice was observed after the challenge, and the results are shown in Figure 3 Compared with the CpG group, the GBSV6 group significantly improved the survival rate of mice (80%), indicating that GBSV6-immunized mice provide immune protection against a lethal dose of B-streptococcus.

[0129] Example 4

[0130] GBSV6 mucosal immunization induces the immune response of Th17 cells. Female ICR mice aged 6-8 weeks were randomly divided into two groups and treated as follows: CpG group: CpG solution was dripped into the nasal cavity on the 1st, 7th and 14th day of the experiment; GBSV6 group: vaccine solution was dripped into the nasal cavity on the 1st, 7th and 14th day of the experiment (the vaccine solution was obtained by mixing the 6 recombinant proteins prepared in Example 1 with 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, cells were extracted from the spleen and reproductive tract tissues, and GBSV6 antigen-specific IL-17+ cells were measured by ELISPOT. The results are shown in Figure 4 Compared with the CpG group, the GBSV6 group significantly induced antigen-specific Th17 cells in the spleen and reproductive tract tissues, indicating that GBSV6 nasal immunization of mice induced Th17 cell responses.

[0131] Example 4

[0132] GBSV6 immunization induced antibody response in mice. Female ICR mice aged 6-8 weeks were randomly divided into three groups and treated as follows: CpG group: CpG solution was instilled into the nasal cavity on the 1st, 7th and 14th day of the experiment; GBSV6 group: vaccine solution was instilled into the nasal cavity on the 1st, 7th and 14th day of the experiment (the vaccine solution was obtained by mixing the 6 recombinant proteins prepared in Example 1 with CpG solution, and each mouse was given 10 μg of each of the 6 recombinant proteins and 10 μg of CpG each time).

[0133] On the 21st day of the experiment, blood and genital tract lavage fluid were collected from the tail of the anesthetized mice, and serum IgG and genital tract lavage fluid IgA were measured by ELIAS. Figure 5 and Figure 6 Compared with the CpG group, the GBSV6 group significantly induced antigen-specific serum IgG and genital IgA.

[0134] Example 5

[0135] GBSV6 was inhaled intranasally or injected intramuscularly to immunize mother mice, which provided cross-immune protection to newborn mice against lethal infection with different serotypes of Streptococcus B. Female ICR mice aged 6-8 weeks were randomly divided into four groups and treated as follows: CpG group: CpG solution was instilled into the nasal cavity on the 1st, 7th and 14th day of the experiment, and the mice were mated with male mice on the 15th day to conceive, and newborn mice were born 4 weeks later; CpG+GBSV6 group: vaccine solution was instilled into the nasal cavity on the 1st, 7th and 14th day of the experiment (the vaccine solution was obtained by mixing the 6 recombinant proteins prepared in Example 1 with CpG solution, and each mouse was given 10 μg and 10 μg of each of the 6 recombinant proteins each time). CpG), and on the 15th day, they were caged with male mice for conception, and newborn mice were born 4 weeks later. Alum group: aluminum adjuvant solution was injected intramuscularly on the 1st, 7th, and 14th day of the experiment, and on the 15th day, they were caged with male mice for conception, and newborn mice were born 4 weeks later. Alum+GBSV6 group: vaccine solution was injected intramuscularly on the 1st, 7th, and 14th day of the experiment (the vaccine solution was obtained by mixing the 6 recombinant proteins prepared in Example 1 with the Alum solution, with each mouse receiving 10 μg of each of the 6 recombinant proteins and 25 μg of Alum each time), and on the 15th day, they were caged with male mice for conception, and newborn mice were born 4 weeks later.

[0136] Two-day-old mice were challenged with GBS1b or III by intraperitoneal injection (the concentration of the bacterial solution was 2×10 6 CFU / 20 μl, 20 μl injected per mouse), and the survival rate of newborn mice in each group was observed. The results are shown in Table 1.

[0137] Table 1. Intranasal or intramuscular inhalation of GBSV6 immunized mother mice provided cross-immune protection to newborn mice against lethal infection with different serotypes of Streptococcus B.

[0138]

[0139] As can be seen from the table above, the GBSV6 group of the present invention not only provides protection against Streptococcus serotype Ib, but also provides protection against Streptococcus serotype III of different serotypes, indicating that GBSV6-immunized mice induce cross-immunity protection against different serotypes of Streptococcus

[0140] The above embodiments and test examples are merely examples for clarity of explanation and are not intended to limit the embodiments. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all embodiments here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A broad-spectrum multi-subunit vaccine for preventing group B streptococcal infection, characterized by: The active ingredients of the vaccine are composed of component A, component B, component C, component D, component E, component H and component H; wherein component A is sortase A, and the sortase A is a protein composed of the amino acid sequence shown in Sequence 3 in the sequence listing; The component B is CspA, which is a protein consisting of the amino acid sequence shown in Sequence 6 in the sequence table; The component C is C5a peptidase, which is a protein consisting of the amino acid sequence shown in Sequence 9 in the sequence table; The component D is FbsB, which is a protein consisting of the amino acid sequence shown in Sequence 12 in the sequence list; The component E is Srr2, which is a protein consisting of the amino acid sequence shown in Sequence 15 in the sequence list; The component is BibA, which is a protein consisting of the amino acid sequence shown in Sequence 18 in the sequence list; It also includes component heptadjuvant 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; The group B streptococcus serotype is specifically group B streptococcus serotype Ib or serotype III.

2. A broad-spectrum multi-subunit vaccine for preventing group B streptococcal infection according to claim 1, characterized in that: The functions of the vaccine are as follows (I) or (II) or (III) or (IV): (I) inhibiting infection by group B streptococci of serotype Ib or serotype III; (II) Prevent human respiratory, reproductive tract and systemic infections caused by serotype Ib or serotype III group B streptococci; (III) Provide immune protection against neonatal group B streptococcal infection and death after maternal immunization.

3. A broad-spectrum multi-subunit vaccine for preventing group B streptococcal infection according to claim 2, characterized in that: The prevention of group B streptococcal infection is the prevention of human infection caused by serotype Ib or serotype III group B streptococci.

4. A broad-spectrum multi-subunit vaccine for preventing group B streptococcal infection according to claim 3, characterized in that: The human infections are infections of the respiratory system, digestive system, urinary system, reproductive system, skin and blood circulation system.

5. A broad-spectrum multi-subunit vaccine for preventing group B streptococcal infection according to claim 1, characterized in that: The vaccine can be used by nasal inhalation, oral administration, subcutaneous injection, intradermal injection, genital tract injection or anal injection.

6. Use of the vaccine according to any one of claims 1 to 5 in the preparation of a medicament for preventing group B streptococcal infection, wherein the serotype of the group B streptococcus is group B streptococcus serotype Ib or serotype III.

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