A streptococcus agalactiae multi-epitope fusion protein containing an intramolecular CTB adjuvant, a vaccine and application thereof
Patent Information
- Application Number
- CN202611030168.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-11
- Publication Date
- 2026-10-09
AI Technical Summary
然而，现有蛋白亚单位疫苗存在单一抗原免疫原性弱、保护率偏低、难以同时激活体液与细胞免疫等缺陷
（1）多靶点协同免疫设计：本发明将无乳链球菌FbsA、PGK和Cfb三种关键毒力因子的优势抗原表位整合于同一融合蛋白中，实现了单一抗原无法达到的多靶点协同免疫保护效果，克服了单一抗原保护率偏低的技术缺陷。
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Figure CN122878451A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedicine and genetic engineering vaccine technology, and in particular to a multi-epitope fusion protein of Streptococcus agalactiae containing CTB intramolecular adjuvant, a vaccine, and its applications. Background Technology
[0002] agalactococcus ( Streptococcus agalactiae Streptococcus agalactiae (SAA) is a Gram-positive bacterium with a wide range of hosts, affecting both humans and animals. It is a major pathogen causing diseases such as mastitis in dairy cows, neonatal sepsis, and meningitis. The World Health Organization has listed vaccination against SAA in pregnant women as an urgent medical need. Traditional antibiotic treatment faces increasingly serious problems with drug resistance; globally, SAA has developed resistance to multiple antibiotics, including β-lactams and macrolides. Vaccine development has become a key focus for prevention and control.
[0003] Currently, there are no commercially available Streptococcus agalactiae vaccines. Existing research on Streptococcus agalactiae vaccines mainly focuses on two directions: capsular polysaccharide vaccines and protein subunit vaccines. Capsular polysaccharide vaccines are limited in application due to the large number of Streptococcus agalactiae serotypes (10 serotypes have been identified so far) and the lack of cross-protection between serotypes. Protein-based subunit vaccines have attracted much attention because they can provide serotype-independent protection. However, existing protein subunit vaccines have drawbacks such as weak immunogenicity of a single antigen, low protection rates, and difficulty in simultaneously activating humoral and cellular immunity. Furthermore, traditional subunit vaccines require the addition of aluminum adjuvants or Freund's adjuvant to enhance the immune response, but these exogenous adjuvants have limitations such as inducing primarily humoral immunity, insufficient activation of cellular immunity, and local adverse reactions, and they cannot achieve active targeted delivery of antigens.
[0004] Therefore, developing a streptococcal vaccine that can induce potent humoral and cellular immunity without or with reduced dependence on exogenous adjuvants and has targeted delivery capabilities has significant clinical application value and industrialization prospects. Summary of the Invention
[0005] To address the aforementioned technical problems, this invention provides a multi-epitope fusion protein of Streptococcus agalactiae containing CTB intramolecular adjuvant, a vaccine, and its applications.
[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution: In a first aspect, this invention provides a Streptococcus agalactiae multiepitope fusion protein containing an intramolecular adjuvant CTB, wherein the fusion protein comprises a cholera toxin B subunit CTB intramolecular adjuvant and a multiepitope antigen sequence fused with the CTB intramolecular adjuvant, wherein the multiepitope antigen sequence is composed of antigenic epitopes of three virulence factors of Streptococcus agalactiae: fibrinogen-binding protein A (FbsA), phosphoglycerate kinase (PGK), and cAMP factor pore-forming toxin (Cfb).
[0007] Further, the multi-epitope antigen sequence comprises at least one antigenic epitope selected from the following MHC-I epitopes, MHC-II epitopes, and B-cell epitopes: MHC-I epitopes include one or more of WKSGKLWLY from FbsA and TAALPTIKY from PGK; MHC-II epitopes include one or more of LGSTIILGSSPVSAMDSV from FbsA, NNSRTISVINKLPKTGDDQ from PGK, ETPERPFVAILGGSK from Cfb, IGVMDAIVKQPGVKS from PGK, and SIQLRNIKDNVQGTD from Cfb; B-cell epitopes include one or more of HSSQGDSNKQSFSKKV from FbsA, DTEGEAVSEGFLGLDI from PGK, KSIIGGGDSAAAAINLGRAD from Cfb, IEAITFSTQHLANKVS from Cfb, and RATIYTKSKLDKEIWN from FbsA.
[0008] Furthermore, the amino acid sequence of the intramolecular adjuvant CTB is shown in SEQ ID NO.1; the full-length amino acid sequence of the fusion protein contains 353 amino acids.
[0009] Furthermore, the intramolecular adjuvant of CTB is linked to the multi-epitope antigen sequence via a flexible linker peptide selected from (GGGGS)2, EAAAK, and combinations thereof.
[0010] Furthermore, the multiple antigenic epitopes in the fusion protein are linked by a linker sequence selected from one or more of AAY, GGPPG, and GGGGS.
[0011] Secondly, this invention provides an agalactia-resistant streptococcal vaccine containing the aforementioned fusion protein as an active ingredient.
[0012] Furthermore, the aforementioned agalactia-associated streptococcal vaccine also contains a pharmaceutically acceptable adjuvant and / or carrier.
[0013] Thirdly, this invention provides the application of the aforementioned fusion protein or vaccine in the preparation of drugs for the prevention of Streptococcus agalactiae infection.
[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) Multi-target synergistic immune design: This invention integrates the dominant antigenic epitopes of three key virulence factors of Streptococcus agalactiae, namely FbsA, PGK and Cfb, into the same fusion protein, achieving the multi-target synergistic immune protection effect that cannot be achieved by a single antigen, and overcoming the technical defect of low protection rate of a single antigen.
[0015] (2) Targeted delivery function of CTB intramolecular adjuvant: This invention utilizes the cholera toxin B subunit as an intramolecular adjuvant to fuse with the antigen epitope for expression. GM1-ELISA detection confirmed that the rCTB-FPC fusion protein maintained its binding ability to the GM1 ganglioside receptor, which can effectively mediate the targeted delivery of antigen, overcoming the limitation of traditional exogenous adjuvants in achieving targeted delivery.
[0016] (3) Fully activate humoral and cellular immunity: Animal immunization experiments show that the vaccine of the present invention can induce high levels of specific antibody titers and effectively activate cellular immune responses, achieving full activation of Th1, Th2 and Th17 immune responses, overcoming the shortcomings of traditional aluminum adjuvants that mainly induce humoral immunity and have insufficient cellular immune activation.
[0017] (4) Highly effective immune protection: The results of the challenge protection experiment showed that the survival rate of the vaccine group was 76.7%, which was significantly higher than that of the negative control group (16.7%). It could also effectively reduce the bacterial load in the spleen, liver and kidneys, indicating that the vaccine of the present invention can provide highly effective immune protection.
[0018] (5) High safety: According to AlgPred 2.0 sensitization analysis, the fusion protein of the present invention is non-sensitizing, has good safety, and no adverse reactions were observed after immunization in mice.
[0019] (6) Simple process and good industrialization prospects: High-purity recombinant protein can be obtained by using the Escherichia coli prokaryotic expression system and affinity chromatography purification. The preparation process is mature, the cost is controllable, and it is suitable for large-scale production. Attached Figure Description
[0020] Figure 1 The following is a schematic diagram of the structure of the recombinant multi-epitope chimeric subunit vaccine rCTB-FPC of the present invention: A is the rCTB-FPC sequence; B is a schematic diagram of the tertiary structure of rCTB-FPC.
[0021] Figure 2 The following are partial schematic diagrams of the interaction interface between rCTB-FPC and receptor molecules: A is a partial schematic diagram of the interaction between rCTB-FPC and TLR2 molecules; B is a partial schematic diagram of the interaction between rCTB-FPC and TLR4 molecules; C is a partial schematic diagram of the interaction between rCTB-FPC and GM1 receptor molecules.
[0022] Figure 3The figure shows the results of GM1-ELISA detection of the binding activity of rCTB-FPC fusion protein to GM1.
[0023] Figure 4 The figure shows the results of indirect ELISA detection of serum-specific IgG antibody levels in immunized mice.
[0024] Figure 5 This figure shows the cytokine secretion levels of spleen cells in immunized mice after stimulation with the rCTB-FPC specific antigen.
[0025] Figure 6 Kaplan-Meier survival curves for mice after challenge with the virus.
[0026] Figure 7 This image shows the bacterial load in different tissues of mice after challenge with the virus. Detailed Implementation
[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.
[0028] Example 1: Construction, preparation and characterization of rCTB-FPC recombinant multi-epitope chimeric subunit vaccine 1) Molecular design of fusion proteins This embodiment constructs a recombinant multi-epitope chimeric subunit vaccine, named rCTB-FPC. Its active ingredient is a recombinant fusion polypeptide, with the structure from the N-terminus to the C-terminus as follows: CTB intramolecular adjuvant-flexible linker-multi-epitope antigen sequence (see...). Figure 1 ).
[0029] The CTB adjuvant sequence is derived from the cholera toxin B subunit, with an amino acid sequence shown in SEQ ID NO:1 (124 amino acids). It is responsible for targeting and binding to the cell surface GM1 receptor, mediating targeted antigen delivery. The flexible linker utilizes... The EAAAK linker peptide ensures that CTB folds independently with the multi-epitope antigen. The multi-epitope antigen sequence integrates dominant antigenic epitopes derived from Streptococcus agalactiae FbsA, PGK, and Cfb proteins, including: MHC-I epitopes WKSGKLWLY (FbsA) and TAALPTIKY (PGK); MHC-II epitopes LGSTIILGSSPVSAMDSV (FbsA), NNSRTISVINKLPKTGDDQ (PGK), ETPERPFVAILGGSK (Cfb), IGMVMDAIVKQPGVKS (PGK), and SIQLRNIKDNVQGTD (Cfb); and B-cell epitopes HSSQGDSNKQSFSKKV (FbsA), DTEGEAVSEGFLGLDI (PGK), KSIIGGGDSAAAAINLGRAD (Cfb), IEAITFSTQHLANKVS (Cfb), and RATIYTKSKLDKEIWN (FbsA). Each tabletop is connected in series with a sequence such as AAY, GGPPG, GGGGS to ensure the correct processing and presentation of the tabletops.
[0030] The complete fusion protein has a total length of 353 amino acids, a theoretical molecular weight of 36,179.59 Da, and an isoelectric point (pI) of 8.83.
[0031] 2) Expression and purification of fusion proteins The nucleotide sequence encoding the above fusion protein was chemically synthesized after codon optimization in E. coli, cloned into the pET-28a(+) expression vector, transformed into E. coli DH5α, screened for positive clones and sequenced to verify, and obtained the recombinant plasmid pET-28a-rCTB-FPC.
[0032] The recombinant plasmid was transformed into the expression host bacterium BL21(DE3), and a single colony was picked and inoculated into LB liquid medium containing kanamycin (100 μg / mL) and cultured at 37°C with shaking until OD. 600 The concentration was 0.6, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 30°C for 6 h. Bacterial cells were collected, sonicated, and centrifuged to obtain the supernatant. The supernatant was purified using a Ni-NTA affinity chromatography column, and the target protein was eluted with elution buffer containing 250 mM imidazole. SDS-PAGE analysis showed that the purified product exhibited a single protein band at approximately 36 kDa, with a purity >90%. The final vaccine protein rCTB-FPC was obtained after cleaving the His tag with enterokinase.
[0033] 3) Physicochemical properties and safety analysis of the fusion protein The secondary structure of rCTB-FPC was predicted using the SOPMA method. The results showed that α-helices accounted for 24.08%, extended chains accounted for 23.51%, and random coils accounted for 52.41%. The high proportion of random coils is beneficial for the flexible exposure of antigenic epitopes and immune recognition.
[0034] The sensitization of rCTB-FPC was predicted using AlgPred 2.0 software. The results showed that the protein sequence did not contain known allergen characteristic motifs and had no sensitization, indicating that the vaccine of this invention has good safety.
[0035] The three-dimensional structure of the rCTB-FPC protein was homology-modeled using AlphaFold Server, and molecular docking simulations were performed with TLR2, TLR4, and GM1 receptors, respectively. The results are as follows: Figure 2 As shown, by Figure 2 It is known that the rCTB-FPC protein can form a stable docking complex with TLR2, TLR4 and GM1 receptors, providing structural biological support for CTB-mediated targeted delivery and the immune mechanism of vaccine activation of the TLR signaling pathway.
[0036] 4) CTB adjuvant function validation (GM1-ELISA) To verify the GM1 receptor binding activity of the CTB component in the rCTB-FPC fusion protein, a sandwich GM1-ELISA method was used. GM1 gangliosides were coated onto an ELISA plate, and serially diluted rCTB-FPC fusion protein was added. Standard CTB protein was used as a positive control, and BSA as a negative control. Detection was performed using anti-CTB antibody and HRP-labeled secondary antibody, and OD was measured. 450 value.
[0037] The results are as follows Figure 3 As shown, by Figure 3 It can be seen that the binding of the rCTB-FPC fusion protein to the GM1 receptor is concentration-dependent; as the protein concentration increases, the OD... 450 The value gradually increases, reaching the highest concentration point OD. 450 The binding activity reached 1.40. This binding activity was comparable to that of the standard CTB positive control, while the negative control BSA showed no significant binding at any concentration. The results indicate that the CTB domain in the vaccine of this invention is correctly folded and maintains complete GM1 receptor binding function, and can effectively mediate targeted antigen delivery as an intramolecular adjuvant.
[0038] Example 2: Immunogenicity evaluation of rCTB-FPC vaccine 1) Laboratory animals and immunization procedures Six- to eight-week-old SPF-grade female BALB / c mice were randomly divided into a vaccine group (rCTB-FPC + Freund's adjuvant) and a negative control group (PBS + Freund's adjuvant), with 10 mice in each group. Subcutaneous injections were administered at multiple sites on days 0, 14, and 28. The vaccine group received a dose of 50 μg per mouse. Serum and spleen samples were collected 14 days after the last immunization (day 42) for immunological analysis.
[0039] 2) Antibody titer testing The level of specific IgG antibodies in the serum of immunized mice was detected by indirect ELISA. Using rCTB-FPC protein as the coating antigen, the serum of each group of mice was diluted 1:1000, and the OD was measured. 450 value.
[0040] The results are as follows Figure 4 As shown, by Figure 4 The results showed that the serum specific IgG antibody titer in the vaccine-treated mice reached its peak on day 42 post-immunization (14 days after the third immunization) and remained at a high level on day 90. The titer in the negative control group (PBS + adjuvant) showed no significant change at any time point. These results indicate that the vaccine of this invention can induce a high level of specific humoral immune response, and the antibody response has good durability.
[0041] 3) Cytokine detection Spleens were harvested from immunized mice, and splenic lymphocytes were isolated and stimulated in vitro with rCTB-FPC protein (10 μg / mL) for 72 h. The supernatant was collected, and the secretion levels of IFN-γ (Th1 marker), IL-4 (Th2 marker), and IL-17a (Th17 marker) were detected using an ELISA kit.
[0042] The results are as follows Figure 5 As shown, by Figure 5 The results showed that after stimulation with specific antigen, the spleen cells of the vaccine-treated mice secreted IFN-γ at 1324.9±93.9 pg / mL, IL-4 at 21.9±0.8 pg / mL, and IL-17a at 1240.0±204.6 pg / mL, all significantly higher than those of the negative control group (P < 0.001). In the negative control group, regardless of antigen stimulation, the secretion levels of IFN-γ, IL-4, and IL-17a remained at baseline levels. These results indicate that the vaccine of this invention can simultaneously activate Th1, Th2, and Th17 immune responses, achieving comprehensive synergy between cellular and humoral immunity.
[0043] Example 3: Evaluation of the protective effect of rCTB-FPC vaccine against viral challenge Fourteen days after the last immunization (day 42), mice in each group were intraperitoneally injected with a lethal dose (1×10⁻⁶). 8Mice were observed for 14 consecutive days with a highly virulent strain of Streptococcus agalactiae (serum type Ia) (CFU / mouse), and the survival status of each group of mice was recorded and Kaplan-Meier survival curves were plotted.
[0044] The results are as follows Figure 6 As shown, by Figure 6 The results showed that the survival rate of the vaccine group (rCTB-FPC + Freund's adjuvant) was 76.7% (23 / 30) within 15 days after challenge; while the negative control group (PBS + Freund's adjuvant) began to die on day 3 after challenge, with a survival rate of 16.7% (5 / 30) on day 15. The Log-rank test showed a highly significant difference in survival curves between the two groups (P < 0.0001). These results indicate that the vaccine of this invention provides highly effective immune protection against lethal challenge with virulent Streptococcus agalactiae strains.
[0045] On day 3 post-challenge, spleen, liver, and kidney tissues were aseptically collected from mice in each group. After weighing, the tissues were homogenized, serially diluted 10-fold, and plated onto blood agar plates. After incubation at 37°C for 24 hours, colony-forming units (CFU) were counted, and the bacterial load per gram of tissue was calculated (log(s)). 10 CFU).
[0046] The results are as follows Figure 7 As shown, by Figure 7 The results showed that after challenge, the mean bacterial load in the spleen, liver, and kidneys of the vaccine group mice was 3.79±0.32, 4.67±0.19, and 3.55±0.19 log CFU, respectively, while the bacterial load in the corresponding tissues of the negative control group was 6.38±0.20, 6.62±0.23, and 4.96±0.07 log CFU, respectively. The difference between the two groups was highly significant (P < 0.001). These results indicate that the vaccine of the present invention can effectively inhibit the colonization and spread of Streptococcus agalactiae in mice and significantly reduce tissue bacterial load.
[0047] The results of the above embodiments show that the rCTB-FPC recombinant multi-epitope chimeric subunit vaccine constructed in this invention has excellent physicochemical properties, is non-allergenic, and has complete CTB adjuvant function; it can induce high-titer and durable specific antibody responses, while fully activating Th1 / Th2 / Th17 cellular immunity; it can effectively improve the body's ability to resist Streptococcus agalactiae infection, has good immunogenicity and protective efficacy, and has the potential to be developed as a candidate vaccine for Streptococcus agalactiae.
[0048] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.
Claims
1. A multi-epitope fusion protein of Streptococcus agalactiae containing CTB intramolecular adjuvant, characterized in that, The fusion protein comprises a cholera toxin B subunit CTB intramolecular adjuvant and a multi-epitope antigen sequence fused to the CTB intramolecular adjuvant, wherein the multi-epitope antigen sequence consists of antigenic epitopes of three virulence factors from Streptococcus agalactiae: fibrinogen-binding protein A (FbsA), phosphoglycerate kinase (PGK), and cAMP factor pore-forming toxin (Cfb).
2. The agalactia-resistant streptococcal multi-epitope fusion protein containing CTB intramolecular adjuvant according to claim 1, characterized in that, The multi-epitope antigen sequence comprises at least one antigenic epitope selected from the following MHC-I epitopes, MHC-II epitopes, and B-cell epitopes: MHC-I epitopes include one or more of WKSGKLWLY from FbsA and TAALPTIKY from PGK; MHC-II epitopes include one or more of LGSTIILGSSPVSAMDSV from FbsA, NNSRTISVINKLPKTGDDQ from PGK, ETPERPFVAILGGSK from Cfb, IGVMDAIVKQPGVKS from PGK, and SIQLRNIKDNVQGTD from Cfb; B-cell epitopes include one or more of HSSQGDSNKQSFSKKV from FbsA, DTEGEAVSEGFLGLDI from PGK, KSIIGGGDSAAAAINLGRAD from Cfb, IEAITFSTQHLANKVS from Cfb, and RATIYTKSKLDKEIWN from FbsA.
3. The agalactia-resistant streptococcal multi-epitope fusion protein containing CTB intramolecular adjuvant according to claim 1, characterized in that, The amino acid sequence of the CTB intramolecular adjuvant is shown in SEQ ID NO.1; the full-length amino acid sequence of the fusion protein contains 353 amino acids.
4. The agalactia-resistant streptococcal multi-epitope fusion protein containing CTB intramolecular adjuvant according to claim 1, characterized in that, The intramolecular adjuvant of CTB is linked to the multi-epitope antigen sequence via a flexible linker peptide selected from (GGGGS)2, EAAAK, and combinations thereof.
5. The agalactia-resistant streptococcal multi-epitope fusion protein containing CTB intramolecular adjuvant according to claim 1, characterized in that, The fusion protein contains multiple antigenic epitopes linked by a linker sequence selected from one or more of AAY, GGPPG, and GGGGS.
6. A non-lactate streptococcal vaccine, characterized in that, It contains the fusion protein according to any one of claims 1-5 as an active ingredient.
7. The agalactiae streptococcal vaccine according to claim 6, characterized in that, It also contains pharmaceutically acceptable adjuvants and / or carriers.
8. Use of a fusion protein as described in any one of claims 1-5 or a vaccine as described in claim 6 or 7 in the preparation of a medicament for the prevention of Streptococcus agalactiae infection.