Improved recombinant bcg vaccine and its use

Abstract

The application provides an improved recombinant bacillus Calmette-Guerin (BCG) and application thereof, and belongs to the technical field of biotechnology. The BCG ManLAM mutant strain is obtained by knocking out the coding gene of a key glycosyltransferase MptC for synthesizing the terminal mannose of ManLAM in the BCG, and is used as the improved BCG. Compared with the wild-type BCG, the improved BCG constructed in the application can promote the functional maturation of dendritic cells and improve the anti-tuberculosis infection immune response, can be used as a new vaccine for preventing tuberculosis or as an active ingredient for preparing a medicine for preventing and treating tuberculosis, and has a wide prospect in the development and clinical application of the new vaccine. In addition, the application provides a new tool and idea for the prevention and treatment of tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an improved recombinant BCG vaccine and its applications. Background Technology

[0002] Tuberculosis is caused by infection with Mycobacterium tuberculosis (M.tb). The 2024 WHO Global Tuberculosis Report shows that in 2023, there were 10.8 million new cases of tuberculosis, resulting in 1.25 million deaths worldwide. Furthermore, the emergence, persistence, and spread of multidrug-resistant strains, delays in diagnosis, and the inability of individual immune systems to control and eliminate the pathogen have made the current situation for tuberculosis prevention and control even more severe.

[0003] Prophylactic immunization is a key strategy for reducing the incidence of tuberculosis (TB), and vaccination can significantly control the morbidity and mortality of infectious diseases. Bacillus Calmette-Guérin (BCG) is currently the only approved vaccine for preventing Mycobacterium tuberculosis infection. Developed by scientists Calmette and Guérin through 13 years of continuous attenuated passage culture of Mycobacterium bovis (BCG) 231 times, BCG's immunogenicity declines with age, thus providing effective protection only in children and having almost no effect on adult pulmonary tuberculosis. Scientists both domestically and internationally are working to develop new TB vaccines, such as subunit vaccines, whole-cell vaccines, and nucleic acid vaccines. Some new vaccines have entered clinical trials, but their protective efficacy in the population is not ideal. Therefore, there is still an urgent need to develop safe and effective new vaccine adjuvants.

[0004] Dendritic cells (DCs), as the most potent antigen-presenting cells, are receiving increasing attention in vaccine development and evaluation. After capturing vaccine molecules such as BCG, DCs migrate to lymph nodes and present antigens to naive T lymphocytes, thereby activating an adaptive immune response to control Mtb infection.

[0005] Unique glycocomplexes exist on the surface of the Mycobacterium tuberculosis cell wall, including lipomannan (LM), lipoarabinomannan (LAM), and mannose-modified lipoarabinomannan (ManLAM). Manose-modified lipoarabinomannan (ManLAM) is a major component of the cell wall surface of virulent Mycobacterium tuberculosis and BCG, and is also an immunosuppressive factor. ManLAM exerts its immunosuppressive effect by binding to the mannose receptor-MR receptor on host cells. Conversely, blocking the binding of ManLAM to MR promotes the activation of macrophages and dendritic cells by activating CD44. Therefore, the cell wall polysaccharides and glycolipids of Mycobacterium tuberculosis have the potential to serve as targets for developing novel recombinant vaccines. Summary of the Invention

[0006] To address the shortcomings of the existing technology, this invention provides an improved recombinant BCG vaccine and its application in the preparation of vaccines or drugs for the prevention and treatment of tuberculosis. The improved recombinant BCG vaccine is a recombinant BCG vaccine with a mutation in the surface glycosaminoglycan ManLAM (Mannose-capped lipoarabinomannan) of the BCG strain. Compared with wild-type BCG, the improved recombinant BCG vaccine of this invention significantly enhances antigen presentation by dendritic cells (DCs) and the immune response and protective effect against tuberculosis infection, and can be used as a novel vaccine for the prevention of tuberculosis or as an active ingredient in the preparation of drugs for the prevention and treatment of tuberculosis.

[0007] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides an improved recombinant BCG vaccine, obtained by knocking out the encoding gene of MptC, a key glycosyltransferase for synthesizing ManLAM terminal mannose, in the BCG-BCG strain, denoted as rBCGΔMptC; the encoding gene of MptC, the key glycosyltransferase for synthesizing ManLAM terminal mannose, is shown in SEQ ID NO:1.

[0009] Furthermore, the protein sequence of the key glycosyltransferase MptC for synthesizing ManLAM terminal mannose is shown in SEQ ID NO:2.

[0010] Furthermore, the gene for MptC, a key glycosyltransferase for synthesizing terminal mannose in ManLAM, was knocked out using phage-mediated homologous recombination technology.

[0011] Secondly, the present invention provides a method for preparing the improved recombinant BCG vaccine, comprising: using phage-mediated homologous recombination technology to knock out the encoding gene of the key glycosyltransferase MptC for synthesizing ManLAM terminal mannose in the BCG-BCG strain, thereby obtaining the improved recombinant BCG vaccine.

[0012] Thirdly, the present invention provides the use of the improved recombinant BCG vaccine in the preparation of vaccines or drugs for the prevention and treatment of tuberculosis.

[0013] Furthermore, the modified recombinant BCG vaccine achieves the effect of preventing and treating tuberculosis by promoting the functional maturation of dendritic cells and enhancing their immune response and protective capacity against tuberculosis infection. Specifically, the modified recombinant BCG vaccine enhances the immune response and protective capacity against tuberculosis infection by promoting the expression of antigen-presenting molecules on dendritic cells.

[0014] Fourthly, the present invention provides a vaccine or drug for the prevention and treatment of tuberculosis, including the modified recombinant BCG vaccine.

[0015] Furthermore, the vaccine or drug may also include a pharmaceutically acceptable adjuvant.

[0016] Furthermore, the dosage form of the vaccine or drug includes injectable or inhaled formulations.

[0017] This invention utilizes molecular cloning technology to knock out the key glycosyltransferase MptC, the terminal mannose-synthesizing enzyme in the ManLAM of BCG, to obtain rBCGΔMptC. The surface ManLAM of wild-type BCG and rBCGΔMptC is extracted, separated, and purified. Biotin-ConA and glycogen staining are used to identify the reduction in mannose on the surface of rBCGΔMptC. The rBCGΔMptC is then used to stimulate mouse bone marrow-derived dendritic cells (BMDCs), and flow cytometry is used to analyze the effect of rBCGΔMptC on the expression of antigen-presenting molecules in BMDCs. The results show that, compared with wild-type BCG, the rBCGΔMptC of this invention can promote the expression of CD80, CD86, and MHC-II in DCs. Furthermore, the safety and immunoprotective effect of rBCGΔMptC were tested by subcutaneously immunizing mice. Results showed that, compared to wild-type BCG, the rBCGΔMptC of this invention increased the serum IgG1 / IgG2c level and Th1 immune response in mice, and there was no significant change in serum transaminase levels after immunization (i.e., no hepatotoxicity). Following immunization, mice were infected with H37Rv via aerosol. Results showed that, compared to wild-type BCG, the rBCGΔMptC of this invention reduced the H37Rv load in mouse lung tissue and significantly alleviated the pathological damage induced by H37Rv infection.

[0018] Compared with the prior art, the advantages of the present invention are:

[0019] This invention provides an improved recombinant BCG vaccine, which is a knockout of MptC, a key glycosyltransferase involved in the synthesis of the ManLAM terminal mannose in BCG. Experiments have demonstrated that the ManLAM-mutant recombinant BCG-rBCGΔMptC constructed in this invention can enhance the expression of dendritic cell antigen-presenting molecules. Compared to wild-type BCG, the rBCGΔMptC of this invention significantly increases the IgG1 / IgG2c level and Th1 immune response in mouse serum, exhibiting good safety. Furthermore, rBCGΔMptC reduces the H37Rv load in mouse lung tissue and significantly alleviates the pathological damage induced by H37Rv infection, indicating that rBCGΔMptC has broad prospects as a novel vaccine for tuberculosis prevention or as an active ingredient in the preparation of drugs for tuberculosis control. In addition, this invention provides new tools and ideas for the prevention and treatment of tuberculosis. Attached Figure Description

[0020] Figure 1 The identification results of rBCGΔMptC in Example 2; wherein, Figure 1 A represents the ManLAM results of Biotin-ConA identification of rBCGΔMptC and wild-type BCG; Figure 1 B shows the results of ManLAM identification of rBCGΔMptC and wild-type BCG using glycogen staining.

[0021] Figure 2 This is the effect of rBCGΔMptC on the expression of DC antigen-presenting molecules in Example 3.

[0022] Figure 3 The results of the immunogenicity and safety experiments of mice immunized with rBCGΔMptC in Example 4 are shown; among them, Figure 3 A is a schematic diagram of the experimental procedure for immunizing mice with rBCGΔMptC. Figure 3 B represents the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in rBCGΔMptC-immunized mice. Figure 3 C represents the detection of the expression level of CD86, a co-stimulatory molecule of DCs, in the lung tissue of rBCGΔMptC-immunized mice; Figure 3 D represents CD4 in the lung tissue of mice immunized with rBCGΔMptC. + IFN-γ expression level and CD8 expression level of T cells + Detection of IFN-γ expression level in T cells; Figure 3 E represents the result of antibody level detection in the serum of mice immunized with rBCGΔMptC.

[0023] Figure 4 The results of the anti-tuberculosis infection immunoprotective efficacy experiment of rBCGΔMptC in Example 5 are shown; among them, Figure 4 A is a schematic diagram of the rBCGΔMptC immunization process and Mtb infection in mice; Figure 4 B represents the H37Rv load in the lung tissue of rBCGΔMptC-immunized mice after infection with H37Rv. Figure 4 C represents the staining results of lung tissue from rBCGΔMptC-immunized mice infected with H37Rv. Detailed Implementation

[0024] To enable those skilled in the art to clearly and completely understand the technical solution of the present invention, the present invention will be further described in detail below with reference to embodiments. Obviously, the embodiments described herein are only for explaining the present invention and are not intended to limit the scope of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the reagents used in the examples are all commercially available, and the operating methods used are conventional operating methods well known to those skilled in the art.

[0026] Example 1: Preparation of rBCGΔMptC

[0027] The upstream and downstream fragments of the gene to be knocked out, BCG-Mptc (the coding gene of MptC is shown in SEQ ID NO:1), were amplified and named the L arm and R arm, respectively; the primers used are as follows:

[0028]

[0029] The L and R arms were constructed into the p0004s vector, and the ligation product was transformed into DH5α. Correct clones were identified and sequenced. The plasmid p0004s-ΔBCGMptC-LR containing the homologous exchange fragment was extracted. The p0004s-ΔBCGMptC-LR plasmid was constructed into the phage plasmid phAE159. The ligation product was packaged, and positive clones were identified after infecting competent HB101 cells. The phAE159-p0004s-ΔBCGMptC-LR plasmid was extracted. The plasmid 9-p0004s-ΔBCGMptC-LR was electroporated into Mycobacterium smegmatis and cultured at 30°C to prepare the temperature-sensitive phage phAE159-p0004s-ΔBCGMptC-LR, which was then amplified to obtain a high-titer version of the phage. BCG was infected with the high-titer phage phAE159-p0004s-ΔBCGMptC-LR and cultured at 37°C for homologous recombination. Positive clones were selected to obtain BCG lacking the MptC gene, which is rBCGΔMptC.

[0030] Example 2: Identification of rBCGΔMptC

[0031] (1) Extraction of ManLAM glycosides from wild-type BCG and rBCGΔMptC: Extraction was performed according to the method described in the reference (Jordi B. Torrelles et al. Journal of Immunology, 2006, 1805-1816). The process is summarized as follows: Collect large quantities of wild-type BCG and rBCGΔMptC by centrifugation and freeze-dry them; resuspend the two bacteria in 10 volumes of chloroform / methanol (volume ratio 2:1) and defatt them at 37 °C for 12 h, centrifuge, collect the precipitate, and repeat the above steps once; centrifuge, collect the defatted bacterial cells and freeze-dry them again; resuspend the dried bacterial cells in PBS containing DNase, RNase, PMSF and 8% Triton X-114, and let them stand at 37 °C for 2 h; centrifuge, take the upper liquid phase and place it in a 37 °C incubator until clear separation occurs; take the lower liquid phase, add 9 volumes of 95% ice ethanol and place it at -20 °C. Calcium temperature increased to ℃ until precipitation occurs; centrifuge, collect the precipitate, freeze-dry, add 2 times the volume of proteinase K solution, incubate in a water bath at 60 ℃ for 2 h, and dialyze overnight at 4 ℃; freeze-dry the liquid in the dialysis bag for later use.

[0032] (2) Biotin-ConA identification method: First, the sample extracted in step (1) was subjected to ordinary SDS-PAGE electrophoresis. After electrophoresis, the target molecule was transferred to the PVDF membrane using a current of 250 mA. After blocking with 5% BSA for 2 h, it was incubated with Biotin-ConA overnight at 4 °C. After washing the membrane 3 times with TBST, HRP-avidin was added and incubated at 37 °C for 2 h. After washing three times with TBST (20 min / time) and developing with ECL chemical reagent, the identification results are as follows: Figure 1 As shown in Figure A.

[0033] (3) Glycogen staining identification method: First, the sample obtained in step (1) was subjected to ordinary SDS-PAGE electrophoresis; the PAGE gel was immersed in a solution containing 30% ethanol, 10% acetic acid, and 7 g / L periodic acid at room temperature for 20 minutes, then rinsed 5 times with double-distilled water for 3 minutes each time, and then immersed in a solution containing Schiff reagent at 4℃ for 30 minutes; rinsed 6 times with clean double-distilled water; the identification results are as follows Figure 1 As shown in B.

[0034] Example 3: Effect of rBCGΔMptC on the expression of antigen-presenting molecules in dendritic cells (DCs)

[0035] Collect induced BMDCs, count them using a cell counting chamber, and adjust the cell concentration to 1×10⁻⁶. 6 Cells were plated at 100 cells / mL for cell experiments. Cells were stimulated for 0 h, 4 h, 8 h, 18 h, and 24 h with wild-type BCG (2.5 MOI), a mixture of wild-type BCG and ManLAM (BCG 2.5 MOI, ManLAM 300 ng / mL), and rBCGΔMptC (2.5 MOI), respectively. Cells were collected, centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in 200 μL PBS. Fc receptors were first blocked with an Fc receptor blocker for 2 min. 2 μL of Fc Receptor Blocking Solution was added per 100 μL to reduce non-specific staining. Anti-mouse FITC-CD11c, PE-CD80, APC-CD86, and BV711-MHC-II were added, and the cells were incubated at 4 °C for 30 min. Unbound antibodies were washed away with PBS, and the cells were centrifuged at 300 g for 5 min at 4 °C. The supernatant was discarded, and the cells were resuspended in 300-500 μL of PBS and filtered into new flow cytometry tubes. The mean fluorescence intensity (MFI) was detected and calculated by flow cytometry. Experimental results are as follows: Figure 2 As shown, the rBCGΔMptC of the present invention can significantly promote the expression of DC antigen-presenting molecule MHC-II, co-stimulatory molecule CD86, and CD80.

[0036] Example 4: Immunogenicity and safety of rBCGΔMptC immunized mice

[0037] like Figure 3 As shown in A, wild-type BCG (5×10) was used. 5 CFU / 100 μL) or rBCGΔMptC (5×10 5 Mice were immunized subcutaneously with CFU / 100μL for 6-8 weeks, and serum was collected from each group of mice on day 14 post-immunization.

[0038] Detection of antibody levels in mouse serum: 2 mL of wild-type BCG cultured to the logarithmic growth phase was inactivated at 65 °C for 2 h. The inactivated bacterial culture was then packaged into ELISA plates treated with poly-L-lysine (100 μL per well, containing 10...). 5 CFU (BCG). Uncoated antigen was washed away with PBST, and the plate was blocked with 5% skim milk powder at 37 °C for 1 h. The plate was washed three times with PBST and patted dry. Post-immunization mouse serum (1:50 dilution) was added at 100 μL per well, and incubated at 37 °C for 2 h. Unbound serum antibodies were washed away with PBST, and rabbit anti-mouse IgG1, IgG2b, IgG2c, IgG3, and IgM were added at a 1:5000 ratio at 100 μL per well. The plate was incubated at 37 °C for 1 h. Unbound antibodies were washed away with PBST, and TMB chromogenic solution (Solution A:Solution B = 1:1) was added at 100 μL per well. The plate was incubated at 37 °C in the dark for 5-15 min, and the reaction was terminated by adding 50 μL of 2 M H2SO4 to each well. The absorbance at 450 nm was measured using a microplate reader. The experimental results are as follows: Figure 3 As shown in Figure E, compared to wild-type BCG, the rBCGΔMptC of this invention can significantly increase the levels of antibodies IgG1 and IgG2c in mouse serum, indicating that rBCGΔMptC can promote specific humoral immune responses.

[0039] Detection of ALT and AST levels in mouse serum: The ALT and AST levels in mouse serum were detected using the Elabscience® ALT / AST colorimetric assay kit. ① Standard wells: Add 5 μL of Reagent 1 (bottom-up) to each of the prepared standard wells. No liquid should adhere to the well walls during any operation. ② Add 20, 18, 16, 14, 12, and 10 μL of Reagent 3 to wells A through F, respectively. ③ Add 0, 2, 4, 6, 8, and 10 μL of Reagent 2 to wells A through F, respectively. After each application of Reagent 2, the pipette tip should be inserted into the liquid in the well and aspirated to mix thoroughly. ④ Sample wells: Add 20 μL of Reagent 3 (pre-warmed for 10 min) to the control and assay wells. Add 5 μL of sample to the assay well and aspirate to mix thoroughly. Incubate the microplate at 37 °C for 30 min. Add 20 μL of Reagent IV to the standard wells, sample control wells, and sample assay wells. Add 5 μL of sample to the sample control wells and pipette into the liquid in the wells to mix. Vortex and mix for 10 s on the microplate reader, then incubate at 37 °C for 20 min. Add 200 μL of Reagent V working solution to each well (use a multichannel pipette, and add at a consistent rate). Vortex and mix for 10 s on the microplate reader, then incubate at room temperature for 15 min. Measure the OD value of each well at 510 nm on the microplate reader. The experimental results are as follows: Figure 3 As shown in Figure B, there was no significant change in serum transaminase levels after immunization of mice with rBCGΔMptC, indicating that rBCGΔMptC has good safety.

[0040] Twenty-eight days after immunization, lung tissue from each group of mice was aseptically isolated, and single-cell suspensions were prepared using collagenase D and counted. The single-cell suspensions from the lung tissue of each group of mice were plated at a cell density of 1 × 10⁻⁶ cells / mL. 7 / well, simultaneously add inactivated BCG (10 MOI) and BFA (1:1000) for re-stimulation for 8 h. Collect cells, centrifuge at 300 g for 5 min, discard supernatant, and resuspend in 200 μL PBS. First, block Fc receptors with Fc receptor blocking agent for 2 min, adding 2 μL of Fc Receptor Blocking Solution per 100 μL to reduce non-specific staining; add APC-cy7-FSV780 to each tube, mix well, and stain at room temperature in the dark for 15 min. Add 2 mL of PBS containing 2% BSA to each tube to wash away excess live and dead stains. Then add anti-mouse PE-cy7-CD8 and PE-CD4, incubate at 4 ℃ for 30 min, and wash away unbound antibodies with PBS. Add 200 μL of fixative to each tube and fix for 30 min. The 10× cell permeabilizing agent was diluted with PBS to a 1× permeabilizing agent beforehand. 2 mL of the 1× permeabilizing agent was added to each tube to wash away the fixative. The tubes were centrifuged at 300 g for 5 min at 4 ℃, and the supernatant was discarded. Each sample was incubated with anti-mouse cytokine antibodies FITC-IFN-γ and Percp-cy5.5-IL-4, respectively, at room temperature for 30 min. After incubation, the cells were centrifuged at 300 g for 5 min at 4 ℃, the supernatant was discarded, and the cells were resuspended in 300-500 μL of PBS and filtered into new flow cytometry tubes. The results were analyzed by flow cytometry. The experimental results are as follows: Figure 3 As shown in C and 3D, compared to wild-type BCG, rBCGΔMptC significantly promoted the expression of CD86, a co-stimulatory molecule of dendritic cells (DCs), in mouse lung tissue, indicating that rBCGΔMptC inoculation promotes the functional maturation of lung DCs. Furthermore, compared to wild-type BCG, rBCGΔMptC significantly increased the secretion of IFN-γ in antigen-specific T cells in mouse lung tissue, indicating that rBCGΔMptC inoculation promotes a lung antigen-specific Th1-type cellular immune response.

[0041] Example 5: The immunoprotective effect of rBCGΔMptC against tuberculosis infection

[0042] like Figure 4 As shown in A, wild-type BCG (5×10) was used. 5 CFU / 100 μL) or rBCGΔMptC (5×10 5Mice were immunized subcutaneously with 100 CFU / 100 μL of H37Rv for 6-8 weeks. On day 28 post-immunization, each mouse was infected with 100 CFU of H37Rv using an aerosol infection device in an ABSL-III laboratory. Mice were euthanized by cervical dislocation 28 days after infection. Lungs were harvested in a clean bench. Half of the lung tissue was fixed in 4% paraformaldehyde and subjected to histopathological examination. The other half of the lung tissue was ground, serially diluted 10-fold with sterile PBS, and evenly spread on 7H10 plates. The plates were incubated at 37 °C for 28 days, and colony counts were performed. The experimental results are as follows: Figure 4 As shown, compared to wild-type BCG, the rBCGΔMptC of the present invention can significantly reduce the H37Rv load in mouse lung tissue ( Figure 4 A) and can significantly reduce pathological damage caused by H37Rv infection ( Figure 4 B).

[0043] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An improved recombinant BCG vaccine, characterized in that, The enzyme was obtained by knocking out the gene encoding MptC, a key glycosyltransferase for synthesizing ManLAM-terminal mannose, in the BCG strain; the gene encoding MptC is shown in SEQ ID NO:

1.

2. The improved recombinant BCG vaccine according to claim 1, characterized in that, The protein sequence of MptC, the key glycosyltransferase for synthesizing ManLAM terminal mannose, is shown in SEQ ID NO:

2.

3. The improved recombinant BCG vaccine according to claim 1, characterized in that, The gene for MptC, a key glycosyltransferase for the synthesis of terminal mannose in ManLAM, was knocked out using phage-mediated homologous recombination.

4. The improved method for preparing recombinant BCG vaccine according to any one of claims 1-3, characterized in that, include: By using phage-mediated homologous recombination technology, the gene encoding the key glycosyltransferase MptC, which synthesizes the terminal mannose of ManLAM, was knocked out in the BCG-BCG strain, resulting in an improved recombinant BCG vaccine.

5. The use of the improved recombinant BCG vaccine according to any one of claims 1-3 in the preparation of a vaccine or drug for the prevention and treatment of tuberculosis.

6. The application according to claim 5, characterized in that, The improved recombinant BCG vaccine achieves the effect of preventing and treating tuberculosis by promoting the functional maturation of dendritic cells and enhancing the immune response and protective capacity against tuberculosis infection.

7. A vaccine or drug for the prevention and treatment of tuberculosis, characterized in that, Including the improved recombinant BCG vaccine as described in any one of claims 1-3.

8. A vaccine or drug for preventing and treating tuberculosis according to claim 7, characterized in that, The vaccine or drug may also include pharmaceutically acceptable adjuvants.

9. A vaccine or drug for preventing and treating tuberculosis according to claim 7, characterized in that, The dosage forms of the vaccine or drug include injectable or inhaled formulations.

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