BCG (Bacillus Calmette Guerin) capsular polysaccharide as well as extraction method and application thereof in tuberculosis subunit vaccine

Abstract

The invention provides an extraction method of bacillus calmette guerin capsular polysaccharide, and a Pickering emulsion based on the bacillus calmette guerin capsular polysaccharide is prepared, and the extraction method comprises the following steps: (1) preparing tuberculosis fusion protein TP and trivalent N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP; (2) preparing GP nano particles TP / GP coated with TP; and (3) dissolving TP / GP, an interferon gene stimulating factor agonist SR717, poly (I: C) and the bacillus calmette guerin capsular polysaccharide in water to serve as a water phase, mixing the water phase with squalene serving as an oil phase, and emulsifying to obtain the Pickering emulsion based on the bacillus calmette guerin capsular polysaccharide. According to the method, the high-purity BCG capsular polysaccharide CPS is successfully extracted. The Pickering emulsion provided by the invention can induce polysaccharide and protein specific immune response in a guinea pig body and induce immune protection against BCG infection in a short time.

Description

TECHNICAL FIELD

[0001] The present application relates to a BCG capsular polysaccharide, an extraction method thereof and an application in a tuberculosis subunit vaccine. BACKGROUND

[0002] The cell wall of Mycobacterium tuberculosis is rich in various glycolipid components, which play an important role in the interaction between bacteria and hosts, not only inducing innate immune response, but also promoting adaptive immune response, and are potential antigens for preventing tuberculosis infection. Capsular polysaccharide (CPS) is located on the surface of bacteria and is closely related to the colonization of bacteria. The CPS of Mycobacterium tuberculosis is mainly composed of alpha-glucan, arabinogalactan (AM) and mannan. There is no related research on the extraction method of BCG capsular polysaccharide in the prior art. SUMMARY

[0003] In order to solve the problems in the prior art, the present application provides a BCG capsular polysaccharide (Capsular polysaccharide, CPS) and an extraction method thereof, and provides a Pickering emulsion based on the BCG capsular polysaccharide and a preparation method and application thereof. The BCG capsular polysaccharide is separated and extracted, and further combined with the Mycobacterium tuberculosis fusion protein (Mycobacterium tuberculosis fusion protein, TP) developed in the laboratory of the applicant in the early stage to construct a Mycobacterium tuberculosis capsular polysaccharide-protein / Pickering emulsion as a Mycobacterium tuberculosis vaccine, and the anti-infection protection induced by the vaccine is preliminarily evaluated.

[0004] The first object of the present application is to provide an extraction method of BCG capsular polysaccharide, comprising the following steps:

[0005] (1) removing protein by a hot phenol method and a Sevag method, and collecting supernatant; in the Sevag method, the volume ratio of chloroform to n-butanol is 4:1;

[0006] (2) removing lipid by using a chloroform-methanol solution, and collecting upper solution; in the chloroform-methanol solution, the volume ratio of chloroform to methanol is 1:1;

[0007] (3) removing nucleic acid by using DNAase and RNAase, and removing residual nucleic acid by using cetyltrimethylammonium bromide, and collecting supernatant;

[0008] (4) alcohol precipitation of the supernatant and collection of the precipitate to obtain BCG capsular polysaccharide.

[0009] The present application establishes a high-efficiency and stable extraction method of BCG capsule polysaccharide through systematic optimization and repeated experiments on the removal method of proteins and nucleic acids in BCG capsule. The present application first removes most of the protein components by combining Sevag method and hot phenol method; then removes nucleic acids by combining RNase and DNase enzymolysis, and further removes nucleic acids by using low-concentration CTAB, thereby significantly improving the purity of polysaccharide; finally, the present application obtains polysaccharide with high purity by ethanol precipitation method. The BCA protein detection kit and Nanodrop One ultramicro spectrophotometer detection show that the concentration of proteins and nucleic acids in the extracted capsule polysaccharide is extremely low.

[0010] As preferred, the method comprises the following steps:

[0011] (1) collecting BCG bacterial bodies;

[0012] (2) resuspending the BCG bacterial bodies with PBS, adding glass beads and oscillating to promote the shedding of the capsule, centrifuging to collect the supernatant and concentrating;

[0013] (3) adding an equal volume of 80% phenol, reacting at 70℃ for 1.5h to remove proteins, and centrifuging to collect the supernatant;

[0014] (4) adding an equal volume of chloroform-n-butanol solution and oscillating to remove residual proteins, and centrifuging to collect the upper solution;

[0015] (5) adding an equal volume of chloroform-methanol solution and oscillating to remove fat, and centrifuging to collect the upper solution;

[0016] (6) adding DNAase and RNAase respectively, and standing at 37℃ for 12-14h to remove nucleic acids, and centrifuging to collect the upper solution;

[0017] (7) adding 0.08% cetyltrimethylammonium bromide, and standing at 4℃ for 12-14h to further remove nucleic acids, and centrifuging to collect the supernatant;

[0018] (8) adding anhydrous ethanol to make the final volume 75%, and adding sodium acetate, and standing at 4℃ for 12-14h to perform alcohol precipitation, and centrifuging to collect the precipitate;

[0019] (9) drying the precipitate to obtain BCG capsule polysaccharide.

[0020] The second object of the present application is to provide a BCG capsule polysaccharide extracted by the above extraction method; as preferred, the purity of the BCG capsule polysaccharide reaches 95.92%.

[0021] The third object of the present application is to provide a preparation method of Pickering emulsion based on BCG capsule polysaccharide, comprising the following steps:

[0022] (1) Preparation of tubercle fusion protein TP and trivalent N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP;

[0023] The tubercle fusion protein TP is obtained by mixing LT33 and LT57;

[0024] The preparation method of the N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP is:

[0025] a. Mix the activated PLGA, NH2-PEG-Alkyen, DMAP, EDC-HCl and triethylamine, and react under inert gas at room temperature to obtain PLGA-PEG-Alkyen;

[0026] b. Mix PLGA-PEG-Alkyen, N3-Tri-GalNAc, Vc-Na and CuSO4 solution, and stir to react at room temperature to obtain N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP;

[0027] (2) Preparation of TP-coated GP nanoparticles TP / GP:

[0028] a. Mix the inner aqueous phase with the oil phase to prepare the initial milk;

[0029] The inner aqueous phase is obtained by dissolving the tubercle fusion protein TP of step (1) in PBS;

[0030] The oil phase is obtained by dissolving the trivalent N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP of step (1) in dichloromethane;

[0031] b. Rapidly add the initial milk of step a to the outer aqueous phase, then perform ice bath ultrasonic treatment under a power of 600W for 8-10min, and then stir for 4h-6h; the outer aqueous phase is a 2% polyvinyl alcohol solution;

[0032] c. Centrifuge to collect the precipitate to obtain TP-coated GP nanoparticles TP / GP;

[0033] (3) Preparation of a Pickering emulsion based on BCG capsular polysaccharide:

[0034] Dissolve TP / GP, interferon gene stimulator agonist SR717, Toll-like receptor 3 agonist polyinosinic-polycytidylic acid (Poly(I:C)) and the above-mentioned BCG capsular polysaccharide in water as the aqueous phase, and mix squalene as the oil phase, emulsify to obtain a Pickering emulsion based on BCG capsular polysaccharide.

[0035] Preferably, the tuberculosis fusion protein TP is obtained by mixing LT33 and LT57 in a weight ratio of 1:1.

[0036] As a preferred embodiment, in step (2), the specific method for preparing the colostrum is as follows: the inner aqueous phase is added to the oil phase, and then ultrasonically treated in an ice bath at a power of 400W for 4-5 minutes.

[0037] Preferably, in step (3), the weight ratio of TP / GP, interferon gene stimulating factor agonist SR717, Toll-like receptor 3 agonist Poly I:C and BCG capsular polysaccharide is 50:5:1.25:0.5.

[0038] The fourth objective of this invention is to provide a Pickering emulsion based on BCG capsular polysaccharide, which is prepared using the method described above.

[0039] The fifth object of the present invention is to provide the application of the Pickering emulsion based on BCG capsular polysaccharide described above in the preparation of Mycobacterium tuberculosis vaccine.

[0040] A sixth object of the present invention is to provide a vaccine comprising the Pickering emulsion based on BCG capsular polysaccharide described above.

[0041] This invention successfully extracted BCG capsular polysaccharide CPS with high purity (very low protein and nucleic acid concentrations). The capsular polysaccharide-protein / Pickering emulsion CPS-TP / P-SP of this invention can induce polysaccharide and protein-specific immune responses in guinea pigs and induce immune protection against BCG infection in a short period of time. Attached Figure Description

[0042] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0043] Figure 1 A flowchart of the immunization program for subunit vaccines in guinea pigs.

[0044] Figure 2 The results are for BCG culture. (A) Colonies after 2 weeks of BCG inoculation on Löwenstein medium. (B) Colonies after 3 weeks of BCG inoculation on Soton medium.

[0045] Figure 3 This is freeze-dried CPS.

[0046] Figure 4Figure 1. Gel chromatography of CPS. (A) Gel chromatography of CPS on Sepharose S-100 column. The detection signals at 206 nm, 280 nm and 260 nm of the extracted CPS were shown. (B) Chemical profile of CPS collected from Sepharose S-100 column.

[0047] Figure 5 Figure 5. Serum CPS-specific IgG antibody levels in TB patients. (A) CPS-specific IgG antibody levels. (B) CPS-specific IgG antibody titers. The numbers represent the GMT values. Data were shown as mean ± SD (n = 5). *p < 0.05.

[0048] Figure 6 Figure 6. Guinea pig lymphocyte proliferation levels. (A) Lymphocyte proliferation levels detected by flow cytometry after in vitro stimulation with CPS. (B) Lymphocyte proliferation levels detected by flow cytometry after in vitro stimulation with LRP. (C) Statistical analysis of the proportion of lymphocyte proliferation after in vitro stimulation with CPS. (D) Statistical analysis of the proportion of lymphocyte proliferation after in vitro stimulation with LRP. Data were shown as mean ± SD (n = 4). *p < 0.05.

[0049] Figure 7 Figure 7. Antigen-specific antibody levels induced by vaccines in guinea pigs. (A) LT57-specific IgG antibody titers. (B) LT57-specific IgM antibody titers. (C) CPS-specific IgG antibody titers. (D) CPS-specific IgM antibody titers. Data were shown as mean ± SD (n = 4).

[0050] Figure 8 Figure 8. Evaluation of the immune protection induced by CPS-TP / P-SP 2 weeks after the last immunization. (A) Panoramic view of the lung of a guinea pig infected with BCG strain after HE staining. (B) Local view of the lung of a guinea pig infected with BCG strain after HE staining. (C) Statistical analysis of the percentage of inflammatory lesion area in the lung of guinea pigs. (n = 4-5) *p < 0.05, **p < 0.01.

[0051] Figure 9 Figure 9. Evaluation of the immune protection induced by CPS-TP / P-SP 6 weeks after the last immunization. (A) Panoramic view of the lung of a guinea pig infected with H37Ra strain after HE staining. (B) Local view of the lung of a guinea pig infected with H37Ra strain after HE staining. (C) Percentage of inflammatory lesion area in the lung of guinea pigs. (D) Statistical analysis of the bacterial load count in the lung of guinea pigs. Data were shown as mean ± SD (n = 3-4). *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0052] The following examples facilitate a better understanding of the present application, but do not limit the present application. The experimental methods in the following examples are conventional methods, unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies, unless otherwise specified. The quantitative tests in the following examples are set up with three repeated experiments, and the results are averaged.

[0053] Example 1

[0054] I. Experimental process

[0055] 1. Extraction of the capsular polysaccharide

[0056] 1.1 Cultivation of BCG

[0057] The stored BCG strain was taken out from the -80°C refrigerator and placed in the 4°C refrigerator to completely thaw. After thawing, 200 μL of the bacterial solution was evenly smeared on the surface of the Lowenstein-Jensen medium, and then placed in a 37°C constant temperature incubator for 2 weeks of cultivation. When the colonies grew to an appropriate density, they were transferred to the Sauton medium and placed back in the 37°C incubator for 3 weeks of further cultivation, and finally the BCG strain was obtained.

[0058] 1.2 Extraction of CPS

[0059] (1) The freshly cultivated BCG bacterial solution was transferred to a centrifuge tube, centrifuged at 3000 rpm for 10 min, and the supernatant was discarded to collect the bacterial bodies.

[0060] (2) 10 g of the bacterial bodies were resuspended with 10 mL of PBS, and 50 g of glass beads were added, and oscillated at 180 rpm for 2 h to promote the shedding of the capsules. The supernatant was collected by centrifugation at 8000 rpm for 30 min. The supernatant was concentrated to 1 / 5 of the original volume using a rotary evaporator, and the supernatant mixture containing the capsular polysaccharide was obtained.

[0061] (3) An equal volume of 80% (100 mL contains 80 g of weight) phenol was added, and the reaction was carried out at 70°C for 1.5 h to remove the protein. The supernatant was collected by centrifugation at 8000 rpm for 30 min.

[0062] (4) An equal volume of chloroform-n-butanol (chloroform and n-butanol in a volume ratio of 4:1) solution was added, and oscillated at 180 rpm for 20 min to remove the remaining protein again. The upper solution was collected by centrifugation at 8000 rpm for 30 min.

[0063] (5) An equal volume of chloroform-methanol (chloroform and methanol in a volume ratio of 1:1) solution was added, and oscillated at 180 rpm for 12 h to remove the fat. The upper solution was collected by centrifugation at 8000 rpm for 30 min.

[0064] (6) Respectively add DNAse (final concentration of 10 μg / mL) and RNAse (final concentration of 10 μg / mL), and let stand overnight at 37℃ to remove nucleic acid. Centrifuge at 8000 rpm for 30 min, and collect the supernatant.

[0065] (7) Add 0.08% (0.08 g in 100 mL) of cetyltrimethylammonium bromide (CTAB), and let stand overnight at 4℃ to further remove residual nucleic acid. The next day, centrifuge at 10000 rpm for 20 min, and collect the supernatant.

[0066] (8) Add anhydrous ethanol to a final volume of 75%, and add 10 mmol of sodium acetate (add ions to promote the precipitation of polysaccharides), and let stand overnight at 4℃. Centrifuge at 8000 rpm for 20 min, and collect the precipitate.

[0067] (9) Freeze-dry to obtain BCG capsular polysaccharide CPS.

[0068] 2. Identification of capsular polysaccharide

[0069] 2.1 Detection of CPS content

[0070] Dissolve the extracted CPS in PBS, and detect the polysaccharide content by the phenol-sulfuric acid method. Specifically, first, take 0, 0.4, 0.8, 1.2, 1.6, and 2.0 mL of a 40 μg / mL glucose standard solution, and supplement with ddH2O to a final volume of 2 mL. Next, add 1 mL of a 6% phenol solution and 5 mL of concentrated sulfuric acid to each tube in turn, mix well, and heat in a boiling water bath for 15 min, and cool to room temperature. Measure the OD value of each tube at a wavelength of 490 nm, and draw a standard curve. Then, dilute the CPS 10-fold with PBS, and determine the OD value in the same way, and calculate the concentration of the CPS according to the standard curve.

[0071] 2.2 Detection of nucleic acid content of CPS

[0072] After dissolving the CPS in PBS, use a Nanodrop One ultramicro spectrophotometer to detect the nucleic acid content in the sample.

[0073] 2.3 Detection of protein content of CPS

[0074] After dissolving the CPS in PBS, use a BCA detection kit to detect the protein content in the sample.

[0075] 2.4 Detection of molecular size distribution of CPS

[0076] The elution position of CPS was detected by Sepharose S-100 column chromatography with PBS as buffer and a flow rate of 1 mL / min. The samples were collected in steps of 4 mL / tube, and the polysaccharide concentration in each tube was detected by phenol-sulfuric acid method. The chromatogram was plotted according to the polysaccharide concentration determined in each tube.

[0077] 3. Detection of serum CPS-specific antibody level in tuberculosis patients

[0078] 3.1 Preparation of solutions

[0079] (1) Coating buffer: 2.93 g of NaHCO3 and 1.59 g of Na2CO3 were weighed and dissolved in 800 mL of ddH2O, and the pH value was adjusted to 9.6 before being diluted to 1 L.

[0080] (2) Washing buffer: 8 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, 2.9 g of KH2PO4·12H2O and 0.5 mL of Tween 20 were weighed and dissolved in 800 mL of ddH2O, and the pH value was adjusted to 7.4 before being diluted to 1 L.

[0081] (3) Diluent: 1 g of bovine serum albumin (BSA) powder was weighed and dissolved in 100 mL of PBS to obtain the diluent.

[0082] (4) Blocking solution: 5 g of BSA powder was weighed and dissolved in 100 mL of PBS to obtain the blocking solution.

[0083] (5) Termination solution: 21.7 mL of concentrated sulfuric acid was added dropwise into 178.3 mL of ddH2O to obtain the termination solution.

[0084] 3. Detection of serum CPS-specific antibody level in tuberculosis patients

[0085] Dilute the BCG capsular polysaccharide CPS extracted in step 1.2 with coating buffer to a final concentration of 5 μg / mL. Add the coating buffer containing the CPS to the ELISA plate at a volume of 100 μL / well, and place it in the 4°C refrigerator for overnight standing. The next day, discard the liquid in the wells, add 300 μL of washing buffer to each well, stand for 1 min, discard the liquid in the wells, and repeat 4 times, and pat dry the residual liquid in the wells on filter paper each time. Add 300 μL of blocking solution to each well, and place it in the 37°C incubator for 1 h of incubation. Discard the liquid in the wells, and wash 4 times with washing buffer, and pat dry the residual liquid in the wells. Start diluting the TB serum and the healthy donor HD serum by the ratio of 1:200 to 1:25600. Add 100 μL of the diluted serum to each well, and place it in the 37°C incubator for 2 h of incubation. Discard the liquid in the wells, and wash 4 times with washing buffer, and pat dry the residual liquid in the wells. Dilute the goat anti-human IgG antibody with the diluent at a ratio of 1:8000, add 100 μL of the diluted antibody to each well, and place it in the 37°C incubator for 1 h of incubation. Discard the liquid in the wells, and wash 4 times with washing buffer, and pat dry the residual liquid in the wells. Add 100 μL of TMB color developing solution to each well, and place it in the 37°C incubator for 10 min of color development. Finally, add 50 μL of stop solution to each well, and detect the OD value with a microplate reader. 450 The antibody titer is the highest serum dilution ratio with which the OD 450 value of the test well is greater than 2.1 of the blank well, and the geometric mean titer (GMT) is calculated. GMT = (X1.X2.X3…Xn)1 / n (n represents the number of samples in each group, and X represents each antibody titer value).

[0086] 4. Preparation and immunization of capsular polysaccharide-protein / Pickering emulsion

[0087] 4.1 Preparation of capsular polysaccharide-protein / Pickering emulsion

[0088] The applicant uses TP-coated GP nanoparticles (TP / GP) as a stabilizer to prepare a capsular polysaccharide-protein / Pickering emulsion.

[0089] 4.1.1 Preparation of tuberculosis fusion protein TP

[0090] 4.1.1.1 Preparation of LT33 (ESAT6-linker-CFP10-Rv1738)

[0091] LT33 is a fusion protein in Chinese patent CN 115920021 A (see paragraphs 33-49 of the patent for the specific preparation method and related sequences).

[0092] LT33 is a fusion of the deleted region antigens ESAT6, CFP10 of the tuberculosis subunit vaccine and the latency antigen Rv1738 of the tuberculosis subunit vaccine.

[0093] 4.1.1.2 Preparation of LT57 (PE25-linker-PPE41-Rv0518):

[0094] LT57 is a fusion protein in Chinese patent CN 119192393 A (see paragraphs 58-67 and 101-107 of the patent for specific preparation methods and related sequences).

[0095] LT57 is a fusion of the PE / PPE family antigens PE25 and PPE41 of Mycobacterium tuberculosis and the cell wall antigen Rv0518 of Mycobacterium tuberculosis.

[0096] 4.1.1.3 Preparation of tuberculosis fusion protein TP

[0097] LT33 and LT57 are mixed in a mass ratio of 1:1 to prepare the tuberculosis fusion protein TP.

[0098] 4.1.2 Preparation of Tri-GalNAc-modified poly lactic-co-glycolic acid-polyethylene glycol (Tri-GalNAc-Poly lactic-co-glycolic acid-polyethylene glycol, Tri-GalNAc-PLGA-PEG, referred to as "GP") in trivalent state

[0099] The preparation of GP is carried out according to the method in the following document: Gong Y, Jia H, Dang W, et al. Enhancing cell-mediated immunity through dendritic cell activation: the role of Tri-GalNAc-modified PLGA-PEG nanoparticles encapsulating SR717 [J]. Frontiers in immunology, 2024, 15: 1490003. Specifically as follows:

[0100] First, poly(lactic-co-glycolic acid) (PLGA) (0.8 g; 0.02 mmol) was dissolved in 8 mL of dichloromethane (DCM), after complete dissolution, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) (38.34 mg; 0.2 mmol) and N-hydroxysuccinimide (NHS) (23 mg; 0.2 mmol) were added, nitrogen was filled, and the reaction was stirred at room temperature for 24 h. The reaction was precipitated with ice-cold ether, and the resulting solid was washed with a mixture of ice-ether and methanol (4:1, v / v) three times, and the solid was collected by centrifugation. The collected solid was completely dried under vacuum, and the activated PLGA was obtained.

[0101] Then, the activated PLGA (0.4 g; 0.01 mmol) was dissolved in 4 mL of DCM, and NH2-PEG-Alkyne (0.102 g; 0.03 mmol) was dissolved in 1 mL of DCM. The completely dissolved NH2-PEG-Alkyne was added dropwise to the activated PLGA, 4-dimethylaminopyridine (DMAP) (6 mg; 0.05 mmol), EDC-HCl (19.17 mg; 0.05 mmol), and triethylamine 50 μL were added, nitrogen was filled, and the reaction was stirred at room temperature for 24 h. The reaction was precipitated with cold ether and washed with a mixture of methanol and ether (4:1, v / v) three times, and the solid was collected by centrifugation. The precipitate was completely dried under vacuum, and the PLGA-PEG-Alkyne was obtained.

[0102] Subsequently, the PLGA-PEG-Alkyne (0.1 g; 0.0025 mmol) was dissolved in 2 mL of DMSO, and N3-Tri-GalNAc (34.5 mg; 0.025 mmol) was dissolved in 1 mL of DMSO. The completely dissolved N3-Tri-GalNAc was added dropwise to the PLGA-PEG-Alkyne, Vc-Na (4.95 mg; 0.025 mmol) was added, and 60 μL of a CuSO4(10 mg / mL) solution was added, and the reaction was stirred at room temperature for 2 h. The reaction was precipitated with ddH2O, and the solid was repeatedly washed with ddH2O, and the solid was collected by centrifugation and completely dried in vacuum to obtain Tri-GalNAc-PLGA-PEG (GP).

[0103] 4.1.3 Preparation of TP-coated GP nanoparticles (TP / GP)

[0104] The tubercle fusion protein TP (1 mg) prepared in step 4.1.1.3 was dissolved in 1 mL of PBS as the inner aqueous phase. 50 mg of GP prepared in step 4.1.2 was dissolved in 2 mL of dichloromethane as the oil phase. The inner aqueous phase was slowly and uniformly poured into the oil phase, and then ice-bath ultrasonic treatment was performed at a power of 400 W for 4 min to prepare the primary emulsion. 2% (2 g of weight in 100 mL) polyvinyl alcohol solution was prepared as the external aqueous phase. The primary emulsion was quickly poured into the external aqueous phase, and then ice-bath ultrasonic treatment was performed at a power of 600 W for 10 min to prepare the multiple emulsion. Stirring was performed in a fume hood for 4 h, the nanoparticles were solidified, and dichloromethane was volatilized. Centrifugation was performed at 12000 rpm for 10 min to collect the nanoparticle precipitate. Washing was performed with ddH2O three times to wash away the excess PVA on the surface of the nanoparticles, and the nanoparticles TP / GP were obtained.

[0105] 4.1.4 Preparation of Capsular polysaccharide-protein / Pickering emulsion

[0106] 50 mg of TP / GP was dissolved in 4.5 mL of ddH2O, 5 mg of Stimulator of Interferon Genes (STING) agonist SR717, 1.25 mg of Toll-like receptors (TLR3) agonist Polyriboinosinic polyribocytidylic acid (Poly(I:C)), and 500 μg of CPS were added and shaken to mix; 500 μL of Squalene (SQ) was added as the oil phase, and ultrasonic treatment was performed at a power of 500 W for 2 min to obtain the Capsular polysaccharide-protein / Pickering emulsion containing SR717 and Poly(I:C) (CPS-TP / GP-SQ-SR717-Poly(I:C), referred to as “CPS-TP / P-SP”).

[0107] 4.1.5 Preparation of TP / P-SP

[0108] In the preparation of TP / P-SP, the only difference compared with the preparation of CPS-TP / P-SP is that no CPS was added, and the rest of the steps and parameters were the same.

[0109] 4.2 Guinea pig vaccination program

[0110] The experimental guinea pigs were divided into four groups, namely PBS group, BCG group, TP / P-SP group and CPS-TP / P-SP group. The immunization dose of the BCG group was 5×10 6 CFU / guinea pig, and the immunization dose of the PBS, TP / P-SP and CPS-TP / P-SP groups was 200 μL / guinea pig.

[0111] The immunization program is divided into two, as follows:

[0112] (1) BCG infection group: the BCG group was immunized once by subcutaneous immunization in the groin at week 4, and the other three groups were immunized three times by subcutaneous immunization in the groin at weeks 0, 2, and 4, respectively. Two weeks after the last immunization, guinea pigs were infected with BCG by nasal instillation, and three weeks after infection, lung tissue was collected and evaluated for lung pathological damage by HE staining (A). Figure 1

[0113] (2) H37Ra infection group: the BCG group was immunized once by subcutaneous immunization in the groin at week 0, and the other three groups were immunized three times by subcutaneous immunization in the groin at weeks 0, 3, and 6, respectively. Six weeks after the last immunization, the immunogenicity was detected, and guinea pigs were infected with the H37Ra strain by nasal instillation, and three weeks after infection, lung tissue was collected for CFU counting and evaluation of lung pathological damage by HE staining (B). Figure 1

[0114] Figure 1 Guinea pig immunization program for subunit vaccine.

[0115] 5. Vaccine immunogenicity evaluation

[0116] 5.1 Detection of spleen lymphocyte proliferation level

[0117] Guinea pig spleen lymphocytes were isolated, and lymphocyte proliferation was detected by the EdU method. Mixed antigens (LT33+Rv0518+PE25-linker-PPE41, LRP, prepared according to the method of step 4.1.1.1, and the preparation methods of LT33, Rv0518, and PE25-linker-PPE41 are as described in Chinese Patent CN119192393A) or CPS were added to stimulate lymphocytes for 3 days, and flow cytometry was used to detect the proliferation proportion of lymphocytes. The specific operation steps of the experiment are as follows:

[0118] The experimental method for detecting the proliferation level of lymphocytes by the EdU method is as follows: 5 x 10 6 ​​Spleen lymphocytes of guinea pigs were inoculated in 24-well plates, 10 μg / mL mixed antigen LRP or CPS was added to each well, and the plates were placed in a cell incubator at 37°C, 5% CO2 for 2 h. Then 1 μL of EdU reagent (10 μM / well) was added to each well, and the plates were placed in a cell incubator at 37°C, 5% CO2 for culture. After 3 days, the cells were collected by centrifugation at 300 x g for 5 min. The cells were washed with 500 μL of flow buffer, and collected by centrifugation at 300 x g for 5 min. The cells were fixed with 500 μL of 4% paraformaldehyde. After 15 min, the cells were collected by centrifugation at 300 x g for 5 min. The cells were broken with PBS containing 0.3% Triton X-100. After 15 min, the cells were collected by centrifugation at 350 x g for 5 min. The Click reaction solution was prepared according to the EdU-647 Cell Proliferation Assay Kit instructions, and 100 μL of the Click reaction solution was used to resuspend the cells, which were reacted in the dark for 20 min. The cells were collected by centrifugation at 350 x g for 5 min. The cells were resuspended with 200 μL of flow buffer, filtered through a 300-mesh sieve, and the proliferation level of the lymphocytes was detected by flow cytometry.

[0119] 5.2 Detection of antigen-specific antibody levels in guinea pig serum

[0120] ELISA plates were coated with LT57 and CPS at a dose of 5 μg / mL, and the antigen-specific antibody levels in guinea pig serum were detected by ELISA, according to the method of step 3.1.

[0121] 6. Preliminary evaluation of vaccine protection effect

[0122] 6.1 Infection of H37Ra strain and detection of bacterial load in guinea pig lungs

[0123] The guinea pigs were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 50 mg / kg. After successful anesthesia, each guinea pig was infected with 5 x 10 6 CFU of H37Ra strain by nasal instillation. After 3 weeks of infection, the lungs of the guinea pigs were carefully separated, accurately weighed, and ground. 1 mL of sterile PBS was added to the ground lung tissue to prepare a lung grinding solution. The lung grinding solution was diluted at a ratio of 1:10. 100 μL of each gradient sample was taken and uniformly coated on 7H10 solid culture medium containing OADC. At the same time, the culture medium was set up and placed in a 37°C incubator for 4 weeks, followed by CFU counting. The final CFU counting result was expressed as log10. CFU = colony number x dilution factor x final volume of resuspension (mL) / tissue weight (g) / coating liquid volume (mL).

[0124] 6.2 Evaluation of lung pathological damage in guinea pigs

[0125] The middle lobe of the lungs of guinea pigs three weeks after infection was harvested and stained with hematoxylin and eosin (HE) to evaluate the pathological damage to the guinea pig lungs.

[0126] 7. Statistical Analysis

[0127] All experimental data in this invention were analyzed and plotted using Graphpad Prism 8.0 software. Data are expressed as mean ± standard deviation (SD). One-way ANOVA and unpaired t-tests were used to compare two groups. Tukey's post-hoc test was used to compare multiple groups. Mann-Whitney U test was used for samples that did not conform to normal distribution or had unequal variances. p < 0.05 was considered statistically significant.

[0128] II. Experimental Results

[0129] 1. Extraction and identification of capsular polysaccharides

[0130] 1.1 BCG culture

[0131] First, the BCG strain, frozen at -80℃, was thawed and inoculated onto Löwenstein-Germain-Latin-Agar medium, then incubated at 37℃. After 2 weeks, cauliflower-like colonies were observed forming on the Löwenstein-Germain-Latin-Agar medium. Figure 2 A). Next, these colonies were transferred to Suton medium for further cultivation. After 3 weeks, a bacterial film formed on the surface of the Suton medium, which was the BCG strain ( Figure 2 B).

[0132] Figure 2 The results are for BCG culture. (A) Colonies after 2 weeks of BCG inoculation on Löwenstein medium. (B) Colonies after 3 weeks of BCG inoculation on Soton medium.

[0133] 1.2 Extraction and chemical characterization of CPS (purity detection of capsular polysaccharides)

[0134] The freeze-dried CPS is milky white. Figure 3 A certain amount of lyophilized CPS was dissolved in PBS, and the polysaccharide concentration was determined to be 1.2 mg / mL using the phenol-sulfuric acid method. The protein concentration was determined to be 18.3 μg / mL using a BCA kit, and the nucleic acid concentration was determined to be 32.7 μg / mL using a Nanodrop One micro spectrophotometer. Therefore, the polysaccharide purity in the CPS prepared in this invention reached 95.92%.

[0135] Figure 3 This is freeze-dried CPS.

[0136] 1.3 CPS Molecular Size Distribution Detection (Physicochemical Property Detection)

[0137] CPS molecular size distribution was detected by Sepharose S-100 column Figure 4 A) As can be seen from the figure, the detection spectrum at 206 nm wavelength showed an absorption peak at about 155 mL. After collecting samples of 4 mL per tube, the polysaccharide content in the samples was detected by phenol sulfuric acid method, and a chromatography chemical map was drawn according to the polysaccharide concentration determined in each sample (4B). As can be seen, the chromatography chemical map drawn corresponds to the absorption peak shape at 206 nm wavelength in the gel chromatogram, further confirming that the peak is the CPS target peak. The peak is relatively symmetrical on both sides, showing a typical "bell-shaped" distribution, indicating that the molecular weight of the extracted CPS is relatively uniform.

[0138] Figure 4 CPS gel chromatogram. (A) Sepharose S-100 column gel chromatogram of CPS, as shown in the figure, the detection signals of the extracted CPS at 206 nm, 280 nm and 260 nm wavelengths. (B) Sepharose S-100 column distribution collection chemical map of CPS.

[0139] 2. Specific IgG antibodies to CPS exist in the serum of tuberculosis patients

[0140] The serum of tuberculosis (TB) patients with positive acid-fast staining of sputum smear and the serum of health donors (HD) were collected. The specific IgG antibodies to CPS in the serum were detected by ELISA, and the antibody geometric mean titer (GMT) was calculated. The results showed that the TB patient serum had a higher level of specific antibodies to CPS than the HD serum ( Figure 5 ).

[0141] Figure 5 The specific IgG antibody levels to CPS in the serum of tuberculosis patients. (A) The specific IgG antibody levels to CPS. (B) The specific IgG antibody titers to CPS, the numbers represent the GMT values. The data are shown as mean ± standard deviation (n = 5). *p < 0.05.

[0142] 3. CPS-TP / P-SP induced the generation of memory cells in guinea pigs

[0143] After antigen stimulation, memory cells can proliferate and differentiate into effector cells. Guinea pig spleen lymphocytes were isolated 6 weeks after the last immunization of subunit vaccine, and stimulated by CPS or LRP in vitro for 3 days, respectively. The proliferation ability of memory cells was analyzed by EdU method. The results showed that the proliferation level of lymphocytes in CPS-TP / P-SP group was significantly higher than that in PBS, BCG and TP / P-SP groups after CPS stimulation (p<0.05). The proliferation level of lymphocytes in CPS-TP / P-SP group was significantly higher than that in PBS group after mixed protein antigen stimulation (p<0.05). Figure 6 ) The above results show that the Pickering emulsion of CPS-TP / P-SP combined protein and polysaccharide antigens can induce the production of memory cells in guinea pigs.

[0144] Figure 6 The proliferation level of guinea pig lymphocytes. (A) Flow cytometry detection of lymphocyte proliferation level after CPS in vitro stimulation. (B) Flow cytometry detection of lymphocyte proliferation level after LRP in vitro stimulation. (C) Statistical analysis of lymphocyte proliferation ratio after CPS in vitro stimulation. (D) Statistical analysis of lymphocyte proliferation ratio after LRP in vitro stimulation. Data are presented as mean ± standard deviation (n=4). *p<0.05.

[0145] 4. CPS-TP / P-SP induces antibody production in guinea pigs

[0146] The levels of LT57 and CPS antigen-specific IgG and IgM antibodies in guinea pig serum were detected at 1, 2 and 6 weeks after the last immunization of subunit vaccine, respectively, and the antibody geometric mean titer (GMT) was calculated. The results showed that the levels of LT57-specific IgM and IgG antibodies in the TP / P-SP and CPS-TP / P-SP groups gradually increased from 1 to 2 weeks after the last immunization of subunit vaccine, and decreased from 2 to 6 weeks after the last immunization. Similarly, the CPS-specific IgM antibody level in the CPS-TP / P-SP group was higher at 2 weeks after the last immunization of subunit vaccine, and decreased at 6 weeks. However, the CPS-specific IgG in the CPS-TP / P-SP group remained at a low level Figure 7 ).

[0147] Figure 7 The levels of antigen-specific antibodies induced by vaccines in guinea pigs. (A) Change trend of LT57-specific IgG antibody titer. (B) Change trend of LT57-specific IgM antibody titer. (C) Change trend of CPS-specific IgG antibody titer. (D) Change trend of CPS-specific IgM antibody titer. Data are presented as mean ± standard deviation (n=4).

[0148] 5. CPS-TP / P-SP immunization 2 weeks later reduced lung pathological damage in guinea pigs

[0149] Two weeks after the last immunization with subunit vaccine, guinea pigs were infected with BCG by intranasal route (5 x 10 6 CFU / guinea pig) and lungs were removed 3 weeks later. Lung pathological damage was evaluated by HE staining. The results showed that significant exudative inflammatory lesions were observed in PBS group. In contrast, lung exudative lesions were reduced in BCG, TP / P-SP and CPS-TP / P-SP groups compared to PBS group (p<0.05). The proportion of inflammatory lesions in CPS-TP / P-SP group was reduced compared to TP / P-SP group (p<0.01). Figure 8

[0150] Figure 8 Evaluation of the immune protection induced 2 weeks after the last immunization with CPS-TP / P-SP. (A) Lung panoramic scan of guinea pigs infected with BCG strain after HE staining. (B) Lung local scan of guinea pigs infected with BCG strain after HE staining. (C) Percentage of lung inflammatory lesion area. (n=4-5) *p<0.05, **p<0.01.

[0151] 6. No difference in lung bacterial load and pathological damage in guinea pigs immunized with TP / P-SP and CPS-TP / P-SP 6 weeks later

[0152] Six weeks after the last immunization with subunit vaccine, guinea pigs were infected with M. tuberculosis attenuated strain H37Ra by intranasal route (5 x 10 6 CFU / guinea pig) and lung H37Ra bacterial load was measured 3 weeks later. The results showed that lung bacterial load was reduced by about 1.1 log 10 CFU (p<0.01) in TP / P-SP group compared to PBS group, and by 1.56 log 10 CFU (p<0.01) in BCG and CPS-TP / P-SP groups. Figure 9 CPS-TP / P-SP group was reduced by about 0.3 log 10 CFU compared to TP / P-SP group. Lung pathological damage was evaluated by HE staining. The results showed that significant exudative inflammatory lesions were observed in PBS group. In contrast, lung exudative lesions were reduced in TP / P-SP and CPS-TP / P-SP groups compared to PBS group, mainly in the form of lymphoproliferative inflammatory changes induced by the vaccine. Lung pathological damage was similar in TP / P-SP and CPS-TP / P-SP groups, with no significant difference between the two groups.

[0153] Figure 9 ​To evaluate the induced immune protection effect after 6 weeks of the last immunization of CPS-TP / P-SP. (A) Panoramic scan of guinea pig lungs infected with H37Ra strain after HE staining. (B) Local scan of guinea pig lungs infected with H37Ra strain after HE staining. (C) Area percentage of inflammatory lesions in guinea pig lungs. (D) Statistical analysis of bacterial load count in guinea pig lungs. Data are shown as mean ± standard deviation (n = 3-4). *p < 0.05, **p < 0.01.

[0154] Finally, it should be noted that the above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will still be able to modify the technical solutions described in the foregoing embodiments or make equivalent replacements to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for the extraction of the capsular polysaccharide of BCG, characterized in that: The method comprises the following steps: (1) removing protein by using a hot phenol method and a Sevag method, and collecting supernatant; in the Sevag method, the volume ratio of chloroform to n-butanol is 4:1; (2) removing fat by using a chloroform-methanol solution, and collecting upper solution; in the chloroform-methanol solution, the volume ratio of chloroform to methanol is 1:1; (3) removing nucleic acid by using DNase and RNase, and removing residual nucleic acid by using cetyltrimethylammonium bromide, and collecting supernatant; (4) alcohol precipitation is performed on the supernatant, and a precipitate is collected, so that the BCG capsular polysaccharide is obtained.

2. The extraction method of claim 1, wherein: The method comprises the following steps: (1) collecting BCG bacterial bodies; (2) resuspending the BCG bacterial bodies in PBS, adding glass beads and oscillating to promote the capsular shedding, centrifuging to collect supernatant and concentrating; (3) adding an equal volume of 80% phenol, reacting at 70°C for 1.5 hours to remove protein, and centrifuging to collect supernatant; (4) adding an equal volume of chloroform-n-butanol solution and oscillating to remove residual protein, and centrifuging to collect upper solution; (5) adding an equal volume of chloroform-methanol solution and oscillating to remove fat, and centrifuging to collect upper solution; (6) adding DNase and RNase respectively, and standing at 37°C for 12-14 hours to remove nucleic acid, and centrifuging to collect upper solution; (7) adding 0.08% cetyltrimethylammonium bromide, and standing at 4°C for 12-14 hours to further remove nucleic acid, and centrifuging to collect supernatant; (8) adding anhydrous ethanol to make the final volume 75%, and adding sodium acetate, and standing at 4°C for 12-14 hours to perform alcohol precipitation, and centrifuging to collect precipitate; (9) drying the precipitate, so that the BCG capsular polysaccharide is obtained.

3. A BCG capsular polysaccharide, characterized by: The BCG capsular polysaccharide is obtained by using the extraction method in claim 1 or 2; preferably, the polysaccharide purity of the BCG capsular polysaccharide reaches 95.92%.

4. A method for the preparation of a Pickering emulsion based on a BCG capsular polysaccharide, characterized in that: The method comprises the following steps: (1) preparation of a tubercle fusion protein TP and a trivalent N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP; The tubercle fusion protein TP is obtained by mixing LT33 and LT57; The preparation method of the N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP is as follows: a. mixing activated PLGA, NH2-PEG-Alkyen, DMAP, EDC-HCl and triethylamine, and reacting under inert gas condition at room temperature to obtain PLGA-PEG-Alkyen; b. mixing PLGA-PEG-Alkyen, N3-Tri-GalNAc, Vc-Na and CuSO4 solution, and stirring to react at room temperature to obtain the N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP; (2) preparation of TP / GP nanoparticles in which TP is wrapped: a. mixing an inner water phase and an oil phase to prepare a primary milk; The inner water phase is obtained by dissolving the tubercle fusion protein TP in step (1) in PBS; The oil phase is obtained by dissolving the trivalent N-acetylgalactosamine modified polylactic acid-glycolic acid-polyethylene glycol GP in step (1) in dichloromethane; b. rapidly adding the initial milk of step a into the external water phase, then ice-bath ultrasonic treatment under the power of 600 W for 8-10 min, then stirring for 4 h-6 h; the external water phase is 2% polyvinyl alcohol solution; c. centrifuging to collect the precipitate to obtain TP-coated GP nanoparticles TP / GP; (3) Preparation of the Pickering emulsion based on BCG capsular polysaccharide: TP / GP, interferon gene stimulator agonist SR717, Toll-like receptor 3 agonist polyinosinic-polycytidylic acid (Poly(I:C)), and the BCG capsular polysaccharide of claim 3 are dissolved in water as the water phase, and squalene as the oil phase is mixed to emulsify, to obtain the Pickering emulsion based on BCG capsular polysaccharide.

5. The method of claim 4, wherein: The tuberculosis fusion protein TP is obtained by mixing LT33 and LT57 at a weight ratio of 1:

1.

6. The method of claim 4, wherein: In step (2), the specific method for preparing the initial milk is: adding the internal water phase into the oil phase, then ice-bath ultrasonic treatment under the power of 400 W for 4 min-5 min.

7. The method of claim 4, wherein: In step (3), the weight ratio of TP / GP, interferon gene stimulator agonist SR717, Toll-like receptor 3 agonist polyinosinic-polycytidylic acid (Poly(I:C)), and BCG capsular polysaccharide is 50:5:1.25:0.

5.

8. A Pickering emulsion based on a BCG capsular polysaccharide characterised in that: is prepared by the method of any one of claims 4-7.

9. Use of the Pickering emulsion based on BCG capsular polysaccharide of claim 8 in the preparation of a Mycobacterium tuberculosis vaccine.

10. A vaccine, characterized in that: The vaccine comprises the Pickering emulsion based on BCG capsular polysaccharide of claim 8.

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