A tuberculosis subunit vaccine containing natural polysaccharides

Abstract

The present invention provides a tuberculosis subunit vaccine, which comprises natural polysaccharides extracted from Mycobacterium tuberculosis, and comprises one, more or all of the proteins ESAT6, Ag85b, HspX, Rv1738 and Rv2626; and is combined with aluminum adjuvant and polyIC. The polysaccharides extracted from Mycobacterium tuberculosis have both antigenic and adjuvant functions; ESAT6, Ag85b, HspX, Rv1738 and Rv2626 cover highly antigenic tuberculosis proteins and antigenic proteins for latent infection, enabling the vaccine to not only prevent Mycobacterium tuberculosis infection but also prevent tuberculosis in infected individuals, playing a role in preventive treatment.

Description

Technical Field

[0001] The present invention belongs to the field of vaccines. Specifically, the present invention relates to a tuberculosis subunit vaccine containing natural polysaccharides. Background Art

[0002] Due to factors such as the ineffectiveness of the conventional BCG vaccine in preventing pulmonary tuberculosis, the emergence of tuberculosis drug-resistant strains, and the co-infection of tuberculosis caused by HIV infection, the incidence and mortality of pulmonary tuberculosis are currently extremely high, and the situation is very serious. According to the estimation of the World Health Organization (WHO), currently about 1.7 billion people globally are infected with Mycobacterium tuberculosis, accounting for about 23% of the global total population. Among the people infected with Mycobacterium tuberculosis (Latent Tuberculosis Infection, LTBI), about 10%-15% of people will develop tuberculosis at some point in their lives. Therefore, the development of new tuberculosis preventive vaccines and therapeutic vaccines is extremely urgent.

[0003] Tuberculosis vaccines can be classified into whole-bacteria vaccines, DNA vaccines, subunit vaccines, etc. according to their types. Subunit vaccines can specifically induce humoral immunity and cellular immunity, and are safe to use, which is an ideal vaccine form. The principle of subunit vaccines is to isolate and purify the proteins or polypeptides with immunoprotective effects secreted during the growth of Mycobacterium Tuberculosis (MTB) as antigens, and combine them with adjuvants to make vaccines. Currently, the candidate antigens of tuberculosis subunit vaccines are mainly ESAT6, CFP10, MPT32, MPT64, HSP65, etc. Due to the genetic diversity in the population, although a single protein antigen has certain immunoprotective effects, the antigen spectrum is narrow and not sufficient to cause effective immune protection. Therefore, most scholars agree that the combination of multiple proteins is better than a single protein, and the fusion expression of multiple antigens or polypeptides helps to improve the immune protection rate of the vaccine.

[0004] Currently, all tuberculosis subunit vaccines entering the clinical trial stage are composed of multiple antigens; they are mainly based on the protective antigens of Mycobacterium tuberculosis, including mainly the secreted proteins of Mycobacterium tuberculosis (mostly encoded by genes in the RD1 region, the RD1 region is an important protective antigen lost during the long-term passage of MTB, only existing in pathogenic mycobacteria, and lost in BCG and environmental mycobacteria), cell wall proteins, and antigenic substances expressed by dormant Mycobacterium tuberculosis, etc. In addition, tuberculosis subunit vaccines need the assistance of effective adjuvants to induce an ideal immune response, and different immune adjuvants can induce different directions of immune responses, Th1 or Th2.

[0005] Therefore, when developing new tuberculosis subunit vaccines, it is necessary to consider from two aspects: antigens and adjuvants. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention aims to provide a novel tuberculosis subunit vaccine, which uses latent infection proteins as antigens and combines mycobacterial protective antigens, and adds naturally extracted mycobacterial polysaccharides and combines commonly used aluminum adjuvants and Poly IC to achieve higher immunogenicity and more lasting protective effects.

[0007] The technical solution of the present invention is as follows.

[0008] On the one hand, the present invention provides a tuberculosis subunit vaccine, which includes natural polysaccharides extracted from mycobacterium tuberculosis and includes one, more or all of the proteins ESAT6, Ag85b, HspX, Rv1738, and Rv2626.

[0009] Among them, the proteins ESAT6, Ag85b, HspX, Rv1738, and Rv2626 exist independently or in the form of a fusion protein. According to a specific embodiment of the present invention, the proteins ESAT6, Ag85b, and HspX form a recombinant fusion protein; and / or, the proteins Rv1738 and Rv2626 form a recombinant fusion protein, which is included in the vaccine of the present invention.

[0010] In the present invention, the term "natural polysaccharides extracted from mycobacterium tuberculosis" refers to polysaccharides extracted from the cell wall of mycobacterium tuberculosis, especially the outermost layer of the cell wall; it can be used interchangeably with the terms "natural polysaccharides of mycobacterium tuberculosis", "mycobacterial natural polysaccharides", "mycobacterial polysaccharides", etc.

[0011] Preferably, the natural polysaccharides are obtained by a method including the following steps:

[0012] (1) Degreasing:

[0013] Add acetone to mycobacterium tuberculosis, reflux at 60 - 80 °C for 2 - 4 hours, and then stop the reaction; after the reaction system cools to 20 - 30 °C, filter by suction and add acetone to wash the filter cake, and then dry overnight at 20 - 25 °C to obtain a solid;

[0014] (2) Cell wall breaking:

[0015] Add NaOH to the solid obtained in step (1), stir at 60 - 80 °C for 3 - 5 hours, and cool down;

[0016] (3) Sedimentation:

[0017] When the reaction system cools to 20 - 35 °C, adjust the pH value to 4 - 5, and then let it stand at 20 - 25 °C for 10 - 20 hours; then filter by suction and wash the filter cake with water to obtain a solution;

[0018] (4) Purification:

[0019] Remove impurities with molecular weights above 50 kDa and below 3 kDa in the solution by ultrafiltration, and then concentrate the resulting solution;

[0020] (5) Decolorization:

[0021] Decolorize the solution to obtain a colorless and transparent solution;

[0022] (6) Freeze-drying:

[0023] Concentrate the colorless and transparent solution under reduced pressure and then freeze-dry it to obtain a solid.

[0024] In step (1), preferably, add acetone at a ratio of 1 g (weight of inactivated wet bacteria): 2 mL, and then heat under reflux in a 70 °C oil bath under nitrogen protection for 3 hours. Preferably, after the reaction system is cooled to room temperature, filter by suction and add acetone to wash the filter cake at a ratio of 1 g (weight of inactivated wet bacteria): 0.4 mL, and then air-dry at room temperature overnight.

[0025] In step (2), preferably, add 0.5 N NaOH at a ratio of 1 g (weight of inactivated wet bacteria): 4 mL. Preferably, stir in a 71 °C oil bath under nitrogen protection for 4 hours and cool in a cold water bath.

[0026] In step (3), preferably, when the reaction system is cooled to 30 °C, add 6 M HCl with stirring to adjust the pH value to 4.5, and then stand at room temperature overnight under nitrogen protection. Preferably, filter the solution by suction the next day and add deionized water to wash the filter cake at a ratio of 1 g (weight of inactivated wet bacteria): 8 mL.

[0027] In step (4), preferably, use a hollow fiber ultrafiltration membrane for ultrafiltration. First, remove impurities with molecular weights above 50 kDa, and after washing the filter membrane with water, then remove impurities with molecular weights below 3 kDa. Preferably, concentrate the solution to 1 / 10 of its volume before ultrafiltration.

[0028] In step (5), preferably, use an anion exchange resin for decolorization, use water as the eluent, and collect the sugar-containing eluate.

[0029] In step (6), preferably, concentrate the colorless and transparent solution to 1 / 3 of its volume at 45 - 50 °C, preferably 45 - 48 °C, under reduced pressure, and then freeze-dry it to obtain a white flocculent solid.

[0030] In the tuberculosis subunit vaccine provided by the present invention, the proteins ESAT6, Ag85b, HspX, Rv1738, and Rv2626 can exist independently or in the form of a fusion protein. The amino acid sequences of these proteins are shown as follows.

[0031] Protein ESAT6 (SEQ ID NO:1; Uniprot Accession No.: P9WNK7):

[0032] MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA

[0033] Protein Ag85b (SEQ ID NO:2; Uniprot Accession No.: P9WQP1):

[0034] FSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG

[0035] Protein HspX (SEQ ID NO:3; Uniprot Accession No.: P9WMK1):

[0036] ATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN

[0037] Protein Rv1738 (SEQ ID NO:4; Uniprot Accession No.: P9WLS3):

[0038] MCGDQSDHVLQHWTVDISIDEHEGLTRAKARLRWREKELVGVGLARLNPADRNVPEIGDELSVARALSDLGKRMLKVSTHDIEAVTHQPARLLY

[0039] Protein Rv2626 (SEQ ID NO:5; Uniprot Accession No.: P9WJA3):

[0040] MTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALAS

[0041] The proteins ESAT6, Ag85b, HspX, Rv1738 and Rv2626 can exist in the subunit vaccine of the present invention in the form of fusion proteins formed by 2, 3, 4 or 5 kinds. Preferably, the proteins ESAT6, Ag85b, HspX form a recombinant fusion protein; and / or, the proteins Rv1738 and Rv2626 form a recombinant fusion protein.

[0042] In the recombinant fusion protein formed by ESAT6, Ag85b, HspX, the order of the three can be N'-ESAT6-Ag85b-HspX-C'. And / or, in the recombinant fusion protein formed by the proteins Rv1738 and Rv2626, the order of the two can be N'-Rv1738-Rv2626-C'.

[0043] In the recombinant fusion protein provided by the present invention, the amino acid sequences of each protein can be connected via a linker sequence. Thus, the structure of the recombinant fusion protein formed by the proteins ESAT6, Ag85b, HspX can be shown as N'-ESAT6-linker-Ag85b-linker-HspX-C'. The structure of the recombinant fusion protein formed by the proteins Rv1738 and Rv2626 can be shown as N'-Rv1738-linker-Rv2626-C'.

[0044] According to the specific embodiments of the present invention, a flexible linker can be adopted in the recombinant fusion protein, such as a flexible linker rich in GS. For example, the linker sequence contains one or more GGGSG, such as (GGGSG)n, where n is an integer from 1 to 5, preferably n = 1, 2 or 3, more preferably n = 2 or 3.

[0045] According to the specific embodiments of the present invention, the recombinant fusion protein formed by ESAT6, Ag85b, HspX ("ESAT6-Ag85b-HspX", which can be abbreviated as "EAH" for short) contains the following amino acid sequence (SEQ ID NO:6):

[0046] N’-MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA GGGSGGGGSGGGGSG FSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG GGGSGGGGSG ATTLPVQRHPRSLFPEFSELFAAFPSFAGLRPTFDTRLMRLEDEMKEGRYEVRAELPGVDPDKDVDIMVRDGQLTIKAERTEQKDFDGRSEFAYGSFVRTVSLPVGADEDDIKATYDKGILTVSVAVSEGKPTEKHIQIRSTN-C’

[0047] or comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:6.

[0048] The recombinant fusion protein formed by Rv1738 and Rv2626 (“Rv1738-Rv2626”, which may be abbreviated as “1738-2626” for short) comprises the following amino acid sequence (SEQ ID NO:7):

[0049] N’-MCGDQSDHVLQHWTVDISIDEHEGLTRAKARLRWREKELVGVGLARLNPADRNVPEIGDELSVARALSDLGKRMLKVSTHDIEAVTHQPARLLY GGGSGGGGSGMTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALAS-C’

[0050] or comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:7.

[0051] The term "identity" as used in the context of the present invention refers to the similarity between two amino acid sequences (or nucleotide sequences hereinafter). "Identity" can be determined by comparing the two sequences using algorithms or software well known in the art, and is expressed as a percentage (%). The term "at least 85% identity" refers to an identity percentage of any value not less than 85% (not limited to integers), such as at least 86% identity, at least 87% identity, at least 88% identity, at least 89% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, and even 100% identity. Up to 25% amino acid sequence differences resulting from "at least 85% identity" may be present in the linker sequence.

[0052] Preferably, in the tuberculosis subunit vaccine provided by the present invention, the recombinant fusion protein ESAT6-Ag85b-HspX and the recombinant fusion protein Rv1738-Rv2626 are mixed to form the vaccine antigen. Preferably, in the vaccine, the mass ratio of the two recombinant fusion proteins is 0.2-10:0.2-10, preferably 0.2-2:0.2-2, and more preferably 1:1.

[0053] Furthermore, the tuberculosis subunit vaccine provided by the present invention further comprises an aluminum adjuvant and poly IC. Preferably, the aluminum adjuvant is aluminum hydroxide (Al(OH) 3 ) adjuvant and / or aluminum phosphate (AlPO 4 ) adjuvant, preferably aluminum hydroxide adjuvant. In the present invention, Poly IC refers to Polyinosinic–polycytidylic acid (Poly(I)·Poly(C)).

[0054] Preferably, in the tuberculosis subunit vaccine provided by the present invention, the mass ratio of the natural polysaccharide, aluminum adjuvant and Poly IC is 0.2-1:0.2-1:0.2-1; preferably 1:1:1.

[0055] In the context of the present invention, the content or concentration of the aluminum adjuvant is based on the content or concentration of Al 3+ by content or concentration.

[0056] Compared with the prior art, the present invention provides a novel tuberculosis subunit vaccine.

[0057] The tuberculosis subunit vaccine provided by the present invention contains the MTB protective antigen proteins Ag85b and ESAT6 with strong immunogenicity, and at the same time adds three latent infection proteins HspX, Rv1738 and Rv2626. The protein HspX is encoded by Rv2031c. Under hypoxic conditions, its expression can reach 25% of the total protein expression in the latent infection state of MTB; Rv1738 is a protein with unknown function composed of 94 amino acids. Under hypoxic conditions, Rv1738 is also one of the proteins with significantly increased expression. HspX, Rv1738 and Rv2626 are all important proteins for latent infection. Selecting these three latent infection proteins to play a synergistic role with the highly immunogenic ESAT6 / Ag85b will play an important role in the treatment of patients with latent infection.

[0058] The tuberculosis subunit vaccine provided by the present invention also contains natural polysaccharides extracted from Mycobacterium tuberculosis. Without being limited to any theory, experiments have proved that the natural polysaccharides provided by the present invention have both the functions of antigen and adjuvant. In addition, the tuberculosis subunit vaccine provided by the present invention also contains Poly IC (an agonist of TLR3) that is easy to synthesize on a large scale and a conventional aluminum adjuvant as a combined adjuvant.

[0059] Therefore, the tuberculosis subunit vaccine proposed by the present invention uses five tuberculosis proteins to form an antigen complex, which includes both the highly immunogenic ESAT6 and Ag85b, and three important latent infection proteins of Mycobacterium tuberculosis, enabling the vaccine to not only prevent Mycobacterium tuberculosis infection, but also prevent tuberculosis in infected individuals infected with Mycobacterium tuberculosis, playing a role in preventive treatment; in addition, adding natural polysaccharides naturally present in Mycobacterium tuberculosis to this tuberculosis subunit vaccine and using them in combination with these tuberculosis proteins can significantly increase the secretion of IFN-γ. In terms of the protective efficacy of the tuberculosis vaccine, the role of cellular immunity is much higher than that of humoral immunity. Experiments have proved that the natural polysaccharides extracted from Mycobacterium tuberculosis can significantly promote the secretion of cytokines and chemokines of the Th1-type immune response, inducing the innate immune and adaptive immune systems of the body to produce Th1-type cell-mediated and humoral immune responses, thereby improving the immunogenicity of the vaccine and having good safety.

[0060] Therefore, compared with the existing tuberculosis vaccine compositions undergoing clinical tests, the tuberculosis subunit vaccine of the present invention will have higher immunogenicity, and the protective effect of the vaccine on the population will be more durable and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

[0062] Figure 1 Shows the titers of specific binding antibodies produced by different vaccine compositions consisting of EAH and Rv1738-Rv2626.

[0063] Figures 2 to 4 Shows IFN-γ per million cells produced by different vaccine compositions. Figure 2 The proteins used were EAH and Rv1738-Rv2626; Figure 3 were EA and Rv1738-Rv2626; Figure 4 were EAH and Rv1813-RpfD; Specific Embodiments

[0064] The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

[0065] The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

[0066] Example 1 Preparation of Recombinant Fusion Proteins ESAT6-Ag85b-HspX and RV1738-RV2626

[0067] (1) Plasmid construction: First, the amino acid sequences of the fusion proteins ESAT6-Ag85b-HspX (EAH) and RV1738-RV2626 were codon-optimized for E. coli codons and constructed into the vector pET-28A-SUMO. The resulting recombinant plasmids were named PET-28A-SUMO-EAH and PET-28A-SUMO-1738-2626.

[0068] (2) The above plasmids were transformed into the E. coli strain BL21(DE3), and the strains were named BL21_SUMO-EAH and BL21_SUMO-1738-2626.

[0069] (3) Inoculate the above engineering bacteria into 5 ml of LB medium containing 50 mcg / ml kanamycin sulfate respectively, incubate overnight in a constant temperature shaker at 37°C, then inoculate at an inoculation amount of 1% into 400 ml of LB medium containing 50 mcg / ml kanamycin sulfate, and culture in a constant temperature shaker at 37°C until the OD600 reaches 0.4 - 0.5. Add IPTG with a final concentration of 0.1 mmol / L and continue to induce at 16°C for 16 hours.

[0070] (4) Collect the bacterial cells, add lysis buffer (PBS + 0.3 M NaCl + 30 mM imidazole + 2 mM PMSF), after ultrasonic disruption, centrifuge at high speed (25,000 g, 30 minutes), collect the supernatant, and then add it to a HIS60 nickel column for purification. After washing away the impurities with the washing buffer (PBS + 0.3 M NaCl + 30 mM imidazole), elute SUMO-EAH or SUMO-1738-2626 with the elution buffer (25 mM Tris pH8.0, 0.5 M NaCl, 10% glycerol, 0.25 M imidazole). Add SUMO protease to the above eluted protein and digest overnight, then add it back to the HIS60 nickel column. The SUMO tag, incompletely digested SUMO-EAH or SUMO-1738-2626, and SUMO protease all contain HIS tags, but the EAH protein and 1738-2626 do not contain any tags, so they are in the flow-through fraction. Collect the flow-through fraction to obtain the purified fusion proteins EAH and 1738-2626.

[0071] Example 2 Preparation of Mycobacterium tuberculosis natural polysaccharide

[0072] (1) Defatting

[0073] Take 125 g of inactivated H37Ra wet bacteria (ATCC) in a 1000 mL eggplant-shaped flask, add 250 mL of acetone, heat under reflux in a 70°C oil bath under nitrogen protection for 3 hours, then stop stirring and heating. After cooling to room temperature, filter by suction and wash the filter cake with 50 mL of acetone, dry at room temperature overnight to obtain 24.5 g of a khaki solid.

[0074] (2) Cell wall breaking

[0075] Put the above obtained dry bacteria in a 2 L three-necked flask, add 500 mL of 0.5 N NaOH, stir in a 71°C oil bath under nitrogen protection for 4 hours, and then cool in a cold water bath.

[0076] (3) Sedimentation

[0077] When the temperature of the reaction system drops to 30 °C, 6M HCl is added under stirring to adjust the pH value to 4.5 (a total of 85 mL of 6M HCl is added), and then the mixture is left standing at room temperature overnight under nitrogen protection. The next day, the solution is filtered by suction, and the filter cake is rinsed with deionized water to obtain about 1 L of solution in total.

[0078] (4) Ultrafiltration fiber column separation and purification

[0079] The above 1 L of solution is passed through a hollow fiber ultrafiltration membrane to remove impurities with a molecular weight above 50 kDa, and then rinsed with 6.5 L of water to obtain 7.5 L of solution in total. Then, the solution is passed through a hollow fiber ultrafiltration membrane again to remove impurities with a molecular weight below 3 kDa, and the solution is concentrated to obtain 100 mL of solution.

[0080] (5) Decolorization

[0081] The above solution is passed through an anion exchange resin (Q-Sepharose Fast Flow), and water is used as the eluent. Collection starts after the sugar-containing solution flows out. Collection is stopped when the effluent is basically sugar-free, and about 300 mL of colorless and transparent solution is obtained in total.

[0082] (6) Freeze-drying

[0083] The 300 mL of solution is concentrated under reduced pressure to 100 mL at 45 - 48 °C and freeze-dried to obtain 127 mg of white flocculent solid, and the yield is 0.1% (calculated based on the weight of wet bacteria).

[0084] The polysaccharide content is determined by the phenol-sulfuric acid method, and the purity is 78.3%.

[0085] Example 3 Preparation of tuberculosis vaccine composition

[0086] Vaccine compositions with or without tuberculosis polysaccharide are prepared, and the composition of each composition is shown in Table 1.

[0087] The specific method is as follows: Take 2.5 μg each of ESAT6-Ag85b-HspX and RV1738-RV2626, totaling 5 μg. Add aluminum adjuvant ( adjuvant, InvivoGen), mix, and gently shake overnight at room temperature; the next day, add CpG ODN2395 (synthesized by GenScript) or Poly IC (Sigma, P1530, 042424-50-0), and mix well. Natural extracted tuberculosis polysaccharide is also added to the Poly IC group. PBS is added to each group to make the total volume of the composition up to 100 μl.

[0088] Table 1. Vaccine composition

[0089] Number Antigen (μg) Alhydrogel (μg) CpG (μg) PolyIC (μg) Tuberculosis polysaccharide (μg) 1 5 50 50 0 0 2 5 50 0 50 0 3 5 50 0 50 50

[0090] Example 4 Immunological Study of Vaccine Compositions

[0091] BAL / C mice aged 6 - 8 weeks were selected as the animal model, and immunological studies were conducted on tuberculosis vaccine compositions with different compositions. There were 5 mice in each group, and the immunogens injected are shown in Table 1; 100 μl of the preparation was injected into the inner thigh muscle, and immunization was carried out once at 0, 3, and 5 weeks. The mice were dissected at the 7th week, and blood was taken and spleens were aseptically removed.

[0092] ELISA was used to detect the antibody titer of anti-tuberculosis protein in the serum:

[0093] The coated antigen was diluted to 4 μg / ml with the coating solution (Solarbio C1055), 100 μl was coated in each well, and incubated overnight at 4°C. The sera of the mice immunized 3 times were diluted starting from 1:1,000, and the dilution factors are shown in Figure 1 . The enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP, Solarbio SE313) was diluted 1:5,000, 100 μl was added to each well, and then incubated at room temperature for 1 hour. 100 μl of the reaction solution (Solarbio PR1200) was added to each well for color development, and then 50 μl of the reaction termination solution (Solarbio C1058) was added, and the reading was taken at 450 nM.

[0094] ELISPOT was used to detect the expression of INF-gamma in spleen cells:

[0095] The ELISPOT kit was purchased from Mouse IFN-γ ELISPOT Basic kit (ALP), MABTECH 3321-2A. The spleens of the mice were aseptically removed, and the spleens were ground with 4 mL of mouse lymphocyte separation solution (DKW33-R0100) to isolate spleen lymphocytes. After passing through a 100 μm cell sieve (CORNING 431752), the separation solution containing spleen cells was immediately transferred to a 15 mL centrifuge tube, covered with 1 ml of RPMI 1640 medium (Solarbio 31800), and centrifuged at 800 xg for 60 min. The middle lymphocyte layer was taken, the cells were washed with the same medium, and then centrifuged at 250 xg for 10 min. Then it was diluted to 5.0x10 6 / ml with complete medium containing serum (10% FBS-RPMI 1640 culture medium).

[0096] Using an ELISpot plate, add 100 μl of coating antibody (AN18) (1.5 μg / well) to each well according to the kit instructions, and incubate overnight at 4°C. After washing the plate 5 times with PBS, add 10% FBS-RPMI 1640 medium identical to the cell suspension, 200 μl / well. Discard the medium after incubating for 30 minutes, and add the extracted lymphocytes. Set 3 wells for the spleen of each mouse, and take 100 μL of cell suspension from each well. In one well, add no stimulant (negative control well), in one well, add 5 μL of antigen stimulant (0.15 mg / ml, final concentration 7.5 μg / ml) (antigen-stimulated well), and in one well, add 1 μL of CoA (0.5 mg / ml, final concentration 5 μg / ml, positive control well). Incubate in an incubator at 37°C, 5% CO 2 for 24 h and then develop color. The color development is carried out according to the kit instructions. Perform spot counting. Subtract the spots in the negative control well from the spots in the antigen-stimulated well as the number of antigen-specific spots. The spots in the positive well basically cover the whole well, indicating normal cell activity.

[0097] The results are shown in Figure 1 and Figure 2 . The results show that the addition of mycobacterial natural polysaccharide did not change the titer of specific tuberculosis antibodies produced by the vaccine composition; however, the inventors surprisingly found that it significantly increased the secretion of IFN-γ, from an average of 432.6 / million cells (aluminum adjuvant, poly IC) to 567.6 / million cells (aluminum adjuvant, poly IC, polysaccharide), an increase of more than 30%.

[0098] Example 5 Preparation and Immunological Study of Tuberculosis Vaccine Compositions Containing Other Antigen Proteins

[0099] Using the method described in Example 1, the control recombinant fusion proteins ESAT6-Ag85b (abbreviated as "EA" for short) and RV1813-RpfD (abbreviated as "1813_RpfD" for short) were prepared.

[0100] ESAT6-Ag85b contains the following amino acid sequence (SEQ ID NO:8):

[0101] N’-MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA GGGSGGGGSGGGGSGFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGA-C’

[0102] RV1813-RpfD contains the following amino acid sequence (SEQ ID NO:9):

[0103] N’-HLANGSMSEVMMSEIAGLPIPPIIHYGAIAYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKVVSRFTRCGAVAYNGSKYQGGTGLTRRAAEDDAVNRLEGGRIVNWACN GGGSGGGGSG LSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD-C’

[0104] Using the method described in Example 3, a control tuberculosis vaccine composition was prepared, and the composition is shown in Table 2.

[0105] Table 2. Control vaccine composition

[0106]

[0107]

[0108] The immunological study of the vaccine composition in Table 2 was carried out using the method described in Example 4.

[0109] The results are shown in Figure 3 and Figure 4The results showed that when a vaccine composition with a similar polysaccharide composition extracted from nature was added to the antigen complex composed of EA and 1738-2626, the addition of polysaccharide did not increase the secretion of IFN-γ when immunizing mice. When the antigen protein was replaced with the complex composed of ESAT6-Ag85b-HspX and RV1813-RpfD, the addition of polysaccharide not only did not increase the secretion of IFN-γ, but on the contrary, decreased the secretion of IFN-γ.

[0110] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention. As long as they do not depart from the spirit of the present invention, they shall fall within the scope of the appended claims of the present invention.

Claims

1. A tuberculosis subunit vaccine, which comprises the proteins ESAT6, Ag85b, HspX, Rv1738 and Rv2626, as well as aluminum adjuvant, poly IC and natural polysaccharides extracted from the cell wall of Mycobacterium tuberculosis; wherein the proteins ESAT6, Ag85b, HspX form a recombinant fusion protein N’ -ESAT6-Ag85b-HspX- C’ ; the proteins Rv1738 and Rv2626 form a recombinant fusion protein N’ -Rv1738-Rv2626- C’ ; and The natural polysaccharide is obtained by a method comprising the following steps: (1) Defatting: Add acetone to Mycobacterium tuberculosis, heat under reflux at 60 - 80°C for 2 - 4 hours, and then stop the reaction; after the reaction system cools to 20 - 30°C, filter by suction and add acetone to wash the filter cake, then dry overnight at 20 - 25°C to obtain a solid; (2) Cell wall breaking: Add NaOH to the solid obtained in step (1), stir at 60 - 80°C for 3 - 5 hours, and cool down; (3) Sedimentation: When the reaction system cools to 20 - 35°C, adjust the pH value to 4 - 5, then let it stand at 20 - 25°C for 10 - 20 hours; then filter by suction and wash the filter cake with water to obtain a solution; (4) Purification: Remove impurities with molecular weight above 50 kDa and impurities with molecular weight below 3 kDa in the solution by ultrafiltration, and then concentrate the obtained solution; (5) Decolorization: Decolorize the solution to obtain a colorless and transparent solution; (6) Freeze - drying: Concentrate the colorless and transparent solution under reduced pressure, and then freeze - dry to obtain a solid.

2. The tuberculosis subunit vaccine according to claim 1, wherein, in step (1), add acetone at a mass - volume ratio of inactivated wet bacteria weight to acetone of 1 g:2 mL, then heat under reflux in a 70°C oil bath under nitrogen protection for 3 hours; after the reaction system cools to room temperature, filter by suction and add acetone to wash the filter cake at a mass - volume ratio of inactivated wet bacteria weight to acetone of 1 g:0.4 mL, then dry at room temperature overnight.

3. The tuberculosis subunit vaccine according to claim 1, wherein, in step (2), add 0.5N NaOH at a mass - volume ratio of inactivated wet bacteria weight to 0.5N NaOH of 1 g:4 mL, stir in a 71°C oil bath under nitrogen protection for 4 hours, and cool in a cold water bath.

4. The tuberculosis subunit vaccine according to claim 1, wherein, in step (3), when the reaction system cools to 30°C, add 6M HCl under stirring to adjust the pH value to 4.5, then let it stand overnight at room temperature under nitrogen protection; the next day, filter the solution by suction and add deionized water to wash the filter cake at a mass - volume ratio of inactivated wet bacteria weight to deionized water of 1 g:8 mL to obtain a solution.

5. The tuberculosis subunit vaccine according to claim 1, wherein, in step (4), use a hollow - fiber ultrafiltration membrane for ultrafiltration, first remove impurities with molecular weight above 50 kDa, after washing the filter membrane with water, then remove impurities with molecular weight below 3 kDa; Concentrate the solution to 1 / 10 of the volume before ultrafiltration.

6. The tuberculosis subunit vaccine according to claim 1, wherein, in step (5), use an anion - exchange resin for decolorization, use water as the eluent, and collect the sugar - containing eluent.

7. The tuberculosis subunit vaccine according to claim 1, wherein, in step (6), concentrate the colorless and transparent solution under reduced pressure to 1 / 3 volume at 45 - 50°C, and then freeze - dry to obtain a white flocculent solid.

8. The tuberculosis subunit vaccine according to claim 7, wherein, in step (6), concentrate under reduced pressure at 45 - 48°C.

9. The tuberculosis subunit vaccine according to claim 1, wherein, the natural polysaccharide is obtained by a method comprising the following steps: (1) Defatting: Acetone is added at a ratio of 1 g: 2 mL of the mass - volume ratio of inactivated wet bacteria weight to acetone, and then heated under reflux in a 70 °C oil bath under nitrogen protection for 3 hours; after the reaction system is cooled to room temperature, it is filtered by suction and acetone is added at a ratio of 1 g: 0.4 mL of the mass - volume ratio of inactivated wet bacteria weight to acetone to wash the filter cake, and then air - dried at room temperature overnight; (2) Cell wall breaking: 0.5 N NaOH is added at a ratio of 1 g: 4 mL of the mass - volume ratio of inactivated wet bacteria weight to 0.5 N NaOH, stirred in a 71 °C oil bath under nitrogen protection for 4 hours, and cooled in a cold water bath; (3) Sedimentation: When the reaction system is cooled to 30 °C, 6 M HCl is added under stirring to adjust the pH value to 4.5, and then left standing at room temperature overnight under nitrogen protection; the next day, the solution is filtered by suction, and deionized water is added at a ratio of 1 g: 8 mL of the mass - volume ratio of inactivated wet bacteria weight to deionized water to wash the filter cake to obtain a solution; (4) Purification: Ultrafiltration is carried out using a hollow - fiber ultrafiltration membrane. First, impurities with a molecular weight above 50 kDa are removed. After washing the filter membrane with water, impurities with a molecular weight below 3 kDa are removed; the solution is concentrated to 1 / 10 of its original volume before ultrafiltration; (5) Decolorization: Anion - exchange resin is used for decolorization, with water as the eluent, and the sugar - containing eluent is collected; (6) Freeze - drying: The colorless and transparent solution is concentrated under reduced pressure to 1 / 3 of its volume at 45 - 48 °C and freeze - dried to obtain a white flocculent solid.

10. The tuberculosis subunit vaccine according to any one of claims 1 to 9, wherein, in the recombinant fusion protein, the amino acid sequences of each protein are connected via a linker sequence.

11. The tuberculosis subunit vaccine according to claim 10, wherein, the linker is a flexible linker.

12. The tuberculosis subunit vaccine according to claim 11, wherein, the linker sequence is (GGGSG)n, where n is an integer from 1 to 5.

13. The tuberculosis subunit vaccine according to claim 12, wherein, n = 1, 2 or 3.

14. The tuberculosis subunit vaccine according to claim 10, wherein, The recombinant fusion protein N’ -ESAT6-Ag85b-HspX- C’ has the amino acid sequence shown in SEQ ID NO: 6; and, The recombinant fusion protein N’ -Rv1738-Rv2626- C’ has the amino acid sequence shown in SEQ ID NO:

7.

15. The tuberculosis subunit vaccine according to any one of claims 11 to 14, wherein, in the tuberculosis subunit vaccine, the mass ratio of the two recombinant fusion proteins is 0.2 - 10:0.2 - 10.

16. The tuberculosis subunit vaccine according to claim 15, wherein, in the tuberculosis subunit vaccine, the mass ratio of the two recombinant fusion proteins is 0.2 - 2:0.2 - 2.

17. The tuberculosis subunit vaccine according to claim 15, wherein, in the tuberculosis subunit vaccine, the mass ratio of the two recombinant fusion proteins is 1:

1.

18. The tuberculosis subunit vaccine according to claim 1, wherein, The aluminum adjuvant is aluminum hydroxide (Al(OH) 3 ) adjuvant and / or aluminum phosphate (AlPO 4 ) adjuvant.

19. The tuberculosis subunit vaccine according to claim 1, wherein, The aluminum adjuvant is an aluminum hydroxide adjuvant.

20. The tuberculosis subunit vaccine according to claim 1, wherein, in the tuberculosis subunit vaccine, the mass ratio of the natural polysaccharide, aluminum adjuvant, and Poly IC is 0.2-1:0.2-1:0.2-1.

21. The tuberculosis subunit vaccine according to claim 1, wherein, the mass ratio of the natural polysaccharide, aluminum adjuvant, and PolyIC is 1:1:

1.

22. The tuberculosis subunit vaccine according to claim 20 or 21, wherein, The aluminum adjuvant is aluminum hydroxide (Al(OH) 3 ) adjuvant and / or aluminum phosphate (AlPO 4 ) adjuvant.

23. The tuberculosis subunit vaccine according to claim 20 or 21, wherein, the aluminum adjuvant is an aluminum hydroxide adjuvant.

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