Adjuvants containing mycobacterium bovis surface polysaccharides and uses thereof

Abstract

The application discloses an adjuvant containing Mycobacterium vaccae surface polysaccharide and application thereof, and the adjuvant comprises: Mycobacterium vaccae surface polysaccharide, wherein the Mycobacterium vaccae surface polysaccharide comprises: LAM polysaccharide, and LM polysaccharide and / or PIMs polysaccharide. The adjuvant of the application can improve the immunization effect of a vaccine antigen component and has good safety. Therefore, the adjuvant of the application can further improve the prevention and / or treatment effect of a vaccine, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine. Specifically, the present application relates to an adjuvant containing Mycobacterium bovis surface polysaccharide and its application. BACKGROUND

[0002] Tuberculosis (TB) is a chronic and debilitating infectious disease caused by Mycobacterium tuberculosis (M.tb). Bovine tuberculosis (BCG) is a chronic infectious disease caused by Mycobacterium bovis (M.bovis) that is shared by humans and animals, characterized by tuberculous nodular granulomas and caseous, calcified necrotic lesions in tissues and organs. BCG (Bacillus Calmette-Guerin) is the only vaccine approved by WHO for the prevention of tuberculosis, which has a protective effect on infants and adolescents, but its protective effect on adults is not clear. Therefore, there is an urgent need to develop more effective new tuberculosis vaccines and immunological adjuvants.

[0003] Purified BCG glycolipids were used as coating antigens in ELISA detection methods to detect specific IgG in CSF of TB patients, and the sensitivity was high. There is no report on the use of BCG glycolipids as vaccine adjuvants. Therefore, there is an urgent need to develop new adjuvants to enhance the immune effect of vaccines. SUMMARY

[0004] The present application aims to at least partially solve at least one of the technical problems existing in the prior art. To this end, the present application proposes an adjuvant containing Mycobacterium bovis surface polysaccharide and its application. The adjuvant containing Mycobacterium bovis surface polysaccharide of the present application can improve the immune effect of the vaccine antigen component and has good safety. Therefore, the adjuvant of the present application can further improve the preventive and / or therapeutic effect of the vaccine, and has a wide application prospect.

[0005] It should be stated that the present application is based on the following work of the inventors:

[0006] The inventors obtained BCG surface polysaccharide extract from BCG bacterial bodies by delipidation, deproteinization, DNA, RNA, etc. The BCG surface polysaccharide extract was identified as a high-purity extract containing LAM, LM, and PIMs three polysaccharides. The BCG surface polysaccharide extract was used to enhance the immune effect of BCG (Bacillus Calmette-Guerin) and AL (tuberculosis subunit vaccine). It was unexpectedly found that the survival of mice after immunization was not affected, the safety was high, and the bacterial load of Mycobacterium bovis in the lungs of mice was reduced.

[0007] Further, the inventors cross-link the BCG surface polysaccharide extract with an aluminum adjuvant to obtain a composite adjuvant, and immunize mice with the composite adjuvant, and find that the immune effect of BCG is significantly improved, and the survival of the test mice is not affected, and the safety is good.

[0008] Therefore, in a first aspect of the present application, the present application provides an adjuvant. According to an embodiment of the present application, the adjuvant comprises: a Mycobacterium bovis surface polysaccharide, the Mycobacterium bovis surface polysaccharide comprising: a LAM polysaccharide, and a LM polysaccharide and / or a PIMs polysaccharide. The adjuvant according to the embodiment of the present application can improve the immune effect of the vaccine antigen component, and has good safety, and thus the adjuvant of the present application can further improve the preventive and / or therapeutic effect of the vaccine, and has a wide application prospect.

[0009] In a second aspect of the present application, the present application provides a use of the aforementioned Mycobacterium bovis surface polysaccharide in preparing an adjuvant, the Mycobacterium bovis surface polysaccharide comprising: a LAM polysaccharide, and a LM polysaccharide and / or a PIMs polysaccharide. The Mycobacterium bovis surface polysaccharide according to the embodiment of the present application can improve the immune effect of the vaccine antigen component, and thus can be used to develop a new vaccine adjuvant.

[0010] In a third aspect of the present application, the present application provides a use of the aforementioned adjuvant in preparing a vaccine.

[0011] It can be understood by those skilled in the art that the features and advantages described above for the adjuvant also apply to the use, and will not be repeated here.

[0012] In a fourth aspect of the present application, the present application provides a subunit vaccine. According to an embodiment of the present application, the subunit vaccine comprises: a fusion protein comprising an Ag85A fragment; and the aforementioned adjuvant. The subunit vaccine according to the embodiment of the present application has high immune protection effect and good safety; and the subunit vaccine can further strengthen the immune effect of the BCG vaccine.

[0013] In a fifth aspect of the present application, the present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises: the aforementioned subunit vaccine.

[0014] It can be understood by those skilled in the art that the features and advantages described above for the adjuvant and the subunit vaccine also apply to the pharmaceutical composition, and will not be repeated here.

[0015] In a sixth aspect of the present application, the present application provides a vaccine. According to an embodiment of the present application, the vaccine comprises: an antigen; and the aforementioned adjuvant, the aforementioned subunit vaccine, or the aforementioned pharmaceutical composition.

[0016] It will be appreciated by persons skilled in the art that the features and advantages described hereinabove for the adjuvant, subunit vaccine also apply to the vaccine, and are not repeated here.

[0017] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the description or be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0018] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0019] Figure 1 Figure (A) is the SDS-PAGE electrophoresis silver staining result of BCG surface polysaccharide extract; and Figure (B) is the Western blot detection result of BCG surface polysaccharide extract. In Figures (A) and (B), "M" represents the protein Marker lane, "S" represents the sample lane, "(KDa)" and the following numbers represent the standard protein molecular weight corresponding to each electrophoresis band of the protein Marker lane, "LAM" represents BCG surface polysaccharide LAM, "LM" represents BCG surface polysaccharide LM, and "PIMs" represents BCG surface polysaccharide PIMs.

[0020] Figure 2 Figure is the schematic diagram of the mouse immunization scheme in Example 2 of the present application.

[0021] Figure 3 Figure is the result graph of the detection of the bacterial load in the organs of the mice in each test group after challenge in Example 2 of the present application. In Figure (A), the bacterial load in the lungs of the mice in each test group; and in Figure (B), the bacterial load in the spleen of the mice in each test group. *, **, ***, and **** represent P<0.05, P<0.01, P<0.001, and P<0.0001, respectively.

[0022] Figure 4 Figure is the result graph of the detection of the serum antibody titer of the mice in each group after challenge in Example 2 of the present application. In Figure (A), IgG; in Figure (B), IgG1; in Figure (C), IgG2c; and in Figure (D), IgG3.

[0023] Figure 5 Figure is the result graph of the investigation of the effect of the serum of the mice in Example 2 of the present application on the phagocytosis of bacteria by macrophages. DETAILED DESCRIPTION

[0024] Embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the present application.

[0025] It should be noted that the terms "first", "second" are used only for descriptive purpose, and cannot be construed as indicating or implying relative importance or an indicated number of technical features. Thus, the features defined with "first", "second" can include one or more of the features explicitly or implicitly. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0026] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The ranges or values should be construed to be roughly about the ranges or values. For ranges, the endpoints are included as well as intervening points which are included in the ranges. For values, the values are included as well as intervening values which are included in the values.

[0027] Terms and definitions

[0028] In the present text, the terms "comprising" or "including" are open-ended expressions that are to be construed to mean including, but not limited to, the recited steps or options thereof.

[0029] In the present text, the terms "optionally", "optional" or "option" generally mean that the subsequently described event or circumstance can or can not occur, and the description includes instances where the event or circumstance occurs and instances where it does not.

[0030] In the present text, the term "BCG" is equivalent to "Bacillus Calmette-Guerin", which is a live bacterial vaccine made from a suspension of attenuated live bacteria of Mycobacterium bovis.

[0031] In the present text, the term "fusion protein" refers to a new protein formed by fusion of at least two proteins or polypeptides, which can be achieved by genetic engineering or other techniques, for example, the expression product of two genes recombined by DNA recombination technology. In the present text, it refers to a fusion protein comprising an Ag85A fragment.

[0032] In the present text, the term "fragment" refers to a protein fragment, which can include a full-length fragment of a protein, or a partial fragment of a protein. Exemplarily, the Ag85A fragment can be a full-length fragment of Ag85A protein, or a partial fragment of Ag85A protein; the LpqH fragment can be a full-length fragment of LpqH protein, or a partial fragment of LpqH protein.

[0033] In the present text, the term "pharmaceutically acceptable" means that the substance or composition must be compatible chemically and / or toxicologically, with the other ingredients comprising a formulation, and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" means approved by a regulatory agency or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0034] In the present text, the term "pharmaceutically acceptable excipient" can include any solvent, solid or liquid excipient, diluent, or other vehicle, etc., suitable for the particular target dosage form. Except insofar as any conventional excipient is incompatible with the compounds of the application, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application.

[0035] In the present text, the term "pharmaceutically acceptable excipient" can include any solvent, solid or liquid excipient, diluent, or other vehicle, etc., suitable for the particular target dosage form. Except insofar as any conventional excipient is incompatible with the compounds of the application, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application.

[0036] The present application provides an adjuvant, use, subunit vaccine, pharmaceutical composition and vaccine, which will be described in detail respectively as follows.

[0037] Adjuvant

[0038] The present application provides an adjuvant. According to an embodiment of the present application, the adjuvant comprises: a Mycobacterium bovis surface polysaccharide, the Mycobacterium bovis surface polysaccharide comprising: a LAM polysaccharide, and a LM polysaccharide and / or a PIMs polysaccharide. The adjuvant according to the embodiment of the present application can improve the immune effect of the vaccine antigen component, and has good safety, thereby the adjuvant of the present application can further improve the preventive and / or therapeutic effect of the vaccine, and has a wide application prospect.

[0039] According to an embodiment of the present application, the molecular weight of the LAM polysaccharide is 35-40 KDa. According to the embodiment of the present application, the molecular weight of the LAM polysaccharide is within the range, which can improve the immune effect of the vaccine antigen component.

[0040] According to an embodiment of the present application, the molecular weight of the LM polysaccharide is 15-20 KDa. According to the embodiment of the present application, the molecular weight of the LM polysaccharide is within the range, which can improve the immune effect of the vaccine antigen component.

[0041] According to an embodiment of the present application, the PIMs polysaccharide has a molecular weight of 2-10 KDa. According to an embodiment of the present application, the PIMs polysaccharide has a molecular weight in this range, which can improve the immune effect of the vaccine antigen component.

[0042] In the present text, the term "LAM polysaccharide" is equivalent to "lipoarabinomanna".

[0043] In the present text, the term "LM polysaccharide" is equivalent to "lipoarabinomanna".

[0044] In the present text, the term "PIMs polysaccharide" is equivalent to "phosphatidyl-myo-inositol mannosides".

[0045] According to an embodiment of the present application, the Mycobacterium bovis is an attenuated strain of Mycobacterium bovis. In this way, the safety of the adjuvant is further improved.

[0046] According to an embodiment of the present application, the attenuated strain of Mycobacterium bovis is from Bacillus Calmette-Guerin (BCG). BCG is the only vaccine approved by WHO to prevent tuberculosis at present, which has a protective effect on infants and adolescents. In this way, the new adjuvant according to the embodiment of the present application has high safety, is easy to obtain, and is used for developing vaccines with enhanced immune effect, and has a wide application prospect.

[0047] In a specific embodiment of the present application, the Mycobacterium bovis surface polysaccharide is obtained by the following method:

[0048] The inactivated Mycobacterium bovis is subjected to delipidation treatment to obtain delipidated bacteria, and the delipidated bacteria are subjected to deproteinization, DNA and RNA treatment to obtain the Mycobacterium bovis surface polysaccharide.

[0049] According to an embodiment of the present application, the delipidation treatment comprises: contacting the inactivated Mycobacterium bovis with an organic solution, incubating for 1-3 hours, centrifuging to discard the supernatant, and collecting the bacteria.

[0050] According to an embodiment of the present application, the above delipidation treatment is repeated 2-4 times until the bacteria are completely delipidated.

[0051] According to an embodiment of the present application, the organic solution is a chloroform:methanol solution with a volume ratio of 1:2.

[0052] According to an embodiment of the present application, the completely delipidated bacteria are subjected to crushing treatment before deproteinization, DNA and RNA treatment; the crushing treatment is ultrasonic crushing, and the ultrasonic program of the ultrasonic crushing is: 2s ultrasonic, 3s pause, and the time of the ultrasonic crushing is 4-16 min.

[0053] According to an embodiment of the present application, the organic solution in the defatted mycobacterium is completely volatilized before ultrasonic crushing.

[0054] According to an embodiment of the present application, the deproteinization, DNA and RNA treatment comprises: contacting the solution of the crushed mycobacterium with DNase, RNase, PMSF and Triton X-114, standing at 4°C for more than 8 hours, taking the supernatant after the first centrifugation, standing and layering the supernatant, taking the organic layer, adding 7-11 times volume of 95% ethanol, standing at -80°C for more than 8 hours, discarding the supernatant and taking the precipitate after the second centrifugation, contacting the precipitate with proteinase K, incubating at 56°C for 1-3 hours, and obtaining the BCG surface polysaccharide extract after proteinase K inactivation and PBS dialysis.

[0055] According to an embodiment of the present application, the final concentration of the proteinase K is 2 mg / mL.

[0056] According to an embodiment of the present application, the first centrifugation is performed at 32000 x g at 4°C for 1 hour.

[0057] According to an embodiment of the present application, the second centrifugation is performed at 10000 rpm for 5 minutes.

[0058] According to an embodiment of the present application, the DNase and RNase are added in an amount of 1000 U / mL of the bacterial solution, the final concentration of the PMSF is 3 mM, and the final concentration of the Triton X-114 is 8%.

[0059] The mycobacterium surface polysaccharide component obtained by the method according to an embodiment of the present application is determined, has high purity, and can be further used as an adjuvant to improve the immune effect of a vaccine antigen component.

[0060] According to an embodiment of the present application, the adjuvant is a composite adjuvant. In this way, the performance of enhancing the immune effect is further improved.

[0061] According to an embodiment of the present application, the adjuvant further comprises at least one of an aluminum adjuvant, a Poly IC adjuvant, a Freund's adjuvant, CpG ODN, CpG 1018, MF59, AS03, AS04, AF03, AS01 B , DDA, MPLA, IC31, and an alum adjuvant. The composite adjuvant comprising the aforementioned mycobacterium surface polysaccharide according to an embodiment of the present application has further improved performance of enhancing the immune effect of an antigen component.

[0062] According to an embodiment of the present application, the adjuvant further comprises an aluminum adjuvant. The inventors have determined through a large number of screening tests that the composite adjuvant comprising the aforementioned mycobacterium surface polysaccharide and the aluminum adjuvant has better performance of enhancing the immune effect of an antigen component.

[0063] It is to be noted that "aluminum adjuvant" generally refers to an immunological adjuvant comprising aluminum, which at least includes, but is not limited to, one or more of aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum, and potassium alum. For example, it can be a suspension of aluminum hydroxide and magnesium hydroxide.

[0064] According to an embodiment of the present application, the aluminum adjuvant is an aluminum hydroxide adjuvant and / or an aluminum salt adjuvant.

[0065] According to an embodiment of the present application, the mass ratio of the Mycobacterium bovis surface polysaccharide to the aluminum adjuvant is 2:(5-15); preferably 2:5. The inventors have determined this preferable mass ratio through a large number of screening experiments, whereby the immunopotentiating performance of the composite adjuvant according to the embodiment of the present application for the vaccine antigen component is further improved.

[0066] Use

[0067] The present application provides use of the aforementioned Mycobacterium bovis surface polysaccharide in the preparation of an adjuvant, the Mycobacterium bovis surface polysaccharide comprising: LAM polysaccharide, and LM polysaccharide and / or PIMs polysaccharide. The Mycobacterium bovis surface polysaccharide according to the embodiment of the present application can improve the immunopotentiating performance of the vaccine antigen component, and thus can be used for the development of a new vaccine adjuvant.

[0068] According to an embodiment of the present application, the molecular weight of the LAM polysaccharide is 35-40 KDa.

[0069] According to an embodiment of the present application, the molecular weight of the LM polysaccharide is 15-20 KDa.

[0070] According to an embodiment of the present application, the molecular weight of the PIMs polysaccharide is 1-10 KDa.

[0071] According to an embodiment of the present application, the adjuvant is used for enhancing the immunopotentiating performance of a Mycobacterium vaccine.

[0072] According to an embodiment of the present application, the Mycobacterium is Mycobacterium tuberculosis. Exemplarily, the Mycobacterium tuberculosis vaccine includes, but is not limited to: a recombinant BCG (rBCG) vaccine, an attenuated M.tb vaccine, an adjuvant subunit protein vaccine, a viral vector vaccine, a whole cell vaccine, a DNA vaccine, an RNA vaccine, and the like.

[0073] In some specific embodiments of the present application, the Mycobacterium tuberculosis vaccine is Bacillus Calmette-Guerin (BCG).

[0074] According to an embodiment of the present application, the adjuvant is a composite adjuvant.

[0075] The present application also provides use of the aforementioned adjuvant in the preparation of a vaccine.

[0076] According to embodiments of the present application, the vaccine is a mycobacterial vaccine.

[0077] According to embodiments of the present application, the mycobacterium is Mycobacterium tuberculosis.

[0078] In some specific embodiments of the present application, the Mycobacterium tuberculosis vaccine is Bacillus Calmette-Guerin (BCG).

[0079] It is understood by those skilled in the art that the features and advantages described above for the adjuvant apply equally to this use, which will not be repeated here.

[0080] Subunit vaccine

[0081] The present application provides a subunit vaccine. According to embodiments of the present application, the subunit vaccine comprises: a fusion protein comprising an Ag85A fragment; and the aforementioned adjuvant. The subunit vaccine according to embodiments of the present application has good immunoprotective effect and high safety; and the subunit vaccine can further enhance the immunization effect of a tuberculosis vaccine (such as BCG).

[0082] According to embodiments of the present application, the fusion protein comprises: an Ag85A fragment and an LpqH fragment; further, the Ag85A fragment and the LpqH fragment are connected by a connecting peptide.

[0083] According to embodiments of the present application, the amino acid sequence of the Ag85A fragment is shown in SEQ ID NO: 1.

[0084] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA (SEQ ID NO: 1).

[0085] According to embodiments of the present application, the amino acid sequence of the LpqH fragment is shown in SEQ ID NO: 2.

[0086] VKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTAAGTTASPGAASGPKVVIDGK DQNVTGSVVCTTAAGNVNIAIGGAATGIAAVLTDGNPPEVKSVGLGNVNGVTLGYTSGT GQGNASATKDGSHYKITGTATGVDMANPMSPVNKSFEIEVTCS (SEQ ID NO: 2).

[0087] According to an embodiment of the present application, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO: 3.

[0088] GGGGSGGGGSGGGGS (SEQ ID NO: 3).

[0089] According to an embodiment of the present application, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 4.

[0090] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSVKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTAAGTTASPGAASGPKVVIDGKDQNVTGSVVCTTAAGNVNIAIGGAATGIAAVLTDGNPPEVKSVGLGNVNGVTLGYTSGTGQGNASATKDGSHYKITGTATGVDMANPMSPVNKSFEIEVTCSHHHHHH (SEQ ID NO: 4).

[0091] According to an embodiment of the present application, the mass ratio of the fusion protein and the adjuvant is 1:(1-8). The present inventors have determined the above mass ratio through a large number of experiments. Within the above mass ratio range, the fusion protein containing the Ag85A fragment and the adjuvant can synergize to further improve the immune effect of the subunit vaccine, and the vaccine is safe.

[0092] Pharmaceutical composition

[0093] The present application provides a pharmaceutical composition. According to an embodiment of the present application, the pharmaceutical composition comprises the aforementioned subunit vaccine. The pharmaceutical composition according to the present application can further improve the immune effect of the body against Mycobacterium tuberculosis, and can also be used as an immune booster to improve the immunity of the body.

[0094] According to an embodiment of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0095] According to an embodiment of the present application, the excipient comprises one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0096] According to an embodiment of the present application, the pharmaceutical composition is an injection.

[0097] Those skilled in the art can understand that the features and advantages described above for the adjuvant, the subunit vaccine also apply to the pharmaceutical composition, which will not be described herein again.

[0098] The pharmaceutical composition of the present application contains a safe and effective amount of the subunit vaccine of the present application and a pharmaceutically acceptable excipient. Such excipients include, but are not limited to, physiological saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.

[0099] Vaccine

[0100] The present application provides a vaccine. According to an embodiment of the present application, the vaccine comprises an antigen; and the aforementioned adjuvant, the aforementioned subunit vaccine or the aforementioned pharmaceutical composition. The vaccine according to the embodiment of the present application has good immune effect and high safety.

[0101] According to an embodiment of the present application, the antigen is attenuated live Mycobacterium bovis.

[0102] According to an embodiment of the present application, the vaccine further comprises a pharmaceutically acceptable excipient.

[0103] According to an embodiment of the present application, the excipient comprises one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0104] According to an embodiment of the present application, the vaccine is an injection. In some specific embodiments of the present application, the injection is a subcutaneous injection.

[0105] The pharmaceutically acceptable adjuvant described in the present application includes (but is not limited to) water, saline, liposome, lipid, protein, peptide, cellulose, nanogel, or a combination thereof. The selection of the adjuvant should be matched with the administration mode, which is well known to those skilled in the art.

[0106] Those skilled in the art can understand that the features and advantages described above for the adjuvant and the subunit vaccine also apply to the vaccine, which will not be repeated here.

[0107] In the embodiments of the present application, the content of BCG surface polysaccharide is determined as follows:

[0108] 1. Prepare a 1 mg / mL L-arabinose standard solution.

[0109] 2. Prepare a gradient L-arabinose solution: dilute the L-arabinose standard solution with distilled water to 0 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL.

[0110] 3. Prepare a 6% phenol solution: dissolve 6 g of phenol in 100 mL of distilled water.

[0111] 4. Polysaccharide concentration determination: take 20 μL of each of the above gradient L-arabinose solution sample and the sample to be tested, first add 20 μL of 6% phenol, mix well, then add 200 μL of concentrated sulfuric acid and mix well. Read the OD490 on the microplate reader, use the OD value and concentration of the L-arabinose solution to draw a standard curve, and then substitute the OD of the polysaccharide to calculate the concentration of the polysaccharide.

[0112] The schemes of the present application will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained commercially.

[0113] Example 1: Extraction and identification of BCG surface polysaccharide

[0114] In this example, the BCG surface polysaccharide is extracted from the BCG bacterial body according to the following method, and then the purity of the BCG surface polysaccharide extract is identified by SDS-PAGE combined with silver staining, and further identified by Western blot to confirm the type of BCG surface polysaccharide. The specific scheme is as follows:

[0115] I. Extraction of BCG surface polysaccharide

[0116] The method is as follows:

[0117] 1. Material pretreatment: collect the bacteria after heat inactivation of BCG Pasteur 1173P2 strain cultured in 7H9 medium.

[0118] 2. Defatting: prepare solution A of chloroform:methanol (v / v=1:2) in a fume hood, put the heat-inactivated bacteria into solution A, and incubate at 37°C for 2 h with shaking, and then centrifuge at 8000 r / min to discard the supernatant, thereby completing one defatting. Repeat the defatting for three times, and then volatilize the organic reagents in the fume hood, and perform the next step.

[0119] 3. Grinding and crushing: add PBS to the defatted bacteria to suspend, and perform ice bath ultrasonic crushing according to the procedure of 2 s ultrasonic and 3 s pause, and the ultrasonic crushing is performed for a total of 8 min.

[0120] 4. De-proteinization, DNA and RNA: add 1000 U of DNase, RNase, 3 mM of PMSF and Triton X-114 with a final concentration of 8% to the ultrasonically crushed bacterial solution, and place in a 4°C refrigerator overnight. Centrifuge at 32000 x g at 4°C for 1 h, and carefully aspirate the supernatant. Place in a 50 mL centrifuge tube, and stand at 37°C until obvious layering. Take the lower organic layer, add 9 times the volume (pre-cooled at -20°C) of 95% ethanol, and stand at -80°C overnight. Centrifuge at 10000 rpm for 5 min, and discard the supernatant to obtain the precipitate. Resuspend the precipitate with 2 mL of PBS, and add 2 mg / mL of proteinase K thereto. First, incubate at 56°C for 2 h to enzymatically hydrolyze the protein, and then inactivate the proteinase K at 95°C for 10 min. PBS dialysis is performed overnight, and then centrifuge to obtain the supernatant, thereby obtaining the BCG surface polysaccharide extract, which is stored in a -20°C refrigerator for standby.

[0121] II. Purity identification of BCG surface polysaccharide extract

[0122] The purity of the BCG surface polysaccharide extract is identified by SDS-PAGE (polyacrylamide gel electrophoresis) combined with silver staining, and the specific steps are as follows:

[0123] 1. SDS-PAGE electrophoresis: prepare protein electrophoresis gel with a concentration gel of 5% and a separation gel of 12%, and add 20 μL of sample per hole after boiling the sample with an equal volume of loading buffer for 3 min. The working voltage of the concentration gel is 80 V, and the working voltage of the separation gel is 135 V. Stop the electrophoresis when the bromophenol blue reaches the bottom of the gel.

[0124] 2. After the SDS-PAGE electrophoresis is completed, perform silver staining with high iodine nitric acid, and the specific steps are as follows:

[0125] (1) After electrophoresis, the gel was transferred to a petri dish containing 100 mL of 40% (v / v) methanol, 10% (v / v) acetic acid in deionized water (fixative A) and incubated at room temperature for 45 min with shaking.

[0126] (2) Fixative A was discarded and 100 mL of fixative A containing 0.7% periodic acid was added and incubated at room temperature for 7 min with shaking.

[0127] (3) The periodic acid solution was discarded and 100 mL of 5% (v / v) methanol, 7% (v / v) acetic acid in deionized water (fixative B) was added and incubated at room temperature for 5 min with shaking.

[0128] (4) Fixative B was discarded and 50 mL of 2.5% glutaraldehyde was added and incubated at room temperature for 5 min with shaking.

[0129] (5) The glutaraldehyde was carefully removed and the gel was washed 4 times with 100 mL of distilled water for 10 min each time.

[0130] (6) 100 mL of 0.0025% DTT solution was added and incubated for 6 min.

[0131] (7) The DTT solution was discarded and 100 mL of 0.1% silver nitrate solution was added and incubated at room temperature for 5 min with shaking.

[0132] (8) The gel was quickly rinsed with 100 mL of distilled water.

[0133] (9) The gel was developed in 200 mL of 3% sodium carbonate solution containing 100 μL of 37% formaldehyde.

[0134] (10) When the contrast between the sample bands and the background of the polyacrylamide gel was moderate, the development was stopped by adding 10 mL of 50% citric acid.

[0135] The results of the purity identification of the BCG surface polysaccharide are shown in Figure 1 A.

[0136] III. Identification of the polysaccharide type of the BCG surface polysaccharide extract

[0137] Using Western blot, LAM in the BCG surface polysaccharide was selected for identification of the polysaccharide type

[0138] After SDS-PAGE electrophoresis, another gel was used for Western blot immunoblotting. The protein bands on the SDS-PAGE gel were transferred to a nitrocellulose membrane using a semi-dry transfer system, and Western blot immunoblotting was performed using 2F12 (an antibody against LAM) as the first antibody.

[0139] The results of the identification of the BCG surface polysaccharide are shown inFigure 1 B.

[0140] The results show that: 1) the BCG surface polysaccharide extract obtained in this example contains LAM, LM and PIMs, wherein the molecular weight of the LAM polysaccharide is 35-40 KDa, the molecular weight of the LM is 15-20 KDa, and the molecular weight of the PIMs is 2-10 KDa. 2) The BCG surface polysaccharide extract obtained in this example has high purity, and can be further used to investigate the immune response effect of the BCG surface polysaccharide composed of LAM, LM and PIMs on tuberculosis vaccine in mice.

[0141] Example 2: Effect of BCG surface polysaccharide on immune response of tuberculosis vaccine in mice

[0142] In this example, the effect of the BCG surface polysaccharide of Example 1 on the serum antibody titer of the immune mice and the phagocytosis of the mouse serum on macrophages was further investigated. The test animals were 6-8 week-old female SPF C57BL / 6J mice purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.; the immune animals used BCG were BCG Pasteur 1173P2 strain; the aluminum salt adjuvant was purchased from Thermo Scientific TM Imject TM , Catalog No.: 77161.

[0143] I. Immunization scheme and antibody titer detection

[0144] The test animals were divided into 5 groups, and the mice were immunized according to the immunization scheme shown in Figure 2 . Two weeks after the completion of immunization, the antibody titer was detected, and the immunization method was subcutaneous injection (s.c.). Among them, the BCG surface polysaccharide was the BCG surface polysaccharide extract of Example 1, and before immunizing the animals, the bacteria were removed, the endotoxin was removed, the pyrogen was removed, and the necessary sugar concentration was quantified.

[0145] Before performing the immunization scheme of Figure 2 , the inventors explored the immunization dose of the BCG surface polysaccharide. The specific scheme is as follows:

[0146] The inventors selected different doses (20 μg, 40 μg, 60 μg) of the BCG surface polysaccharide extract of Example 1, and immunized 6-8 week-old female SPF C57BL / 6J mice according to the experimental scheme of the PBS group. Figure 2 The test found that the mice had normal viability after the last immunization, and none of them died.

[0147] The above experimental results show that: when mice are immunized with LAM polysaccharide alone, the side effects are obvious and the safety is low; when mice are immunized with the BCG surface polysaccharide complex containing LAM polysaccharide, LM polysaccharide and PIMs polysaccharide, the survival of the mice is not affected and the safety is high.

[0148] Therefore, the inventors selected 40 μg of the BCG surface polysaccharide extract of Example 1 as an adjuvant for boosting the tuberculosis subunit.

[0149] The specific immunization scheme of each test group is as follows:

[0150] The PBS group: immunized four times, once every two weeks, 100 μL of PBS per mouse each time.

[0151] The BCG group: initially immunized once, 10 6 CFU BCG per mouse.

[0152] The AL-Alum (ALA) group: initially immunized once, 10 6 CFU BCG per mouse, immunized once every two weeks after four weeks, a total of three times of boosting, 20 μg of AL (tuberculosis subunit) and aluminum salt adjuvant mixture per mouse each time. Among them, the mixing ratio of AL and aluminum salt adjuvant is 20 μg: 100 μg by mass ratio.

[0153] The AL-Alum-BCG polysaccharide (ALS) group: initially immunized once, 10 6 CFU BCG per mouse, immunized once every two weeks after four weeks, a total of three times of boosting, 20 μg of AL (tuberculosis subunit) and the complex adjuvant containing aluminum salt adjuvant and BCG surface polysaccharide mixture per mouse each time. Among them, the mass ratio of BCG surface polysaccharide and aluminum salt adjuvant is 40 μg: 100 μg by mass ratio, and after mixing, it is mixed at 300 rpm for 30 min at 4°C on a shaker to make it uniformly cross-linked to form a complex adjuvant; the mixing ratio of AL and the complex adjuvant is 20 μg: 140 μg.

[0154] It should be noted that the dose of each mouse each time is 20 μg in terms of the protein content of AL.

[0155] AL is a tuberculosis subunit, which is prepared according to Chinese Patent CN115850520A (Invention title: Preparation method and application of novel tuberculosis subunit vaccine AL). Among them, the tuberculosis subunit is a fusion protein containing an Ag85A fragment. The fusion protein includes: an Ag85A fragment and an LpqH fragment, and the Ag85A fragment and the LpqH fragment are connected by a connecting peptide.

[0156] The amino acid sequence of the Ag85A fragment is shown in SEQ ID NO: 1.

[0157] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA (SEQ ID NO: 1).

[0158] The amino acid sequence of the LpqH fragment is shown in SEQ ID NO: 2.

[0159] VKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTAAGTTASPGAASGPKVVIDGK DQNVTGSVVCTTAAGNVNIAIGGAATGIAAVLTDGNPPEVKSVGLGNVNGVTLGYTSGT GQGNASATKDGSHYKITGTATGVDMANPMSPVNKSFEIEVTCS (SEQ ID NO: 2).

[0160] The amino acid sequence of the linker peptide is shown in SEQ ID NO: 3.

[0161] GGGGSGGGGSGGGGS (SEQ ID NO: 3).

[0162] The amino acid sequence of the fusion protein comprising the Ag85A fragment is shown in SEQ ID NO: 4.

[0163] MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAGGGGSGGGGSGGGGSVKRGLTVAVAGAAILVAGLSGCSSNKSTTGSGETTTAAGTTASPGAASGPKVVIDGKDQNVTGSVVCTTAAGNVNIAIGGAATGIAAVLTDGNPPEVKSVGLGNVNGVTLGYTSGTGQGNASATKDGSHYKITGTATGVDMANPMSPVNKSFEIEVTCSHHHHHH (SEQ ID NO: 4).

[0164] The mouse immunization experiment results show that after three times of booster immunization of the test mice in the ALS group, the viability is not affected compared with the BCG group and the ALA group, the BCG surface polysaccharide extract of Example 1 is used for boosting the immunization effect of BCG and AL, and the effect is good and the safety is high.

[0165] II. Challenge experiment

[0166] After the antibody titer detection is completed, the challenge is carried out to investigate the protection effect of each experimental group on the test mice.

[0167] The challenge dose is 200 CFU, the strain is M. bovis C68004, and the challenge is carried out by aerosol infection. After four weeks, the mice are euthanized, and the bacterial load of the lung and spleen is detected. Figure 3 ).

[0168] The challenge results show that compared with the ALA group, the bacterial load of the organs is significantly reduced after the challenge of the ALS group. The results show that the composite adjuvant formed by the aluminum salt adjuvant and the BCG surface polysaccharide can further enhance the immunization effect of BCG and effectively prevent and treat tuberculosis.

[0169] III. Detection of serum antibody titer of mice

[0170] After the mice were challenged for 4 weeks, the mice in each test group were sacrificed, and the mouse serum was taken to detect the antibody titer. The specific detection method is as follows:

[0171] M. bovis was plated, resuspended after high-pressure sterilization, and adjusted to an OD value of 1.0. 100 μL of M. bovis suspension with an OD value of 1.0 was added to each well of a 96-well plate, which was placed in a 60°C oven to dry. After drying, the plate was fixed with ice-cold methanol for 2 h. The plate was washed with PBS four times, 5 min each time. The plate was blocked with 5% skim milk powder PBS solution at 37°C for 2 h. The plate was washed with PBS three times, 5 min each time. The serum was diluted 1:1000, and 100 μL was added to the 96-well plate, which was incubated at 37°C for 1 h. The plate was washed with PBS three times, 5 min each time. The HRP-labeled goat anti-mouse IgG, IgG1, IgG2c, IgG3 (Abeam) was diluted 1:10000, and 100 μL was added to the 96-well plate, which was incubated at 37°C for 1 h. The plate was washed with PBS three times, 5 min each time. 100 μL of TMB (Solarbio, Beijing) was added to each well for color development, and 50 μL of 2M H2SO4 was added to each well to stop the color development after 20 min. The OD450 was read by a microplate reader.

[0172] The results are shown in Figure 4 .

[0173] The results show that compared with the ALA group, the IgG1 and IgG3 antibody titers in the serum of the mice in the ALS group challenged for 4 weeks were significantly increased.

[0174] Four, the effect of mouse serum on the phagocytosis of macrophages

[0175] After the mice were challenged for 4 weeks, the mice in each test group were sacrificed, and the mouse serum was taken to detect the antibody titer. The specific detection method is as follows:

[0176] 1. The Raw264.7 cells (mouse monocyte macrophage leukemia cells) were cultured to a good state, counted, and 5x10 5 cells were plated in each well of a 24-well plate, and 1 mL of DMEM (without FBS and double antibodies) cell culture solution was added. The cells were cultured in an incubator for 8 hours.

[0177] 2. The FITC fluorescent probe was dissolved in CBC buffer (0.05M carbonate bicarbonate buffer, pH 9.6) at a concentration of 50 mg / mL for standby.

[0178] 3. Take BCG (Pasteur 1173P2 strain) to measure OD600, add FITC (FITC staining final concentration 1 mg / mL) and incubate at 37°C for 2 hours, centrifuge and discard the supernatant, wash with PBS for three times, resuspend with DMEM containing mouse antibody (serum) (without FBS and double antibody), then add BCG at a MOI of 5:1, mix and add to the well plate, add an amount of 10% (v / v) of the amount of cell culture medium per well, and infect Raw264.7 cells for 2 hours.

[0179] 4. Wash with pre-cooled PBS for three times after 2 hours of infection.

[0180] 5. Add 1 mL of cell fixation solution to each well to fix the cells, and incubate at room temperature for 2 hours.

[0181] 6. Take out the cells and use BD flow cytometer to detect cell phagocytosis.

[0182] The results are shown in Table 1. Figure 5

[0183] The results show that compared with the ALA group, the serum of the mice in the ALS group can significantly improve the bacterial phagocytosis activity of macrophages after 2 weeks of bacterial attack. Thus, the ability of macrophages to kill tumor cells is further enhanced.

[0184] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.​

Claims

1. An adjuvant, characterized in that, Comprise: a Mycobacterium bovis surface polysaccharide comprising: LAM polysaccharide, and LM polysaccharide and PIMs polysaccharide; and an aluminum adjuvant; the LAM polysaccharide has a molecular weight of 35-40 KDa; the LM polysaccharide has a molecular weight of 15-20 KDa; the PIMs polysaccharide has a molecular weight of 1-10 KDa; the Mycobacterium bovis surface polysaccharide is obtained by the following method: inactivating BCG Pasteur 1173P2 strain to obtain defatted bacteria, and then deproteinizing, DNA and RNA treating the defatted bacteria to obtain the Mycobacterium bovis surface polysaccharide; the defatting treatment comprises: contacting the inactivated BCG Pasteur 1173P2 strain with an organic solution, incubating for 1-3 hours, centrifuging to discard the supernatant, and collecting the bacteria; the defatting treatment comprises: contacting the inactivated BCG Pasteur 1173P2 strain with an organic solution, incubating for 1-3 hours, centrifuging to discard the supernatant, and collecting the bacteria; the deproteinization, DNA and RNA treatment comprises: contacting the bacteria solution after the crushing treatment with DNase, RNase, PMSF and Triton X-114, standing at 4°C for 8 hours or more, taking the supernatant after the first centrifugation, standing and layering the supernatant, taking the organic layer, adding 7-11 times the volume of 95% ethanol, standing at -80°C for 8 hours or more, discarding the supernatant after the second centrifugation, and taking the precipitate, contacting the precipitate with proteinase K, incubating at 56°C for 1-3 hours, PBS dialysis after the inactivation of proteinase K, and obtaining the Mycobacterium bovis surface polysaccharide.

2. The adjuvant of claim 1, characterized in that, The Mycobacterium bovis is an attenuated strain of Mycobacterium bovis.

3. The adjuvant of claim 2, characterized in that, The attenuated strain of Mycobacterium bovis is from BCG.

4. The adjuvant of claim 1, characterized in that, The aluminum adjuvant is an aluminum hydroxide adjuvant and / or an aluminum salt adjuvant.

5. The adjuvant of claim 1, wherein, The mass ratio of the Mycobacterium bovis surface polysaccharide to the aluminum adjuvant is 2:(5-15).

6. The adjuvant of claim 1, wherein, The mass ratio of the Mycobacterium bovis surface polysaccharide to the aluminum adjuvant is 2:

5.

7. Use of Mycobacterium bovis surface polysaccharide for the preparation of an adjuvant, characterized in that, The Mycobacterium bovis surface polysaccharide comprises: LAM polysaccharide, and LM polysaccharide and PIMs polysaccharide; the LAM polysaccharide has a molecular weight of 35-40 KDa; the LM polysaccharide has a molecular weight of 15-20 KDa; the PIMs polysaccharide has a molecular weight of 1-10 KDa; the adjuvant is a complex adjuvant comprising the Mycobacterium bovis surface polysaccharide and the aluminum adjuvant; the Mycobacterium bovis surface polysaccharide is obtained by the following method: inactivating BCG Pasteur 1173P2 strain to obtain defatted bacteria, and then deproteinizing, DNA and RNA treating the defatted bacteria to obtain the Mycobacterium bovis surface polysaccharide; the defatting treatment comprises: contacting the inactivated BCG Pasteur 1173P2 strain with an organic solution, incubating for 1-3 hours, centrifuging to discard the supernatant, and collecting the bacteria; Before deproteinization, DNA and RNA treatment, the completely defatted bacteria are subjected to a crushing treatment; the crushing treatment is ultrasonic crushing, and the ultrasonic program of the ultrasonic crushing is 2 s ultrasonic and 3 s pause, and the time of the ultrasonic crushing is 4-16 min; The deproteinization, DNA and RNA treatment comprises the following steps: contacting the bacteria solution after the crushing treatment with DNase, RNase, PMSF and Triton X-114, standing at 4 ℃ for more than 8 hours, taking the supernatant after the first centrifugal treatment, taking the organic layer after the supernatant is allowed to stand and stratify, adding 7-11 times the volume of 95% ethanol, standing at-80 ℃ for more than 8 hours, discarding the supernatant and taking the precipitate after the second centrifugal treatment, contacting the precipitate with proteinase K, incubating at 56 ℃ for 1-3 h, PBS dialysis after the proteinase K is inactivated, and obtaining the Mycobacterium bovis surface polysaccharide; The adjuvant is used for enhancing the immune effect of the mycobacterium vaccine; The mycobacterium is Mycobacterium tuberculosis.

8. Use of the adjuvant according to any one of claims 1-6 in the preparation of a vaccine; The vaccine is a mycobacterium vaccine; The mycobacterium is Mycobacterium tuberculosis.

9. A subunit vaccine characterized in that, Comprises: a fusion protein comprising an Ag85A fragment and an LpqH fragment; and the adjuvant according to any one of claims 1-6; the Ag85A fragment and the LpqH fragment are connected by a connecting peptide.

10. The subunit vaccine according to claim 9, characterized in that, The amino acid sequence of the Ag85A fragment is shown in SEQ ID NO:

1.

11. The subunit vaccine according to claim 9, characterized in that, The amino acid sequence of the LpqH fragment is shown in SEQ ID NO:

2.

12. The subunit vaccine according to claim 9, characterized in that, The amino acid sequence of the connecting peptide is shown in SEQ ID NO:

3.

13. The subunit vaccine of claim 9, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

4.

14. The subunit vaccine of claim 9, characterized in that, The mass ratio of the fusion protein to the adjuvant is 1: (1-8).

15. A pharmaceutical composition comprising, Comprises: the subunit vaccine according to any one of claims 9-14.

16. A vaccine, characterized in that, Comprises: an antigen, which is attenuated live Mycobacterium bovis; and the adjuvant according to any one of claims 1-6, the subunit vaccine according to any one of claims 9-14 or the pharmaceutical composition according to claim 15.

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