CN107970444A - Composite adjuvant and the vaccine containing the composite adjuvant - Google Patents

Abstract

The invention discloses a kind of immunity composite adjuvant and preparation method thereof, and the purposes of immunity composite adjuvant enhancing vaccine immunity effect.The invention further relates to the vaccine combination comprising the immunity composite adjuvant, and purposes of the vaccine combination in relevant disease is treated.

Description

Compound adjuvant and vaccine containing the compound adjuvant

technical field

The invention relates to a composite adjuvant and a preparation method thereof. Compared with a single adjuvant, the composite adjuvant induces a Th1-type immune response drift, and the composite adjuvant enhances the immune response against intracellular bacteria such as Mycobacterium tuberculosis and viruses.

Background technique

The first-generation vaccines are generally attenuated live vaccines or inactivated whole-bacteria vaccines. These vaccines are highly immunogenic, but in addition to inducing protective immune responses, they also induce immunopathological responses. The new vaccines studied today are mainly protective antigens or epitopes. Compared with the first-generation vaccines, these antigens are relatively weak in immunogenicity, and adjuvants are needed to enhance their immunogenicity. Adjuvants are used as components of vaccines to enhance the immune response induced by antigens in vivo.

The safety of protein vaccines has been recognized because of their clear ingredients, but because the antigen itself is weak in immunogenicity, adjuvant is usually needed for assistance. Therefore, a protein vaccine generally consists of an adjuvant and an antigen. The adjuvant is injected together with the antigen or the adjuvant is pre-injected into the body to enhance the body's immune response to the antigen or change the type of immune response.

There are many kinds of adjuvants, which can be divided into inorganic adjuvants (such as aluminum hydroxide, alum, etc.), organic adjuvants (such as microorganisms and their components, including BCG, Corynebacterium pumilus, muramyl dipeptide, BCG CpG, BCG polysaccharide nucleic acid, etc.) and synthetic adjuvants (such as antimicrobial peptides, synthetic TLR agonists, etc.). However, the number of adjuvants approved for clinical research is very limited. MF59, AS03, AS04 and liposomes are adjuvants approved for use in humans after aluminum adjuvants [Mbow ML et al., Curr OpinImmunol, 2010, 22:411-416. ].

The adjuvant used in the preventive vaccines recorded in the third part of the "Pharmacopia of the People's Republic of China" published in 2010 is basically aluminum hydroxide (edited by the National Pharmacopoeia Committee, "Pharmacopia of the People's Republic of China", P21-132, Chemical Industry Press, 2010) . Aluminum hydroxide is a kind of aluminum adjuvant, which mainly enhances the antigen-induced humoral immune response, while for intracellular bacteria, such as Mycobacterium tuberculosis, the body mainly clears them through cellular immune response. The effect of aluminum adjuvant as an adjuvant for intracellular mycoprotein vaccines such as tuberculosis vaccine is difficult to meet people's requirements.

Water-oil microspheres enhance the natural immune response in a unique way. MF59 is an oil-in-water nano-emulsion. The vaccine composed of the adjuvant and the antigen is mainly used for intramuscular immunization. The MF59 adjuvant regulates the temporary expression of genes related to immune activation in muscle cells and induces the transient release of endogenous ATP to promote cell Recruitment, rapidly recruit neutrophils, monocytes, macrophages, and CD11b+ cells to the injection site to form an immune-enhancing microenvironment, and at the same time, the recruited antigen-presenting cells take up antigens and transport them to the draining lymph nodes to present to T cells to initiate acquisition Sexual immune response [Seubert A et al., J Immunol, 2008, 180:5402-5412; Vono M et al., Proc Natl Acad Sci USA, 2013, 110: 21095-21100], the cellular immunity induced by vaccines with MF5 as adjuvant generally starts at Th2-type immunity is mainly manifested by IgG1 antibodies in the serum and increased levels of antigen-specific IL-4+IL-13+CD4+T cells in splenocytes [Caproni E et al., J Immunol, 2012,188:3088- 3098]. However, intraperitoneal injection of high-dose human papillomavirus type 16 antigen E2 with MF59 adjuvant produced Th1 type immune response drift and cytotoxic activity [Heinemann L et al., Viral Immunol, 2008, 21:225-233]. Although experiments have proved that TLR4 agonist E6020, TLR9 agonist CpG ODN and MF59 combined adjuvants can enhance the drift of Th1 immune response [Baudner BC, et al., Pharm Res, 2009, 26:1477-1485; Singh M, et al., Hum Vaccin Immunother, 2012, 8: 486-890; Yang M, et al., Int Immunopharmacol, 2012, 13: 408-416], but there are also experiments that prove that the combination of MF59 and TLR agonists only improves the humoral immune response, such as MF59/CpG/ The HIV polyvalent vaccine only enhances the humoral immune response [Burke B, et al., Virology, 2009, 387:147-156]. The composition of GLA-SE and MF59-E6020 is similar, and the adjuvant is also very similar. SE as an adjuvant of Mycobacterium tuberculosis recombinant protein ID93 mainly induces Th2 type immune response, and the same protein is adjuvanted with GLA-SE or GLA-SE/CpG Agents induce Th1-type immune responses and protective immune responses, but both initiate innate immunity through different signaling pathways [Bertholet S, et al., Sci Transl Med, 2010, 2:53ra74; Orr MT, et al., PLoS ONE, 2014, 9 :e83884; Desbien AL, et al., Eur J Immunol, 2015, 45:407-417; Orr MT, et al., Eur J Immunol, 2013, 43:2398-2408].

Contents of the invention

The present invention relates to the following items:

1. An immune complex adjuvant comprising sorbitan trioleate, squalene, surfactant and heat-inactivated Bacillus Calmette-Guerin (hBCG) or high-pressure homogeneously broken BCG.

2. The composite adjuvant of item 1, wherein the amount of said sorbitan trioleate is 0.1% to 1% (w/v).

3. The composite adjuvant according to item 1 or 2, wherein the squalene is 1-10% (v/v).

4. The composite adjuvant according to any one of items 1-3, wherein the surfactant is a nonionic surfactant; preferably Tween; more preferably Tween 80.

5. The composite adjuvant of any one of items 1-4, which also includes a buffer; preferably the buffer is a sodium citrate buffer, a phosphate buffer, a histidine buffer; more preferably the buffer is Concentration is 5-10mM, pH is 6-7.

6. The composite adjuvant according to any one of items 1-5, wherein the amount of said sorbitan trioleate is 0.3% to 0.7%; preferably 0.4-0.6%, more preferably 0.5%.

7. The composite adjuvant according to any one of items 1-6, wherein the squalene is 3-7%; preferably 4-6%; more preferably 5%.

8. The composite adjuvant according to any one of items 1-7, wherein the concentration of the surfactant is 0.3-0.7% (w/v); preferably 0.4-0.6%; more preferably 0.5%.

9. The composite adjuvant according to any one of items 1-8, wherein the amount of heat-inactivated Bacillus Calmette-Guerin (hBCG) or high-pressure homogeneously broken BCG is 5-500 μg, more preferably 25-500 μg; more preferably 50-500 μg; more preferably Preferably 50-450 μg; more preferably 50-400 μg; more preferably 50-400 μg; more preferably 50-300 μg; most preferably 50-250 μg.

10. The method for preparing the composite adjuvant of any one of items 1-9, comprising

1) Add a surfactant to the buffer, stir evenly, and use it as the water phase;

2) Weighing sorbitan trioleate, adding squalene and fully mixing, as the oil phase;

3) Add the oil phase to the water phase and mix well;

4) Fully emulsify to form water-oil microspheres of 160-220nm, preferably 170-210nm, more preferably 180-200nm, add heat-inactivated Bacillus Calmette-Guerin (hBCG) or high-pressure homogeneously crushed BCG after sterilization.

11. The method of item 10, wherein the amount of sorbitan trioleate is 0.1% to 1% (w/v).

12. The method according to item 10 or 11, wherein the squalene is 1-10% (v/v).

13. The method of any one of items 10-12, wherein the surfactant is a nonionic surfactant; preferably Tween; more preferably Tween 80.

14. The method according to any one of items 10-13, which also comprises a buffer; preferably the buffer is sodium citrate buffer, phosphate buffer, histidine buffer; more preferably the buffer has a concentration of 5 -10mM, pH 6-7.

15. The method of any one of items 10-14, wherein the amount of sorbitan trioleate is 0.3% to 0.7%; preferably 0.4-0.6%, more preferably 0.5%.

16. The method of any one of items 10-15, wherein said squalene is 3-7%; preferably 4-6%; more preferably 5%.

17. The method of any one of items 10-16, wherein the concentration of the surfactant is 0.3-0.7% (w/v); preferably 0.4-0.6%; more preferably 0.5%.

18. The method according to any one of items 10-17, wherein the amount of heat-inactivated BCG (hBCG) or high-pressure homogeneously broken BCG is 5-500 μg, more preferably 25-500 μg; more preferably 50-500 μg; more preferably 50 μg - 450 μg; more preferably 50-400 μg; more preferably 50-400 μg; more preferably 50-300 μg; most preferably 50-250 μg.

19. The present invention also relates to a Mycobacterium tuberculosis antigen or a fusion protein with Mycobacterium tuberculosis immunoreactivity selected from the group consisting of:

(a) comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence to SEQ ID NO:4 identity and still be immunogenic;

(b) one or more amino acids are substituted, deleted, inserted and/or added in the amino acid sequence shown in SEQ ID NO: 4, and still have immunogenicity;

(c) is encoded by a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence to SEQ ID NO:3 identity;

(d) is encoded by a nucleotide sequence that substitutes, deletes, inserts and/or adds one or more nucleotides in the nucleotide sequence shown in SEQ ID NO:3; and

(e) is encoded by a nucleotide sequence that is at least moderately stringent, at least medium-highly stringent, at least highly stringent, or at least very highly stringent with the multinuclear DNA shown in SEQ ID NO:3 nucleotide hybridization.

20. The present invention also relates to a nucleic acid comprising or consisting of:

(a) a polynucleotide whose nucleotide sequence has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:3;

(b) a polynucleotide whose nucleotide sequence is substituted, deleted, inserted and/or added with one or more nucleotides in the nucleotide sequence shown in SEQ ID NO:3;

(c) a polynucleotide whose nucleotide sequence hybridizes to the polynucleotide shown in SEQ ID NO:3 under at least moderately stringent conditions, at least medium-high stringent conditions, at least high stringent conditions, or at least very high stringent conditions;

(d) a polynucleotide encoding a fusion protein whose amino acid sequence is at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:4 % sequence identity;

(e) a polynucleotide encoding a fusion protein that substitutes, deletes, inserts and/or adds one or more amino acids in the amino acid sequence shown in SEQ ID NO: 4; or

(f) A polynucleotide that encodes SEQ ID NO:4 but differs from SEQ ID NO:3 due to codon degeneracy.

21. The present invention also relates to a method for preparing the Mycobacterium tuberculosis antigen of Item 19 or a fusion protein having Mycobacterium tuberculosis immunoreactivity, comprising expressing the nucleic acid of Item 20 in a suitable host cell.

The present invention further relates to a nucleic acid construct or expression vector comprising the above polynucleotide, which is preferably pET28a-PstS1-LEP. The present invention further relates to a host cell comprising the above-mentioned construct or expression vector, which is preferably an Enterobacteriaceae cell, more preferably an Escherichia coli cell, and still more preferably an Escherichia coli BL21 (DE3) cell.

In another aspect, the present invention also provides a method for preparing the above-mentioned fusion protein with Mycobacterium tuberculosis immunoreactivity or the above-mentioned nucleic acid construct or expression vector (preferably PstS1-LEP), which comprises using the polynucleotide of the present invention, Nucleic acid construct, expression vector or host cell. Specifically, the method comprises (a) cultivating the host cell of the invention under conditions conducive to production of the fusion protein; and (b) recovering the fusion protein.

More preferably, the above-mentioned preparation method comprises the steps of:

1) obtaining a linear vector-PstS1 recombinant plasmid, preferably a linear PET28a-PstS1 recombinant plasmid;

2) obtaining and synthesizing the LEP amino acid coding nucleic acid sequence shown in SEQ ID NO: 2, linking the synthesized LEP gene to a carrier, preferably a PUC19 carrier, to form a carrier-LEP recombinant plasmid;

3) Use the obtained carrier-LEP recombinant plasmid to transform a suitable host cell, preferably Escherichia coli, screen and pick a single clone colony for cultivation, extract the plasmid for enzyme digestion and obtain the LEP gene fragment, preferably after double enzyme digestion The LEP gene fragment was recovered by electrophoresis;

4) connecting the linear vector-PstS1 recombinant plasmid and the LEP gene fragment, preferably by T4DNA ligase; introducing the connected vector-PstS1-LEP recombinant plasmid into a suitable host cell, preferably an Escherichia coli host cell, more preferably Escherichia coli cells BL21 (DE3);

5) cultivating the host cell under conditions conducive to the expression of the vector-PstS1-LEP sequence; and

6) recovering, purifying, annealing and/or identifying the expressed fusion protein.

22. The present invention also relates to a pharmaceutical composition comprising any one of items 1-9 immune complex adjuvant and antigen, preferably the antigen is an intracellular bacterial or viral antigen, more preferably Mycobacterium tuberculosis antigen or macrophage A cellular virus antigen, more preferably the antigen or fusion protein of item 19 or cytomegalovirus (CMV) gB antigen (CMV gB).

23. The pharmaceutical composition of item 22, which is a vaccine, further, it is a prophylactic and/or therapeutic vaccine, further, it is a prophylactic and/or therapeutic vaccine for intracellular bacterial infection or viral infection, more preferably It is a prophylactic and/or therapeutic vaccine for Mycobacterium tuberculosis infection or cytomegalovirus infection.

24. Use of the immune complex adjuvant of any one of items 1-9 in improving the immune reactivity of the body, preferably the immunoreactivity is for intracellular bacteria or viruses, preferably the immunoreactivity is Immunoreactivity against Mycobacterium tuberculosis or Cytomegalovirus.

25. Use of the immune complex adjuvant of any one of items 1-9 in the preparation of a pharmaceutical composition or vaccine, preferably the pharmaceutical composition or vaccine is a preventive and/or therapeutic drug combination against intracellular bacteria or viruses Drugs or vaccines, preferably prophylactic and/or therapeutic pharmaceutical compositions or vaccines against Mycobacterium tuberculosis or cytomegalovirus.

26. Use of the immune complex adjuvant according to any one of items 1-9 for preventing and/or treating intracellular bacterial or viral infection, preferably preventing and/or treating Mycobacterium tuberculosis or cytomegalovirus infection.

27. A method for preventing and/or treating intracellular bacterial or viral infection, comprising administering a prophylactically or therapeutically effective amount of the pharmaceutical composition or vaccine according to item 22 or 23 to a patient.

28. In the use and method of the above items, the pharmaceutical composition or vaccine is administered by subcutaneous or intramuscular injection.

29. The above-mentioned fusion protein of the present invention is a multi-epitope antigen with good immunoreactivity and can be used for the diagnosis of tuberculosis. Therefore, the present invention also relates to the use of the above fusion protein in the diagnosis of tuberculosis. In one embodiment, the fusion protein is used to detect serum IgG to diagnose pulmonary tuberculosis; in another embodiment, the fusion protein is used to detect serum IgM to diagnose extrapulmonary tuberculosis. Both assays have high sensitivity and specificity. In addition, in one embodiment, the fusion protein can diagnose pulmonary tuberculosis and extrapulmonary tuberculosis by detecting IgG and IgM in the same well.

30. The present invention relates to a tuberculosis diagnostic kit comprising the antigen or fusion protein described in item 19 above.

31. The present invention also relates to the use of the antigen or fusion protein described in item 19 above in the preparation of a tuberculosis diagnostic kit.

The compound adjuvant disclosed by the present invention and the composition containing the compound adjuvant and antigen overcome the generally insufficient immunogenicity of Mycobacterium tuberculosis and the generally weak effect of existing adjuvants on enhancing the body's immune response against intracellular bacteria or viruses shortcoming. By using the composite adjuvant of the present invention, the immune reactivity and immune effect against intracellular bacteria or viruses, especially mycobacterium tuberculosis or cytomegalovirus are effectively improved, and the effective prevention and/or treatment of tuberculosis is promoted.

The "intracellular bacterium" in the present invention is also called "intracellular parasite" or "intracellular bacterium", which is a pathogenic bacterium that stays in the host cell for most of the time after invading the human body and reproduces. For example, Mycobacterium tuberculosis, Bacillus leprae, Brucella, etc. all belong to this category. Since antibodies cannot enter cells, the effect of humoral immunity on such bacterial infections is limited, and the defense against intracellular infections mainly depends on cellular immunity. For example, when the body is infected with Mycobacterium tuberculosis for the first time, since cellular immunity has not yet been established, although phagocytes can engulf them, they cannot digest and kill them effectively. Therefore, pathogenic bacteria are likely to spread and spread in the body with phagocytes, causing systemic infection. However, in the process of infection, the body gradually forms cellular immunity under the stimulation of pathogenic bacteria, and activates phagocytes by sensitizing various lymphokines released by lymphocytes, which can greatly enhance their phagocytosis and digestion ability, and inhibit the survival of pathogenic bacteria in phagocytes, thereby Gain immunity against reinfection by the same species of pathogenic bacteria.

The term "Mycobacterium tuberculosis" in the present invention can also be referred to as "Mycobacterium tuberculosis" for short.

The "virus" mentioned in the present invention is a kind of microorganism without cell structure, but with life characteristics such as heredity and replication. They are smaller than bacteria, have no cellular structure, are composed of proteins and nucleic acids, and can only proliferate in living cells, which is similar to intracellular bacteria.

"BCG (Bacillus Calmette-Guérin, referred to as BCG, the Chinese name comes from its inventor Kashi-Jieshi) is a vaccine used to prevent tuberculosis, using live Mycobacterium bovis in a special artificial culture Basically, after in vitro 230 generations of culture attenuation, it loses its pathogenicity to humans, but still maintains a sufficiently high immunogenicity. Therefore, BCG is a live attenuated vaccine. Inoculation of BCG in the prevention of tuberculosis, especially possible Serious types of tuberculosis that endanger children's lives, such as tuberculous meningitis, miliary tuberculosis, etc., have a very obvious effect. "hBCG" (heat-aggregated bacille Calmette-Guerin) is the abbreviation of heat-inactivated BCG, so "hBCG", "Heat-inactivated BCG" and "heat-inactivated BCG hBCG" are used interchangeably.

"High-pressure homogeneous crushing BCG" is a cell-free product produced by high-pressure homogeneous crushing after heat inactivation of BCG. For example, the method of Example 1 of the present invention can be used to heat inactivate the collected BCG at 80°C for 1 hour, crush it with a low-temperature ultra-high pressure continuous flow cell crusher (JN3000Plus), and cycle it five times under the condition of a power of 1500-1700 Bar; fully crush the bacteria Centrifuge at 5000 rpm for 10 minutes to remove unbroken BCG cells, sterilize with cobalt 60 irradiation, and the product is high-pressure homogeneously broken BCG.

"Heat-inactivated Bacillus Calmette-Guerin (hBCG)" is heat-inactivated BCG that is structurally intact avirulent Mycobacterium bovis, while "High Pressure Homogenized Broken BCG" is the crushed product of inactivated avirulent Mycobacterium bovis .

The term "fusion protein" used in the present invention refers to the fusion of one protein region to the N-terminal or C-terminal region of another protein. Fusion proteins are typically produced by fusing a polynucleotide encoding one protein to a polynucleotide encoding another protein. Techniques for producing fusion proteins are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion protein is under the control of the same promoter and terminator. Fusion proteins can also be constructed using intein technology, where fusion proteins are produced post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). Fusion proteins can also contain a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, Martin et al., 2003, J.Ind.Microbiol.Biotechnol.3:568-576; Svetina et al., 2000, J.Biotechnol.76:245-251; , Appl.Environ.Microbiol.63:3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochem.25:505-512 ; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and the positions disclosed in Stevens, 2003, Drug Discovery World 4:35-48 point.

The term "codon degeneracy" used in the present invention refers to the phenomenon that one amino acid is encoded by more than one triplet codon.

The term "immunoreactivity" used in the present invention refers to the ability of an antigen molecule to specifically bind to the corresponding immune response product (antibody or sensitized lymphocyte) in vitro or in vivo, also known as antigenicity.

The term "composite adjuvant" used in the present invention refers to a new adjuvant composed of two or more adjuvants with different action mechanisms.

The term "antigenic epitope" used in the present invention is also called an antigenic determinant, and refers to a chemical group in an antigen molecule that determines antigen specificity. It is capable of specifically binding to T cell antigen receptor TCR or B cell antigen receptor BCR. the basic unit of .

The term "immunogenicity" used in the present invention refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It also refers to the characteristics that antigens can stimulate specific immune cells, activate, proliferate, and differentiate immune cells, and finally produce immune effector antibody and sensitized lymphocytes.

The term "host cell" as used herein includes any cell type susceptible to transformation, transfection, transduction, etc., using a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.

The term "expression vector" as used in the present invention is defined as a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the present invention linked to other nucleotides for its expression. The term "construct" or "nucleic acid construct" refers to a single- or double-stranded nucleic acid molecule, either isolated from a naturally occurring gene or modified to contain a non-naturally occurring gene, the construct may contain Regulatory sequences required for the expression of the coding sequences of the invention.

The term "sequence identity" or "identity" as used in the present invention is used to describe the relatedness between two nucleotide sequences or amino acid sequences, which can be compared by sequence alignment methods known in the art, using conventional algorithms and penalties. Derived from the sub-rule, expressed as a percentage.

The term "substitution" used in the present invention means to replace the nucleotide/amino acid occupying a certain position with a different nucleotide/amino acid; "deletion" means to remove the nucleotide/amino acid occupying a certain position; "insertion" means Refers to the addition of nucleotides/amino acids in the middle of the sequence adjacent to and immediately after the nucleotide/amino acid occupying a certain position; "addition" means the addition of nucleotides/amino acids at both ends of the sequence.

The terms "very high stringency conditions", "high stringency conditions", "medium-high stringency conditions" and "moderate stringency conditions" used in the present invention mean that for probes with a length of at least 100 nucleotides, at 42°C, Prehybridization and prehybridization were performed according to standard Southern blotting in 5X SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 50%, 50%, 35%, and 35% formamide, respectively. Hybridize for 12 to 24 hours. The support material was finally washed three times with 2X SSC, 0.2% SDS at 70°C, 65°C, 60°C and 55°C for 15 minutes each.

The term "kit" used in the present invention refers to a complete set of reagents prepared by using the protein of the present invention to complete the detection and diagnosis of tuberculosis. The kit is used for the diagnosis of tuberculosis, including pulmonary tuberculosis and extrapulmonary tuberculosis. The kit includes a container containing the fusion protein of the present invention. The fusion protein of the present invention can be packaged in any convenient and suitable packaging manner. The kit of the present invention may also include a plurality of other containers, which may respectively contain standards used for detection, antibodies or labeled antibodies, enzymes, substrates or buffers and the like. In this kit, a label and a package insert are also included to provide instructions for using the kit. Other materials such as microtiter plates and the like may also be included to suit the needs of the user.

The term "adjuvant" in the present invention refers to a class of non-specific immune enhancer, which is usually inoculated before vaccine immunization alone or mixed with the vaccine at the same time in order to enhance the animal body's immune response to antigenic substances or change its immune response. The type of immune response, especially after the second immunization (second immunization), its immune enhancement effect is more obvious. The term "composite adjuvant" refers to the combination of two or more adjuvants or an adjuvant with other agents to induce a stronger immune response.

The term "tuberculosis" in the present invention refers to a chronic infectious disease caused by Mycobacterium tuberculosis infection. The term "tuberculosis" refers to an infectious disease caused by Mycobacterium tuberculosis infection of the lungs. The term "extrapulmonary tuberculosis" refers to tuberculosis that occurs in various parts of the human body other than the lungs due to the spread of pulmonary lesions to various organs of the human body through the blood or lymphatic system.

The term "vaccine" in the present invention refers to prophylactic biological products used for vaccination of subjects in order to prevent and control the occurrence and/or prevalence of infectious diseases, specifically including the use of microorganisms or their toxins, enzymes, human or animal serum , cell preparations for prevention, diagnosis and treatment.

Brief description of the drawings

Figure 1 shows the restriction map of the recombinant plasmid. In the figure, lane 1 is the λDNA/HindIII marker; lane 2 is pET-28a-PstS1-LEP digested with NcoI; lane 3 is pET-28a-PstS1-LEP digested with BamHI; lane 4 is pET-28a-PstS1- LEP was digested with NcoI+BamHI; lane 5 was digested with NcoI+HindIII for pET-28a-PstS1-LEP; lane 6 was digested with BamHI+HindIII for pET-28a-PstS1-LEP; lane 7 was a 100bp DNA marker ( where the bands indicate 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1500 bp, respectively). As shown in Figure 1, the recombinant plasmid NcoI and BamHI double-digestion shows about 1.1kb of the target gene and about 5.9kb of linear plasmid DNA, NcoI and HindIII double-digestion of about 1.7kb of the target gene and 5.3kb of linear plasmid DNA, BamHI and HindIII double-enzyme digestion showed about 0.6kb target gene and 6.4kb linear plasmid DNA, and NcoI and BamHI single-enzyme digestion respectively showed about 7.0kb linear plasmid DNA. The target gene fragment of NcoI and BamHI double digestion is the PstS1 coding gene, and the target gene fragment of BamHI and HindIII double digestion is the polyepitope protein LEP coding gene, so the DNA sequence encoding the protein is successfully connected to the carrier at the accurate restriction site, The recombinant plasmid contains the fusion gene of the pre-expressed protein, and the size of the inserted foreign gene is consistent with the theory.

Figure 2 shows the SDS-polyacrylamide gel electrophoresis patterns of the recombinant engineered bacteria and the purified protein and the immunoblotting pattern of the purified refolded protein. In Figure 2a: M1 is the standard molecular weight of the protein; 1 is the pET-28a-PstS1-LEP/BL21(DE3) host bacteria induced without IPTG; 2 is the pET-28a-PstS1-LEP/BL21(DE3) host induced by IPTG Bacteria; 3 is the ultrasonic supernatant of IPTG-induced pET-28a-PstS1-LEP/BL21(DE3) host bacteria; 4 is the ultrasonic precipitation of IPTG-induced pET-28a-PstS1-LEP/BL21(DE3) host bacteria; 5 is anion Chromatographic column purification of protein; 6 is refolding protein. In Fig. 2b: M2 is the standard molecular weight of prestained protein; 1 is the reaction of PstS1-LEP protein and anti-PstS1 monoclonal antibody; 2 is the reaction of PstS1-LEP protein and serum IgG of tuberculosis patients; 3 is the reaction of PstS1-LEP protein and serum Ig of tuberculosis patients ( G+M) reaction; 4 is the reaction between PstS1-LEP protein and serum IgM of tuberculosis patients.

Figure 2a shows that the engineered bacteria express the recombinant protein under the induction of IPTG, and express it in the form of inclusion bodies, and the engineered bacteria hardly express the recombinant protein without IPTG induction. Chromatographic column purification and renaturation to obtain a relatively pure recombinant protein. Figure 2b shows that the refolded recombinant protein positively reacts with the anti-PstS1 protein monoclonal antibody, IgG, IgM and Ig(G+M) in serum of tuberculosis patients.

Figure 3 shows the particle size detection results of two batches of water-oil microspheres prepared at different times. Figure 3A is the test result of the particle size of the water-oil microspheres prepared in October 2014. The average particle size shown in the figure is 1846.4nm; Figure 3B is the test result of the particle size of the water-oil microspheres prepared in April 2015. Shown is an average particle size of 193 nm.

Figure 4 shows that water-oil microspheres and hBCG alone or in combination stimulate RAW264.7 cells to secrete TNF-α, MCP-1 and IL-1β in vitro, and water-oil microspheres and hBCG alone or in combination cannot stimulate RAW264.7 cells to secrete IL- 1β; except for low-dose hBCG, all of them can stimulate RAW264.7 cells to secrete TNF-α in vitro; only the compound adjuvant water-oil microspheres/hBCG can stimulate RAW264.7 cells to secrete MCP-1. Different letters on the columns in the figure indicate that different combinations stimulate cells in vitro to secrete the same cell/chemokines, and there is a statistical difference, where a indicates a significant difference compared with the unstimulated well; b indicates a significant difference compared with 25 μg/ml hBCG; c indicates a significant difference compared with 25 μg/ml hBCG; 50μg/ml hBCG has a significant difference; d indicates a significant difference compared with 1:250 water-oil microspheres.

Figure 5A shows the percentages of DC cells, macrophages, neutrophils and monocytes recruited into muscle tissue at the injection site after subcutaneous or intramuscular injection of mice immunized with water-oil microspheres/50 μg/ml hBCG for 24 hours. Figure 5B shows the percentages of DC cells, macrophages, neutrophils and monocytes recruited into muscle tissue at the injection site after subcutaneous or intramuscular injection of mice immunized with water-oil microspheres/250 μg/ml hBCG for 24 hours. Both intramuscular and subcutaneous immunization promote the recruitment of immune cells to the muscle tissue at the injection site.

Figure 6 shows that different adjuvants are compatible with the protein PstS1-LEP of the present invention to immunize mice. Compared with the PstS1-LEP immunization group, only the composite adjuvant water oil microspheres/hBCG is compatible with the protein PstS1-LEP of the present invention. The number of cells secreting PstS1-LEP antigen-specific IFN-γ and IL-2 was significantly increased, while the number of splenocytes secreting IL-4 in mice immunized with Al(OH) ₃ /hBCG and the protein PstS1-LEP of the present invention was compatible Significantly increased. Different letters on the columns in Figure 6 indicate that different adjuvant and PstS1-LEP combinations immunized mice, and the comparison of the level of the same cytokine secreted by mouse splenocytes showed statistical significance, where a indicated a significant difference compared with the water-oil microsphere immunized group; b Indicates a significant difference compared with the hBCG immunized group; c indicates a significant difference compared with the PstS1-LEP immunized group.

Different letters on the column in Figure 7 indicate different adjuvant and PstS1-LEP combinations to immunize mice, and the comparison of the same cytokine level in the culture supernatant of mouse splenocytes stimulated in vitro shows statistical significance, where a indicates the comparison with the water-oil microsphere immunized group Significant difference; b indicates significant difference compared with hBCG immunized group; c indicates significant difference compared with PstS1-LEP immunized group. This figure shows that different adjuvants are compatible with the protein PstS1-LEP of the present invention to immunize mice, and compared with the PstS1-LEP immunization group: water-oil microspheres, water-oil microspheres/hBCG are respectively compatible with the protein PstS1-LEP of the present invention. Enhance mouse splenocytes to secrete PstS1-LEP antigen-specific IFN-γ; water-oil microspheres/hBCG and the protein PstS1-LEP of the present invention to immunize mice can significantly enhance mouse splenocytes to secrete PstS1-LEP antigen-specific IL-2.

Different letters on the columns in Figure 8 indicate different adjuvants combined with PstS1-LEP to immunize mice, and the comparison of the same cytokine level in the in vitro stimulation culture supernatant of mouse peritoneal macrophage cells shows statistical significance, where a indicates the combination with water-oil microspheres Significant difference compared with immunization group; b indicates significant difference compared with hBCG immunized group; c indicates significant difference compared with PstS1-LEP immunized group. This figure shows that different adjuvants are compatible with the protein PstS1-LEP of the present invention to immunize mice, compared with the PstS1-LEP immunization group: water-oil microspheres and the protein PstS1-LEP of the present invention are compatible with immunization of mice to significantly enhance the secretion of mouse peritoneal macrophages PstS1-LEP antigen-specific IL-12; hBCG, water-oil microspheres, Al(OH) ₃ /hBCG were respectively compatible with the protein PstS1-LEP of the present invention to immunize mice, which significantly enhanced the secretion of PstS1-LEP antigen-specific by mouse peritoneal macrophages. IL-1β.

Figure 9 shows that mice were immunized with different doses of hBCG alone or in combination with water-oil microspheres. The secretion of BCG-PPD-specific IFN-γ and IL-2 by mouse splenocytes showed an increasing trend with the increase of hBCG dose. The same dose of hBCG, compound The adjuvant water oil microspheres/hBCG is superior to single hBCG. But for IL-4, there is no obvious rule.

Figure 10 shows that mice were immunized with different doses of hBCG alone or in combination with water-oil microspheres, and mouse peritoneal macrophages secreted BCG-PPD-specific IL-12 and IL-1β. Mice immunized with compound adjuvant enhanced the secretion of IL-12 and IL-1β from macrophages, while mice immunized with compound adjuvant composed of high dose of hBCG and water-oil microspheres inhibited the secretion of IL-12 from macrophages.

Figure 11A-C shows water-oil microspheres and/or hBCG, as K6 antigen adjuvant mice, serum antibody IgG subtype detection results show: compared with K6 antigen immunization group, hBCG, water-emulsion microspheres, water-emulsion microspheres/ Compatibility with hBCG, Al(OH) ₃ /hBCG and K6 antigen respectively immunized mice with K6 antigen significantly induced K6-specific IgG1 antibody, while only Al(OH) ₃ /hBCG and K6 antigen compatible immunization with mice significantly induced K6-specific IgG2a antibody. Different letters on the columns in the figure indicate that the comparison between different immunization groups is statistically significant, a indicates a significant difference compared with the water-oil microsphere immunized group (P<0.05); b indicates a significant difference compared with the hBCG immunized group (P<0.05) ; c indicates a significant difference compared with the K6 immunization group (P<0.05).

Figures 12A-C show the induction of IFN-γ, IL-2 and IL-4 secretion in mouse splenocytes by immunizing mice with K6 antigen combined with different adjuvants.

Figure 13A-B shows that hBCG, water-oil microspheres, water-oil microspheres/hBCG, Al(OH) ₃ /hBCG were used as K6 antigen adjuvant to immunize mice, and peritoneal macrophages secrete K6-specific IL-12, IL- The role of 1β. The results showed that, compared with the K6 immunization group, mice immunized with K6 in combination with water-emulsion microspheres, water-oil microspheres/hBCG, Al(OH) ₃ /hBCG, respectively, could enhance the secretion of IL-12 from macrophages. Different letters on the columns in the figure indicate that the comparison between different immune groups is statistically significant, a indicates a significant difference compared with the water-oil microsphere group (P<0.05); b indicates a significant difference compared with the hBCG group (P<0.05); c Indicates a significant difference compared with K6 (P<0.05).

Figure 14 shows that water-oil microspheres and/or hBCG promote the production of anti-CMV gB IgG antibodies induced by CMV gB. Among them, A: mice immunized with water-oil microspheres, B: mice immunized with water-oil microspheres/hBCG, C: mice immunized with CMV gB antigen, D: mice immunized with water-oil microspheres+CMV gB, E : mice immunized with water-oil microspheres/hBCG+CMV gB, F: mice immunized with water-oil microspheres/hBCG+CMV gB. Groups A-D and F were immunized by intramuscular injection, and group E was immunized by subcutaneous injection. ** in the figure: P<0.01

Figures 15a-b show the effects of water-oil microspheres and/or hBCG on CMV gB-specific IFN-γ and IL-4 secretion. Among them, A: mice immunized with water-oil microspheres, B: mice immunized with water-oil microspheres/hBCG, C: mice immunized with CMV gB antigen, D: mice immunized with water-oil microspheres+CMV gB, E : mice immunized with water-oil microspheres/hBCG+CMV gB, F: mice immunized with water-oil microspheres/hBCG+CMV gB. Groups A-D and F were immunized by intramuscular injection, and group E was immunized by subcutaneous injection. In the figure, *: P<0.05, **: P<0.01.

Figure 16A-C shows the situation that the complex adjuvants prepared by different preparation techniques assist the protein PstS1-LEP of the present invention to induce humoral immune response. IgG and IgG1 are absorbance values measured at 1:50000 dilution of serum (A and B); IgG2a are absorbance values measured at 1:2500 dilution of serum (C).

Figure 17A-C shows the situation that the composite adjuvant prepared by different preparation techniques assists the protein PstS1-LEP of the present invention to induce cellular immune response. Shown in the figure is the number of spot-forming cells secreting PstS1-LEP-specific IFN-γ, IL-4, and IL-17 of the protein of the present invention.

Example

Example 1: Preparation of water-oil microsphere composite adjuvant of the present invention

(1) Preparation of water-oil microspheres

Take 456ml of 10mmol/L pH6.5 citric acid buffer, add Tween 80 (0.5%), stir evenly (water phase); weigh 2.5g of sorbitol trioleate, add squalene 21.5ml-25ml, preferably 21.5 ml, mix well (oil phase); magnetically stir the water phase, slowly add the oil phase, and continue to stir for 1h. Ultrasound three times, each time for 30 minutes, and then crushed by a low-temperature ultra-high pressure continuous flow cell crusher (JN3000Plus), with a power of 1500-1700Bar and five cycles; it is milky white, without stratification, without obvious oil droplets, and evenly hangs on the wall. Sterilize by 0.22μm filter, aliquot, store at 4°C (O'Hagan DT, Ott GS, Nest GV, etc. The history of water-oil microspheres adjuvant: a phoenix that arose from theashes. Expert Rev Vaccines, 2013, 12(1):13-30). Valid for 6-12 months. The average particle size measured by the particle size test is 180-200nm, and the particle size test results of two batches of water-oil microspheres prepared in different periods are shown in Figure 3A and Figure 3B.

(2) Preparation of hBCG

The strain is the same as that of BCG for intradermal injection (Chengdu Institute of Biological Products Co., Ltd.), that is, BCG D2PB302. Dissolve a freeze-dried BCG for intradermal injection in 0.2ml of sterile water for injection, inoculate all of it on a slant of a modified Roche medium, and absorb for 30 minutes; culture at 37°C for 4-5 weeks until the BCG colonies cover the slant. The revived BCG was subcultured and expanded in the modified Roche medium, cultured at 37°C for about 3 weeks, and then transferred to the improved Roche medium for passage and expanded culture, and cultured at 37°C for about 3 weeks. The bacteria were collected on the slant, and some of them were suspended in Sutong medium containing 10% glycerol, and were divided into cryopreservation tubes and stored at -70°C as seed bacteria. The remaining bacteria were transferred to Sutong potato medium for culture, cultured at 37°C for 2-3 weeks to collect BCG, ground, washed, adjusted to 100 mg/ml bacteria concentration, heat inactivated at 80°C for 1 hour, and stored at 4°C. Heat-inactivated BCG is the hBCG used in the composite adjuvant of the present invention.

(3) Preparation of compound adjuvant and its compatibility with antigen

Just before use, mix the water-oil microspheres and hBCG, the water-oil microspheres account for 50% (V/V), and the final hBCG concentration is 25-50 μg/ml (in vitro stimulation) or 50-250 μg/0.2ml (immunized mice ), add the corresponding volume of PBS; mix with the tip more than 30 times.

Before use, mix the water-oil microspheres and hBCG, and add the antigen at the same time; the water-oil microspheres in the vaccine account for 50% (V/V), the final concentration of hBCG is 5-250μg/0.2ml, and the final concentration of antigen is 10μg/0.2 ml, add the corresponding volume of PBS; mix with the tip more than 30 times.

Example 2: The composite adjuvant of the present invention stimulates macrophages to secrete monocyte chemotactic protein-1 (monocyte chemotactic protein 1, MCP-1) in vitro

The DMEM high-glucose medium was adjusted to 1×10 ⁶ /ml passaged cell RAW264.7 (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) and distributed to 12-well culture plates, with 1ml of cell suspension per well. Incubate at 37°C, 5% CO ₂ for 5h. Wash off non-adherent cells, add 1ml DMEM high-glucose complete medium (containing 10% high-quality fetal bovine serum, double antibody (i.e. penicillin and streptomycin) to each well of cells, and add 50 μl of stimulator, each stimulator Repeat 2 wells, add 50 μl DMEM high glucose complete medium to the control well. Cultivate at 37°C, 5% CO ₂ for 24 hours, take the cell culture supernatant, and store at -80°C. The stimulant is prepared from DMEM high glucose complete medium, including 1:5 and 1 :12.5 diluted water-oil microspheres, 0.5mg/ml hBCG, 1mg/ml hBCG, 1:5 water-oil microspheres+0.5mg/ml hBCG, 1:5 water-oil microspheres+1mg/ml hBCG, 1:12.5 water Oil microspheres + 0.5mg/ml hBCG, 1:12.5 water-oil microspheres + 1mg/ml hBCG. Sandwich ELISA method to detect cytokine content in cell culture supernatant: follow TNF-α, IL-1β and MCP-1 ELISA kit instructions (BD Bioscience, USA). Water-oil microspheres and hBCG alone or in combination stimulate RAW264.7 to secrete TNF-α, IL-1β and MCP-1 levels as shown in Figure 4.

50μg/ml hBCG, 1:100 water-oil microspheres, 1:250 water-oil microspheres, any dose of compound adjuvant water-oil microspheres/hBCG can effectively stimulate RAW264.7 cells to secrete TNF-α, while only either Only the combination of adjuvant water-oil microspheres/hBCG can effectively stimulate RAW264.7 cells to secrete MCP-1, and any adjuvant and its combination cannot stimulate RAW264.7 cells to secrete IL-1β.

Example 3: Subcutaneous and intramuscular injection of the compound adjuvant of the present invention enhances the recruitment of natural immune cells to muscle tissue at the injection site

(1) Innate immune cells were recruited from the muscle tissue at the injection site by intramuscular injection of a single adjuvant or a compound adjuvant at different time points

BALB/c mice were divided into 8 groups with 9 mice in each group. PBS, water-oil microspheres, 50 μg hBCG, PstS1-LEP, 250 μg hBCG, water-oil microspheres/50 μgh BCG, water-oil microspheres/50 μg hBCG/PstS1-LEP, water-oil microspheres were injected subcutaneously into the rich muscles of the right hind leg /250 μg hBCG. After the injection, 3 mice in each group were sacrificed in 24 hours, 48 hours, and 72 hours, and the muscle tissue at the injection site of the right leg of the mice was taken and cut into pieces of 2-4 mm; Dissolve collagenase according to the instruction manual of Gentle MACS product), add 9μl collagenase A, 50μl collagenase D, 12μl collagenase P, 1.4ml cell culture medium DEME to the C tube of Gentle MACS and mix well. Put the shredded muscle tissue of the mouse into the enzymatic hydrolysis mixture in Gentle MACS C tube, and put it in a water bath at 37°C for 60 minutes with constant slight shaking. After the water bath, use the program muscle-01 of Gentle MACS to treat once; continue at 37°C Water bath for 30 minutes, with constant slight shaking, and Gentle MACS program muscle-01 for another treatment. Add 3ml of DEME culture medium to the muscle homogenate suspension and mix well. The homogenate is filtered through a 200-mesh nylon membrane into a 15ml centrifuge tube, and DMEM is added to 10ml. Collect the cells by centrifugation, wash the cells once with 10ml DMEM, suspend the cell pellet with 5ml DMEM, filter once through a 200 mesh nylon membrane, centrifuge to suspend the cells, wash the cell pellet once with PBS, and suspend the cells with 700μl PBS. 300 μl of cell suspension was labeled with the following fluorescent antibodies: CD11b-FITC, F4/80-APC, Ly6G-PE and CD14-Percp-CY5.5, and another 300 μl of cell suspension was labeled with the following fluorescent antibodies: CD14-Percp-CY5.5 , CD11c-APC, CD45R/B220-FITC, CD83-PE. After washing, the labeled cells were loaded into the flow cytometer for analysis, and the results were analyzed using the software that comes with the instrument (Table 1 and Table 2).

Table 1: Recruitment of DC cells at the injection site

Note: CD11c+ refers to the percentage of CD11c+ positive cells in the number of CD14 positive cells in the first gate, and CD11c+CD83+ refers to the percentage of CD11c+CD83+ positive cells in the number of CD14 positive cells in the first gate.

Table 2: Macrophages, Neutrophils and Monocytes Recruited at the Injection Site

Note: Neutrophils refer to the percentage of Ly6G and CD11b double-positive cells in the first gate, and monocytes refer to the percentage of CD14 single-positive cells in the first gate. Macrophages refer to the percentage of F4/80 and CD11b double-positive cells in the number of CD14 single-positive cells in the first gate.

Table 1 shows: CD11c+ cells and CD11c+CD83+ cells represent two types of dendritic cells (DC) from different sources, respectively, and the DC cells at the injection site show a trend of decreasing with the prolongation of immunization time. Water-oil microspheres/50μg hBCG immunized mice recruited the highest CD11c+ cells in 24 hours, while antigen-immunized mice had the highest CD11c+CD83+ cells in 24 hours.

Table 2 shows: neutrophils and monocytes showed a trend of increasing with the extension of immunization time. After 24 hours of immunization, the percentage of neutrophils recruited at the injection site of mice in the water-oil microspheres/50 μg hBCG/antigen immunization group was higher than PBS, 50 μg hBCG, antigen, 250 μg hBCG and water-oil microspheres/50 μg hBCG immunization group (P<0.05), and the percentage of monocytes recruited at the injection site of antigen and water-oil microspheres/50 μg hBCG immunized mice was significantly higher than The water-oil microsphere immunization group (P<0.05) was slightly higher than the PBS group (P=0.075 and P=0.061). Except that there were statistical differences in the content of neutrophils and monocytes among the above groups, the other There was no statistically significant difference between groups at 24 hours of immunization and at other times. Macrophages showed a relatively stable trend at the three detection time points. At 24 hours, the water-oil microspheres/50 μg hBCG/antigen immunization group had the best effect in recruiting macrophages; at 48 hours, the water-oil microspheres/50 μg hBCG immunization group recruited macrophages. The effect of phagocytes was the best; at 72 hours, the effect of recruiting macrophages was the best in the water-oil microspheres/250μg hBCG immunization group.

Therefore, Tables 1 and 2 show that: after 24 hours of immunization, WOM/50 μg hBCG enhanced the recruitment of DC cells and monocytes, while WOM/50 μg hBCG/antigen enhanced the recruitment of neutrophils and macrophages. After immunization for 48 hours, water-oil microspheres/50μg hBCG enhanced the recruitment of DC cells, macrophages and monocytes.

(2) Water-oil microspheres/hBCG compound adjuvant to recruit natural immune cells in the muscle tissue of the injection site for 24 hours of immunization by two injection routes: intramuscular and subcutaneous

BALB/c mice were divided into 4 groups with 9 mice in each group. Inject immune water-oil microspheres/50μg hBCG, water-oil microspheres/250μg hBCG subcutaneously in the rich muscles of the right hind leg, or intramuscularly inject water-oil microspheres/50μg hBCG, water-oil microspheres/ 250 μg hBCG. Take the muscle tissue at the injection site 24 hours after the injection, enzymatic hydrolysis of the muscle tissue-mechanical separation of single cells and flow cytometry analysis test operations are the same as above.

Figure 5A shows: water-oil microspheres/50μg/ml hBCG subcutaneous injection or intramuscular injection immunized mice, compared with intramuscular injection, DC cells and monocytes recruited by subcutaneous injection site muscle tissue tended to increase, macrophages and neutrophils Granulocytes tended to decrease, but the difference was not statistically significant (P>0.05). Figure 5B shows: water-oil microspheres/250μg/ml hBCG subcutaneous injection or intramuscular injection immunized mice, compared with intramuscular injection, DC cells and monocytes recruited at the subcutaneous injection site tended to increase, neutrophils tended to decrease, But the difference was not statistically significant (P>0.05).

In summary, both intramuscular injection and subcutaneous injection can cause the recruitment of immune cells to the muscle tissue at the injection site, suggesting that the adjuvant of the present invention recruits cells related to innate immunity to the muscle tissue at the injection site, which is beneficial to the presentation of antigens to induce the acquisition of Sexual immune response.

Embodiment 4: the effect of the non-specific anti-mycobacterium tuberculosis infection of the composite adjuvant of the present invention

(1) Guinea pig challenge and immunotherapy: SPF grade Hartley guinea pig (350±50g), provided by China Institute for Food and Drug Control; production license number of experimental animals: SCXK (Beijing) 2014-0013; quality certificate of experimental animals: 11400500008759 . Guinea pig skin test (intradermal injection of 0.1ml TB-PPD/), observe the skin test results in 24 hours and 48 hours, 36 guinea pigs with negative skin test were infected with Mycobacterium tuberculosis 10 ² -10 ³ CFU/, and the guinea pigs were divided into 3 groups. Group, 12 in each group. Immunization on the 3rd, 10th, 24th, and 38th day after Mycobacterium tuberculosis infection, group A was subcutaneously injected with 0.5ml PBS/monkey, group B was subcutaneously injected with 0.5ml water-oil microspheres/50μg hBCG/bird, and group C was injected subcutaneously 0.5ml water-oil microspheres/250μg hBCG/rat; 1 week after the last immunization, 2 rats in each group were pre-dissected, and the lesions were observed; the formal dissection time was determined according to the lesions.

(2) Guinea pig anatomy, visceral lesions and colony counts: kill the guinea pigs, dissect the guinea pigs and observe the lesions of the spleen, liver, and pulmonary tuberculosis of the guinea pigs; get half of the spleen to grind, and dilute the spleen suspension 10 times with 0.05% Tween80 normal saline, each dilution Inoculate 2 tubes, and inoculate 0.1ml of each Roche medium slant. Cultivate at 37°C for 4 weeks, and count the number of colonies (CFU). The liver, lungs and remaining spleen were soaked in neutral formaldehyde solution.

(3) Lesions of the liver, lung, and spleen: the organs were dehydrated, embedded in paraffin, sectioned, stained with HE, and examined under a microscope.

Results The combination of water-oil microspheres and different doses of hBCG had a certain degree of anti-mycobacterium tuberculosis infection, and water-oil microspheres/250 μg hBCG significantly reduced the visceral lesions of guinea pigs infected with Mycobacterium tuberculosis and reduced the amount of bacteria in the spleen of guinea pigs (table 3).

Table 3: Guinea pig liver, spleen and lung comprehensive lesion score and the logarithmic value of the isolated number of spleen bacteria

Summary: The water-oil microsphere/hBCG composite adjuvant has non-specific anti-mycobacterium tuberculosis infection effect. Finding a suitable and compatible immunogenic Mycobacterium tuberculosis antigen will develop a better effect and clinical application. A new generation protein vaccine for tuberculosis with application value.

Embodiment 5: Preparation of PstS1-LEP fusion protein

PstS1 (38kDa or Rv0934) is highly expressed in Escherichia coli and is easily soluble in 8M urea buffer (He Xiuyun et al., Chinese Journal of Tuberculosis and Respiratory Science, 1999, 22(3): 161-163). The PET28a-PstS1-ESAT6 vector is preserved by our laboratory (He Xiuyun et al., Mycobacterium tuberculosis fusion protein and its application, ZL200610000710.X). The PET28a-PstS1-ESAT6 plasmid was digested with BamHI and HindIII. After electrophoresis of the digested product, the agarose block containing the linear plasmid PET28a-PstS1 fragment was cut, and the SanPrep column DNA gel recovery kit (SanGong Bioengineering (Shanghai) was used. ) Co., Ltd.) recovered the linear recombinant plasmid PET28a-PstS1.

Screen 20 Mycobacterium tuberculosis proteins by comparative genomics, analyze the Th linear antigen epitopes of 20 proteins in the online database SYFPEITHI (http://www.syfpeithi.de), and select 12 proteins with high scores and targeting different HLA loci 15 amino acid Th epitope peptides, 12 Th epitope peptides are concatenated into a polypeptide, called linear multi-epitope protein (linemulti-epitope protein, LEP). Translate the LEP amino acid sequence into a base sequence with the translation tool (primer5) and modify it to the codon preferred by E. coli according to the codon degeneracy. The N-terminal and C-terminal of the sequence are respectively introduced into BamHI restriction site and HindIII restriction enzyme site, and a stop codon UAA was added before the HindIII restriction site). The designed LEP protein coding base sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd., and the synthesized LEP gene was cloned into the pUC19 vector and provided as a pUC19-LEP recombinant plasmid. PUC19-LEP was transformed into competent Escherichia coli DH5α (purchased from Tiangen Biotechnology Co., Ltd.), screened by blue and white spots, picked up white colonies for culture, extracted PUC19-LEP plasmid by alkaline lysis, and digested with BamHI and HindIII. There should be two DNA bands in the electrophoresis of the cleaved product, which are consistent with the size of the linear PUC19 and LEP genes. The agarose block of the LEP gene fragment was cut, and the LEP gene fragment was recovered using the SanPrep Column DNA Gel Recovery Kit (Sangon Bioengineering (Shanghai) Co., Ltd.).

The linear recombinant plasmid PET28a-PstS1 and LEP gene digested by BamHI and HindIII were ligated into recombinant plasmid pET28a-PstS1-LEP under the action of T ₄ DNA ligase, and the recombinant plasmid pET28a-PstS1-LEP was transformed into competent Escherichia coli DH5α. Recombinant Escherichia coli colonies, plasmid extraction by alkaline lysis, and plasmid digestion to identify the recombinant plasmid pET28a-PstS1-LEP. It is required that the single and double digestion patterns of the recombinant plasmid pET28a-PstS1-LEP conform to Figure 1. The DNA sequence of the target gene inserted in the recombinant plasmid pET28a-PstS1-LEP was proved to be completely consistent with the designed sequence. The recombinant plasmid pET28a-PstS1-LEP with the correct inserted LEP gene sequence was transformed into competent Escherichia coli BL21 (DE3) (purchased from Tiangen Biotechnology Co., Ltd.), placed on a kanamycin-resistant LB agar plate, and cultured overnight at 37°C; Pick a single colony in kanamycin-resistant LB medium, culture overnight at 37°C, inoculate 1% in kanamycin-resistant LB medium, culture at 37°C until OD _600nm = 0.6-0.8, add final concentration Induced culture with 0.5mmol/L IPTG for 4 hours, centrifuge to collect the cells, suspend the cells in the lysate, sonicate the cells, and centrifuge at 12000rpm/min for 15min. Under this culture induction condition, the target protein is expressed as inclusion bodies with a molecular weight of about 50KD .

Embodiment 6: Purification, renaturation and identification of fusion protein PstS1-LEP of the present invention

IPTG-induced BL21(DE3) cells containing the recombinant plasmid pET28a-PstS1-LEP were added to lysate (1g wet bacteria plus 3ml lysis buffer) to fully suspend the cells, followed by adding lysozyme and PMSF, and bathed in 37°C water for 60min. After low-temperature sonication for 40 minutes (ultrasonic conditions: power 540W, working time 6 seconds, gap 8 seconds), centrifuge at 4°C, 12000 rpm/min for 15 minutes, and discard the supernatant. The precipitate was washed twice with 2% Triton X-100 (product of Sigma Company) cell lysate, once with 20mmol/L Tris.Cl buffer (pH8.5) containing 2M urea, once with 20mmol/L Tris. Cl buffer (pH8.5) washed twice. After each thorough washing, centrifuge at 4°C and 12000rpm/min for 20min, and discard the supernatant. The precipitate is the initially purified inclusion body. The inclusion bodies were fully dissolved in 20mmol/L Tris.Cl buffer solution (pH8.5) containing 8M urea, centrifuged at 12000rpm/min at room temperature for 20min, and the supernatant was filtered through a 0.45μm filter to prepare the sample for chromatography.

Pack the Q-FF anion exchange column filler, equilibrate 5 column volumes with distilled water; After loading the samples, use 8M urea in 20mmol/L Tris.Cl buffer solution (pH8.5) to continue to equilibrate the chromatography column until the UV absorption value drops to the baseline. Gradient elution was carried out with 8M urea 20mmol/L Tris.Cl buffer (pH8.5) containing 0-1mol/L NaCl, and the elution peaks were collected step by step. The target protein is mainly passing through the peak, and the protein passing through the peak passes through the Source-30Q anion exchange packing chromatography column, and the chromatography method is basically the same as that of Q-FF chromatography. The collected protein was detected by SDS-PAGE, and it was found that the target protein with high purity mainly existed in the elution collection tube near 200mmol/L NaCl, and the collection tube containing the target protein and high purity was combined and the protein solution was put into a dialysis bag. For renaturation, the dialysis bag is put into the following renaturation buffer successively, and each buffer solution is dialyzed for 24 hours. 8.5), 50mmol/L NaCl, 5mmol/L EDTA, 5mmol/L reduced glutathione, 1mmol/L oxidized glutathione; buffer II: 4mol/L urea, 50mmol/L Tris-HCl (pH 8.5), 50mmol/L NaCl, 5mmol/L EDTA, 5mmol/L reduced glutathione, 1mmol/L oxidized glutathione; buffer III: 2mol/L urea, 50mmol/L Tris-HCl (pH 8.5), 50mmol/L NaCl, 5mmol/L EDTA, 5mmol/L reduced glutathione, 1mmol/L oxidized glutathione; Buffer IV: 50mmol/L Tris-HCl (pH 8.5), 50mmol/L L NaCl, 5mmol/L EDTA, 5mmol/L reduced glutathione, 1mmol/L oxidized glutathione; buffer V: 50mmol/L Tris-HCl (pH 8.5); buffer VI: 50mmol/L Tris-HCl (pH 8.5). The refolded protein was filtered through a 0.22 μm filter and packaged. The protein concentration was measured by the Lowry method (edited by the National Pharmacopoeia Committee, "Pharmacopoeia of the People's Republic of China", Chemical Industry Press, 2010), and the protein was stored at -30°C.

SDS-PAGE electrophoresis of the purified protein, gel densitometric scanning showed that the protein purity was close to 95% (Fig. 2a). The purified protein was subjected to SDS-PAGE electrophoresis, and then transferred to PVDF membrane. 1×TBS buffer (25mmol/L Tris, 0.1% Tween-20 and 150mmol/L NaCl) containing 5% skim milk was used to block the PVDF membrane for 2 hours at room temperature, and the membrane was fully washed with 1×TBS buffer. Anti-PstS1 monoclonal antibody (Anti-PstS1 rat monoclonal antibody (Anti-PstS1 rat monoclonal antibody), Jackson Immuno Research Laboratories Inc. USA) diluted in 1×TBS buffer of skim milk 1:10000, or mixed with 5% skim milk Dilute tuberculosis patient serum in 1×TBS buffer 1:100, incubate at room temperature for 2 hours; wash the membrane, and incubate with the corresponding horseradish peroxidase-labeled secondary antibody for 1 hour at room temperature (1×TBS buffer 1 containing 5% skimmed milk: 10000 dilute enzyme-labeled secondary antibody, Jackson Immuno Research Laboratories Inc.USA); wash the membrane, under dark room conditions, add chemiluminescence substrate (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific Inc., USA) to PVDF membrane, develop color, Expose the luminescent signal to X-ray film. PstS1-LEP protein was positively reacted with anti-PstS1 monoclonal antibody, serum IgG and IgM of tuberculosis patients ( FIG. 2 b ), and the fusion protein PstS1-LEP of the present invention had immunoreactivity while retaining PstS1 immunoreactivity.

Antibody reaction between PstS1-LEP and serum of tuberculosis patients: 1) Dilute PstS1-LEP to 5 μg/ml in coating solution, add 100 μl to each well, and coat overnight at 4°C.

2) Wash the plate, 5 min/time, for a total of three times (the washing solution is PBS containing 0.05% Tween-20, PBST). 200 μl of blocking solution (PBST containing 1% bovine serum albumin (BSA), PBST-B) was added to each well, and blocked at 37° C. for 1 h.

3) wash the plate, same as above. Serum to be tested was diluted with blocking solution at a dilution ratio of 1:100, 100 μl of diluted serum was added to each well, two replicate wells were set up for each sample, and incubated at 37°C for 2 hours.

4) wash the plate, same as above. Add 100 μl of HRP-labeled rabbit anti-human IgG secondary antibody dilution, IgM secondary antibody dilution or IgG and IgM secondary antibody dilution mixture (diluted according to the dilution of the secondary antibody instruction manual, and the dilution is blocking solution) to each well, and incubate at 37°C for 1 hour .

5) wash the plate, same as above. Add 100 μl of chromogenic solution to each well, place at room temperature in the dark for 30 min, add 50 μl of stop solution (2NH ₂ SO ₄ ) to stop color development, and measure OD450nm absorbance.

The results of anti-PstS1-LEP IgG and IgM in serum from different sources and IgG and IgM in the same well are shown in Table 4.

Table 4 Human serum anti-PstS1-LEP antibody detection

The results in Table 4 show: the positive rate of serum anti-PstS1-LEP IgG, IgG (G+M) and IgG+IgM in patients with tuberculosis is higher than that of patients with extrapulmonary tuberculosis (χ2=38.6, χ2=9.9 and χ2=11.6; all P<0.001). The positive rate of serum anti-PstS1-LEP IgM in patients with tuberculosis was lower than that in patients with extrapulmonary tuberculosis (χ2=14.1, P<0.001). The positive rates of serum anti-PstS1-LEP Ig(G+M) and IgG+IgM in patients with extrapulmonary tuberculosis were higher than those of IgG (χ2=18.8 and χ2=7.4, all P<0.001), while serum anti-PstS1-LEP IgG in patients with pulmonary tuberculosis , Ig(G+M) and IgG+IgM positive rates, the difference was not statistically significant (P>0.05). Comparison of anti-PstS1-LEP Ig(G+M) and IgG+IgM detection: the former increased the positive rate of detection, but did not reduce the specificity, while the latter increased the positive rate of detection, but decreased the specificity.

Example 7: Immunogenicity of recombinant protein of the present invention in combination with different adjuvants

Composite adjuvant water oil microspheres/hBCG enhances the Th1-type immune response induced by the recombinant protein PstS1-LEP of the present invention, which is manifested by the increase of serum IgG2a antibody level and the number of cells secreted by splenocytes secreted by PstS1-LEP in vitro Increased and secreted IL-2 and IFN-γ levels increased.

BALB/c mice were divided into 7 groups with 8 mice in each group. Subcutaneous immunization with water-oil microspheres, hBCG, PstS1-LEP, hBCG+PstS1-LEP, water-oil microspheres+PstS1-LEP, water-oil microspheres/hBCG+PstS1-LEP, Al(OH) ₃ /hBCG+PstS1- LEP. Each mouse in each experimental group was subcutaneously injected with 0.2ml, and boosted immunization once at the 3rd week and 6th week respectively. Three weeks after the last immunization, the eyes were picked for blood collection, sacrificed and dissected.

(1) Detection of serum antibodies: the levels of IgG, IgG1 and IgG2a in the serum of immunized mice against PstS1-LEP were detected by indirect ELISA.

1) Dilute PstS1-LEP to 5 μg/ml in coating solution, add 100 μl to each well, and coat overnight at 4°C.

2) Wash the plate, 5 min/time, for a total of three times (the washing solution is PBS containing 0.05% Tween-20, PBST). 200 μl of blocking solution (PBST containing 1% bovine serum albumin (BSA), PBST-B) was added to each well, and blocked at 37° C. for 1 h.

3) wash the plate, same as above. Serum to be tested was diluted with blocking solution at a dilution ratio of 1:5000, 100 μl of diluted serum was added to each well, two replicate wells were set up for each sample, and incubated at 37°C for 2 hours.

4) wash the plate, same as above. Add 100 μl of HRP-labeled secondary antibody dilution (diluted according to the dilution in the instructions of the secondary antibody, and the dilution is blocking solution) to each well, and incubate at 37°C for 1 hour.

5) wash the plate, same as above. Add 100 μl of chromogenic solution to each well, place at room temperature in the dark for 30 min, add 50 μl of stop solution (2NH ₂ SO ₄ ) to stop color development, and measure OD450nm absorbance.

Different adjuvants were compatible with the antigen PstS1-LEP of the present invention to immunize mice, and the detection results of IgG, IgG1 and IgG2a in mouse serum against PstS1-LEP are shown in Table 5.

Table 5: Detection of serum anti-PstS1-LEP antibody in mice immunized with recombinant protein PstS1-LEP of the present invention and different adjuvants

^* indicates significant difference compared with the water-oil microsphere immunization group,

^# means significant difference compared with hBCG immune group,

^& indicates a significant difference compared with the PstS1-LEP immune group

Mice immunized with PstS1-LEP alone or in combination with adjuvant could produce anti-PstS1-LEP IgG and IgG1 antibodies, but the levels of anti-PstS1-LEP IgG and IgG1 antibodies produced by adjuvant/PstS1-LEP immunized mice were significantly higher than PstS1-LEP alone immunized group (P<0.05). Anti-PstS1-LEP IgG2a was induced in mice immunized with water-oil microspheres and hBCG alone or in combination as PstS1-LEP adjuvant, IgG2a levels of hBCG+PstS1-LEP group and water-oil microsphere/hBCG+PstS1-LEP group Both were significantly higher than the PstS1-LEP group (P=0.001 and P=0.0002); the IgG2a level of the Al(OH) ₃ +hBCG+PstS1-LEP group was significantly higher than that of the hBCG group (P=0.036), while compared with the PstS1-LEP group The difference was not statistically significant (P>0.05).

(2) Spleen cell immune index detection: Aseptically take the spleen, place the 200-mesh nylon mesh on a disposable culture dish, place the spleen on the nylon mesh, cut it into pieces with scissors, grind the spleen with a 5ml syringe piston, and collect the cells filtered by the nylon mesh The suspension was centrifuged at 1500rpm for 10min, the supernatant was discarded, the cells were suspended in 1ml of RPMI-1640 medium, and 2ml of mouse organ lymphocyte tissue separation solution was slowly added to the liquid surface, and centrifuged at 1700rpm for 20min. After centrifugation, draw the second layer of milky white lymphocytes into a 15ml centrifuge tube, and add RPMI-1640 medium to 10ml. Centrifuge at 1500 rpm for 10 min, discard the supernatant, and wash the cells once with RPMI-1640 medium. Lymphocytes were suspended in RPMI-1640 complete medium (containing 10% high-quality fetal bovine serum and double antibody), counted, and the cell concentration was adjusted to 2×10 ⁶ /ml.

Enzyme-linked immunospot (ELISPOT) detection: According to the instructions of the Mouse IFN-γ, IL-2, IL-4 ELISpot PLUS kit (ALP) kit (Mabtech, Sweden), the secretion of IFN-γ, IL-2, IL-4 by spleen lymphocytes was detected. The number of spot-forming cells of 4, specific operations: (1) Coating ELISPOT plate: For non-pre-coated ELISPOT plate, coat ELISPOT plate the night before dissection of mice (pre-coated ELISPIT does not need this operation): 96-well ELISPOT plate First add 15 μl of 35% alcohol to the wells and activate for 1 min. Wash 5 times with 200 μl deionized water, add 100 μl coating antibody (diluted according to instructions) to each well, and coat overnight at 4°C. (2) Stimulation and culture of splenocytes in vitro: Prepare splenocytes as above, take out the microwell plate, discard the liquid, add 200 μl PBS (0.22 μm filter) to each well to wash the plate 5 times, add 200 μl complete medium to each well, and incubate at room temperature for 30 minutes . Discard the medium, add 100 μl of lymphocyte suspension and 5 μl of PstS1-LEP antigen (final concentration: 5 μg/ml), and incubate at 5% CO ₂ at 37° C. for 24-48 hours. (3) Color development: take out the microwell plate, discard the liquid, add 200 μl PBS (0.22 μm filter) to each well to wash the plate 5 times, add 100 μl detection antibody to each well in sequence, and incubate at room temperature for 2 hours. Discard the solution in the well, wash the plate 5 times as above, add 100 μl Streptavidin-ALP to each well in sequence, and incubate at room temperature for 1 h. Discard the solution in the wells, wash the plate 5 times as above, add 100 μl BCIP/NBT substrate solution (filtered at 0.45 μm), and leave it at room temperature for 10-30 minutes. After spots appear, wash each well with distilled water, and place the microporous plate in a ventilated place Protect from light and dry. (4) Detection of the number of spot forming cells: the plate was read by an enzyme-linked immunospot reader (CTL, USA), and the number of spot forming cells (SFC) of IFN-γ, IL-2, and IL-4 was automatically analyzed. The results are shown in Figure 6.

Figure 6 shows: water-oil microspheres/hBCG+PstS1-LEP immunized mice enhanced the splenocytes to secrete PstS1-LEP-specific IFN-γ and IL-2 (Figure 6A and B), while Al(OH) ₃ /hBCG+ PstS1-LEP immunized mice enhanced the secretion of PstS1-LEP-specific IL-4 from their splenocytes (Fig. 6C).

Sandwich ELISA detection of cytokines IFN-γ, IL-2, IL-4 content: (1) splenocyte stimulation culture in vitro: add 1ml 2×10 ⁶ /ml spleen lymphocyte suspension to each well of 24-well cell culture plate, the same mouse Mouse splenocytes were added to four wells, two of which were not stimulated with antigen, and the other two wells were stimulated with 10 μl of PstS1-LEP antigen (final concentration: 5 μg/ml), and cultured at 37° C., 5% CO ₂ for 72 hours. The lymphocyte culture supernatant was collected by centrifugation. (2) ELISA: detect the content of cytokines in the culture supernatant according to the kit instructions (BD Bioscience, USA). Specific operations: 1) Coating: Dilute the coating antibody to the required concentration, and use each well of a 96-well microtiter plate Add 100 μl and coat overnight at 4°C. 2) Sealing: discard the coating solution, and wash the plate 3 times (washing solution is PBST). Add 200 μl of blocking solution (PBST containing 10% high-quality fetal bovine serum) to each well, and block for 1 hour at 37°C. 3) Primary antibody incubation: wash the plate, same as above. Dilute the culture supernatant to be tested with PBST (dilution ratio 1:5), add 100 μl to each well, repeat two wells, and incubate at 37°C for 2h. 4) Secondary antibody incubation: wash the plate 5 times. Add 100 μl of detection antibody to each well and incubate at 37°C for 1 h. 5) Color development: wash the plate for 7 times. Add 100 μl of chromogenic solution to each well, place at room temperature in the dark for 30 min, add 50 μl of stop solution 2N H ₂ SO ₄ to stop color development, and measure OD450nm absorbance. The results are shown in Figure 7.

Water-oil microspheres and hBCG alone or in combination as PstS1-LEP antigen adjuvants immunized mice to enhance splenocytes to secrete PstS1-LEP antigen-specific IFN-γ, while the compound adjuvant Al(OH) ₃ /hBCG could not effectively enhance PstS1-LEP The splenocytes of antigen-immunized mice secreted PstS1-LEP antigen-specific IFN-γ; the comparison between the groups with statistical differences showed that: the hBCG+PstS1-LEP group was significantly higher than the hBCG group (P=0.013), and the water-oil microspheres+ PstS1-LEP group and water-oil microsphere/hBCG+PstS1-LEP group were significantly higher than water-oil microsphere group (P=0.013 and P=0.003), hBCG group (P=0.001 and P=0.0002) and PstS1-LEP groups (P=0.037 and P=0.008); the Al(OH) ₃ /hBCG+PstS1-LEP group was significantly lower than the water-oil microspheres/hBCG+PstS1-LEP group (P=0.016) ( FIG. 7A ).

Water-oil microspheres, water-oil microspheres/hBCG and Al(OH) ₃ /hBCG were used as PstS1-LEP antigen adjuvants to immunize mice to enhance splenocytes to secrete PstS1-LEP antigen-specific IL-2, while hBCG adjuvant could not effectively enhance The splenocytes of mice immunized with PstS1-LEP antigen secreted PstS1-LEP antigen-specific IL-2; the comparison between groups with statistical difference showed that: water oil microspheres+PstS1-LEP group and Al(OH) ₃ /hBCG+PstS1 -LEP group was significantly higher than water-oil microsphere group (P=0.007 and P=0.004), hBCG group (P=0.004 and P=0.002); water-oil microsphere/hBCG+PstS1-LEP group was significantly higher than water-oil microsphere group Ball group (P=0.0001), hBCG group (P=0.0003), PstS1-LEP group (P=0.007) and hBCG+PstS1-LEP group (P=0.013) ( FIG. 7B ).

The level of IL-4 secreted by splenocytes of mice immunized with different immunogens stimulated by PstS1-LEP antigen in vitro: hBCG+PstS1-LEP group was significantly higher than that of water-oil microsphere group (P=0.020), hBCG group (P=0.010) ; There was no significant difference between other groups (P>0.05) (Fig. 7C).

(3) Secretion of IL-1β and IL-12 by mouse peritoneal macrophages: 1 ml of 6% soluble starch broth was intraperitoneally injected 3 days before the mice were dissected. Three days later, the mice were plucked from the eyeballs to collect blood, killed by neck dislocation, and soaked in 75% alcohol for 1 min. Under sterile conditions, cut the skin, expose the peritoneum, take 8ml of ice-cold peritoneal lavage solution with a 10ml syringe, inject it into the abdominal cavity of the mouse, gently adjust the slope of the needle tip downward, gently stir up the peritoneum, and slowly suck Return the peritoneal lavage fluid (containing macrophages) to a 15ml centrifuge tube. Centrifuge at 1500 rpm for 10 min, discard the supernatant, wash the macrophages twice with RPMI-1640 medium, suspend the macrophages in DMEM medium, count them, and adjust the macrophage concentration to 5x10 ⁵ /ml. Add 1ml of macrophage suspension to each well of a 24-well cell culture plate, adhere to the wall at 37°C and 5% _CO2 for 2-4 hours, wash off unattached cells with DMEM medium, add 1ml DMEM complete medium (containing 10% high-quality Fetal bovine serum and double antibody) and 10 μl of PstS1-LEP antigen (final concentration: 5 μg/ml), cultured at 37° C., 5% CO ₂ for 72 hours. Collect the macrophage culture supernatant. The results of detecting the levels of IL-1β and IL-12 in the culture supernatant of macrophages by sandwich ELISA are shown in FIG. 8 , and the detection method is the same as that of the cytokine content detection in the culture supernatant of splenocytes in this example.

hBCG, water-oil microspheres, and Al(OH) ₃ /hBCG were used as PstS1-LEP adjuvants to immunize mice respectively, which enhanced the secretion of PstS1-LEP antigen-specific IL-12 from mouse peritoneal macrophages. The specific difference comparison: hBCG+PstS1 -LEP group and Al(OH) ₃ /hBCG+PstS1-LEP group were significantly higher than water-oil microsphere group (P=0.050 and P=0.033), hBCG group (P=0.011 and P=0.006); The +PstS1-LEP group was significantly higher than the water-oil microsphere group (P=0.002), hBCG group (P=0.0006) and PstS1-LEP group (P=0.017); the water-oil microsphere/hBCG+PstS1-LEP group was significantly lower In hBCG+PstS1-LEP group (P=0.008), water-oil microspheres+PstS1-LEP group (P=0.0003) and Al(OH) ₃ /hBCG+PstS1-LEP group (P=0.004) (Fig. 8A).

hBCG, water-oil microspheres, water-oil microspheres/hBCG, and Al(OH) ₃ /hBCG were used as PstS1-LEP adjuvants to immunize mice respectively, which enhanced the secretion of PstS1-LEP antigen-specific IL-1β from mouse peritoneal macrophages. Comparison of specific differences: hBCG+PstS1-LEP group, water-oil microsphere+PstS1-LEP group and Al(OH) ₃ /hBCG+PstS1-LEP group were significantly higher than water-oil microsphere group (P=0.0002, P=0.0005 and P=0.0004), hBCG group (P=0.0003, P=0.0001 and P=0.0007) and PstS1-LEP group (P=0.001, P=0.005 and P=0.006); water-oil microspheres/hBCG+PstS1-LEP group was significantly higher than that of water-oil microspheres group (P=0.011) and hBCG group (P=0.003) (Fig. 8B).

Example 8: Effect of hBCG dosage on the effect of composite adjuvant water-oil microspheres/hBCG adjuvant

BALB/c mice were randomly divided into 7 groups with 8 mice in each group. They are: water-oil microspheres, low-dose hBCG (5μg/body, hBCG(L)), medium-dose hBCG (50μg/body, hBCG(M)), high-dose hBCG (500μg/body, hBCG(H)), Water-oil microspheres/hBCG(L), water-oil microspheres/hBCG(M), water-oil microspheres/hBCG(H). Each mouse in each experimental group was subcutaneously injected with 0.2ml, and boosted immunization once in the 2nd week and 4th week respectively. Two weeks after the last immunization, the eyeballs were picked for blood collection, sacrificed and dissected.

The indirect ELISA method was used to measure the serum antibody level, the sandwich ELISA was used to detect the cytokine content of the mouse splenocyte stimulation culture supernatant in vitro and the cytokine content of the macrophage stimulation culture supernatant in vitro. The test operation was the same as in Example 7, except that the cells were stimulated in vitro The cultured antigen is a pure protein derivative (BCG-PPD) prepared from the culture supernatant of BCG. For the preparation method, refer to the literature (edited by the National Pharmacopoeia Committee, "Pharmacopia of the People's Republic of China", P21-132, Chemical Industry Press, 2010). Briefly describe the preparation method: BCG was inoculated with Sutong medium, cultured at 37°C for 6 weeks; sterilized at 121°C for 30 minutes, centrifuged at 4°C and 12,000 rpm for 10 minutes, and the supernatant was filtered with a 0.45 μm membrane; 40% trichloroacetic acid was added to the filtrate to The final concentration is 2-4%, mix well, 4°C, precipitate for 6h; 4°C, 12000 rpm/centrifuge for 10min, wash the precipitate with 1% trichloroacetic acid for 3 times; dissolve the precipitate with phosphate buffer (pH8.2), add saturated Ammonium sulfate, precipitate overnight at 4°C; centrifuge at 4°C, 12,000 rpm for 10 minutes, dissolve the precipitate in phosphate buffer (pH 8.2), dialyze the solution, and use PBS as the dialysate; after thorough dialysis, filter aseptically, and detect protein by Lowry concentration, aliquoted, and stored at -20°C.

Different doses of hBCG alone or in combination with water-oil microspheres were used to immunize mice with compound adjuvant. The IgG, IgG1 and IgG2a antibodies in the serum of mice were detected against BCG-PPD, and the OD value was extremely low, which was close to that of the blank control well.

The results of the secretion of IFN-γ, IL-2 and IL-4 by mouse splenocytes are shown in Figure 9: the splenocytes of mice immunized with low, medium and high doses of hBCG secrete BCG-PPD-specific IFN-γ levels with the increase of hBCG dose However, there was an increasing trend, but there was no statistically significant difference among the three groups; the level of BCG-PPD-specific IFN-γ secreted by splenocytes of water-oil microspheres/hBCG immunized mice also showed an increasing trend with the increase of hBCG dose, And the level of IFN-γ produced by the water-oil microsphere/hBCG (H) group was significantly higher than that of the water-oil microsphere/hBCG (L, M) two groups (P=0.0005 and P=0.001) ( FIG. 9A ). There was no significant difference in the level of BCG-PPD-specific IFN-γ secreted by splenocytes of water-oil microspheres, hBCG(L), and water-oil microspheres/hBCG(L) immunized mice; water-oil microspheres, hBCG(M ) and water-oil microspheres/hBCG(M) immunized mice splenocytes secreted BCG-PPD-specific IFN-γ levels had no statistically significant difference; while water-oil microspheres/hBCG(H) immunized mice splenocytes The level of secreted BCG-PPD-specific IFN-γ was significantly higher than that of the water-oil microspheres and hBCG(H) immunized mice groups (P=0.0002 and P=0.001) ( FIG. 9A ).

The level of BCG-PPD-specific IL-2 secreted by splenocytes of mice immunized with low, medium and high doses of hBCG tended to increase with the increase of hBCG dose, and the level of IL-2 in the hBCG(H) immunized group was significantly higher than that of hBCG (L) group (P=0.044); the level of BCG-PPD-specific IL-2 secreted by splenocytes of mice immunized with water-oil microspheres/hBCG compound adjuvant also showed a rising trend with the increase of hBCG dose, but the three groups The difference was not statistically significant (Figure 9B). The level of BCG-PPD-specific IL-2 secreted by splenocytes of water-oil microspheres/hBCG(L)-immunized mice was significantly higher than that of water-oil microspheres or hBCG(L)-immunized mice (P=0.002 and P=0.001) ; The splenocytes of water-oil microspheres/hBCG (M) immunized mice secreted BCG-PPD-specific IL-2 levels significantly higher than water-oil microspheres or hBCG (M) immunized mice groups (P=0.002 and P=0.001 ); the splenocytes secreting BCG-PPD-specific IL-2 levels of water-oil microspheres/hBCG(H) immunized mice were significantly higher than those of water-oil microspheres or hBCG(H) immunized mice groups (P=0.001 and P= 0.005) (Fig. 9B).

There was no significant difference in the levels of BCG-PPD-specific IL-4 secreted by splenocytes of mice in each group ( FIG. 9C ).

Figure 10 shows the results of IL-12 and IL-1β secreted by mouse peritoneal macrophages. The level of IL-12 produced by the peritoneal macrophages of mice immunized with water emulsion microspheres/hBCG(L) or water emulsion microspheres/hBCG(M) stimulated by BCG-PPD antigen was comparable to hBCG(L) or hBCG(M) immunization There was an increasing trend in the mouse group, but there was no significant difference in IL-12 levels among the groups ( FIG. 10A ). The level of IL-1β in the water-oil microsphere/hBCG(L) group was significantly higher than that in the water-oil microsphere group (P=0.05), hBCG(L) group (P=0.022) and hBCG(M) group (P=0.01) , IL-1β levels in the water-oil microsphere/hBCG(M) group were significantly higher than those in the water-oil microsphere group (P=0.016), hBCG(L) group (P=0.006), hBCG(M) group (P=0.003 ) and hBCG(H) group (P=0.048) ( FIG. 10B ).

Comprehensive splenocyte and macrophage immune response: hBCG dose ranges from 5 μg/dose to 500 μg/dose, and the optimal range is 50 μg/dose to 250 μg/dose.

Example 9: Water-oil microspheres/hBCG adjuvant enhances the immunogenicity of Mycobacterium tuberculosis fusion protein

The Mycobacterium tuberculosis fusion protein is PstS1-EAST6, named K6 (ZL200610000710.X). Female BALB/c mice (SPF grade) aged 6-8 weeks (purchased from the Experimental Animal Center of the Academy of Military Medical Sciences of the Chinese People's Liberation Army) were divided into 7 groups, 8 mice in each group. Mice in different groups were immunized with water-oil microspheres, hBCG, K6, hBCG+K6, water-oil microspheres+K6, water-oil microspheres/hBCG+K6, Al(OH) ₃ /hBCG+K6, respectively. Each mouse was subcutaneously injected with 0.2ml, and the immunization dose was K610μg/time/mouse and hBCG 50μg/time/mouse for booster immunization once in the 2nd week and the 4th week. Two weeks after the last immunization, the mice were sacrificed and dissected. ELISA detection of serum antibodies, sandwich ELISA detection of splenocytes and peritoneal macrophages stimulated and cultured in vitro K6 cytokine content detection in the supernatant collected is the same as in Example 7, the difference is the stimulating antigen used in splenocytes and peritoneal macrophages cultured in vitro It is K6 (final concentration is 5 (g/ml).

(1) Humoral immunity

Mice immunized with K6 antigen induced a certain level of anti-K6 IgG, IgG1 and IgG2a antibodies, but the levels of IgG and IgG1 were significantly lower than those of water-oil microspheres+K6, water-oil microspheres/hBCG+K6 or Al(OH) ₃ /hBCG+K6 Immunized mouse group (Fig. 11). Compared with the K6 immunization group, hBCG, water emulsion microspheres and water emulsion microspheres/hBCG as K6 antigen adjuvant mainly induced K6-specific IgG1 antibodies, but not K6-specific IgG2a antibodies; while Al(OH) ₃ /hBCG as a K6 antigen adjuvant induced IgG1 and IgG2a antibodies (Figure 1 IB and C).

(2) Cytokines secreted by splenocytes

Immunization of mice with K6 antigen and different adjuvants could not effectively induce splenocytes to secrete IFN-γ, IL-2 and IL-4, and Al(OH) ₃ /hBCG compound adjuvant K6 vaccine enhanced splenocytes to secrete IL-4 (Figure 12).

(3) Cytokines secreted by mouse peritoneal macrophages

Using water-oil microspheres, water-oil microspheres/hBCG, and Al(OH) ₃ /hBCG as K6 antigen adjuvants to immunize mice enhanced peritoneal macrophages to secrete K6-specific IL-12 instead of IL-1β (Fig. 13).

Example 10 Water-oil microspheres/hBCG adjuvant enhances cytomegalovirus (CMV) virus gB antigen (CMV gB) immunogenicity

CMV virus gB antigen (1 mg/ml) was purchased from Dakowei Biotechnology Co., Ltd., with a purity greater than 95%. BALB/c mice were divided into 6 groups, 6 mice in each group. Groups A-D and Group F were respectively immunized with water-oil microspheres, water-oil microspheres/hBCG, CMV gB, water-oil microspheres+CMV gB, water-oil microspheres/hBCG+CMV gB in muscle; E group was subcutaneously immunized with water-oil microspheres/ hBCG+CMV gB. Each mouse in each experimental group was injected intramuscularly or subcutaneously with 0.2ml, and boosted immunization once at the 2nd week and 4th week respectively, with the immune dose of CMV gB 5 μg/time/mouse, hBCG 50 μg/time/mouse. Two weeks after the last immunization, the eyes were picked for blood collection, sacrificed and dissected.

(1) Serum antibody detection

The serum anti-CMV gB IgG level of immunized mice was detected by indirect ELISA method:

1) Dilute CMV gB to 5 μg/ml in coating solution, add 100 μl to each well, and coat overnight at 4°C.

2) Wash the plate, 5min/time, for a total of three times (washing solution is PBST). Add 200 μl of blocking solution (PBST containing 1% bovine serum albumin (BSA)) to each well, and block at 37° C. for 1 h.

3) wash the plate, same as above. The serum to be tested was diluted with blocking solution, and the dilution ratio was 1:100 and 1:500 for the serum of mice immunized with water-oil microspheres and water-oil microspheres/hBCG, respectively, and 1:500 and 1:500 for CMV gB-immunized mice :2500 and 1:5000 dilutions, water-oil microspheres+CMV gB and water-oil microspheres/hBCG+CMV gBC (including intramuscular and subcutaneous injection) immunized mouse serum were diluted 1:5000, 1:25000 and 1:30000 respectively, Add 100 μl of serum dilution solution to each well, set up two replicate wells for each dilution of each sample, and incubate at 37°C for 2 hours.

4) wash the plate, same as above. Add 100 μl of HRP-labeled secondary antibody dilution to each well and incubate at 37°C for 1 h.

5) wash the plate, same as above. Add 100 μl of chromogenic solution to each well, place at room temperature in the dark for 30 min, add 50 μl of stop solution (2NH ₂ SO ₄ ) to stop color development, and measure OD450nm absorbance.

The maximum dilution greater than the 1:100 dilution of the serum from mice immunized with water-oil microspheres and the average ± 2SD value of the OD was taken as the serum antibody titer. CMV gB immunized mice induced low levels of anti-CMV gB IgG, and water-oil microspheres and composite adjuvant water-oil microspheres/hBCG could significantly enhance the production of anti-CMV gB IgG antibodies induced by CMV gB (Figure 14). The two adjuvants enhanced CMV gB to induce anti-CMV gB IgG antibody titers, and the difference was not statistically significant; the compound adjuvant water oil microspheres/hBCG+CMV gB immunized mice, subcutaneous injection and intramuscular injection, induced anti-CMV gB IgG Antibodies were produced, and there was no statistically significant difference in antibody titers ( FIG. 14 ).

(2) ELISPOT detection of the number of spot-forming cells secreting IFN-γ and IL-4 from splenic lymphocytes

The test operation is the same as in Example 7, and the results show that: the water-oil microspheres/hBCG+CMV gB intramuscular injection immunized the mice to enhance the splenocytes secreting CMV gB specific IFN-γ, and the number of spot-forming cells was significantly higher than that of other groups (P <0.01); water-oil microspheres/hBCG+CMV gB subcutaneously injected immunized mice to enhance splenocytes to secrete CMV gB-specific IFN-γ, and the number of spot-forming cells was significantly higher than that of the adjuvant group. There was no statistically significant difference between the mice in the sphere+CMV gB immunized group (Fig. 15a). The number of spot-forming cells secreting CMV gB-specific IFN-γ in the splenocytes of mice immunized with water-oil microspheres+CMV gB was significantly higher than that of mice immunized with water-oil microspheres (P<0.05), and slightly higher than that of mice immunized with water-oil microspheres. /hBCG immunized mice group (P=0.052) (Fig. 15a).

The number of spot-forming cells secreting CMV gB-specific IL-4 in splenocytes of CMV gB-immunized mice was significantly higher than that of water-oil microspheres, water-oil microspheres/hBCG, water-oil microspheres+CMV gB, or water-oil microspheres/hBCG +CMV gB intramuscular injection (group F) immunized mice (Fig. 15b); water-oil microspheres/hBCG+CMV gB subcutaneously immunized mice (group E) had a significant number of spot-forming cells secreting CMV gB-specific IL-4 Higher than water oil microspheres/hBCG immunized mice (Fig. 15b).

Conclusion: Both water-oil microspheres/hBCG and water-oil microsphere adjuvant can enhance the antibody production in mice immunized with CMV gB, and the water-oil microsphere/hBCG adjuvant + CMV gB intramuscular injection immunization scheme can effectively enhance the induction of Th1 type immune response , especially the secretion of IFN-γ is conducive to the body to clear the virus. Therefore, water-oil microspheres/hBCG enhanced the antiviral effect of CMV gB better than water-oil microspheres.

Example 11 Comparison of adjuvant properties of composite adjuvant water-oil microspheres/hBCG with two different preparation processes

For the preparation process of the composite adjuvant water-oil microsphere/hBCG-1 (abbreviated as adjuvant 1), see the relevant part of Example 1; for the composite adjuvant water-oil microsphere/hBCG-2 (abbreviated as adjuvant 2), the formula is the same /hBCG-1 is exactly the same, and the preparation process is different from the preparation process of the composite adjuvant water-oil microspheres/hBCG-1: the preparation process of water-oil microspheres/hBCG-1 is that the water-oil microspheres are homogenized under high pressure to form a nanoemulsion, 0.22 μm filtration, hBCG is the complete inactivated bacteria, the two are mixed in proportion to form a composite adjuvant water-oil microspheres/hBCG-1; water-oil microspheres/hBCG-2 is water-oil microspheres mixed with hBCG After high-pressure homogenization, hBCG is broken cells.

BALB/c mice were divided into 2 groups, 6 in each group; 0.2ml water-oil microspheres/hBCG-1+PstS1-LEP and water-oil microspheres/hBCG-2+PstS1-LEP were subcutaneously injected, respectively, at the second week and In the 4th week, each booster immunization was given once, and the immune dose was PstS1-LEP 5 μg/time/monkey, hBCG 50 μg/time/bird (water-oil microspheres/hBCG-1 was 50 μg inactivated BCG cells, water-oil microspheres/hBCG- 2 is 50 μg inactivated BCG bacterial cell crush). Two weeks after the last immunization, the eyes were picked for blood collection, sacrificed and dissected.

Serum IgG, IgG1, IgG2a detection method is the same as Example 7 "serum antibody detection". As shown in Figure 16: There was no significant difference in the levels of IgG, IgG1 and IgG2a against PstS1-LEP produced by mice immunized with tuberculosis protein vaccine compatible with PstS1-LEP in adjuvant 1 and adjuvant 2 respectively (P>0.05).

The method for detecting spot-forming cells secreting IFN-γ, IL-4 or IL-17 from splenocytes by ELISPOT is the same as that in Example 7 "Detection of immune indexes of splenocytes".

As shown in FIG. 17 : tuberculosis protein vaccine (adjuvant 2+PstS1-LEP) immunized mice, and induced splenocytes to secrete IFN-γ and IL-17 better than tuberculosis protein vaccine (adjuvant 1+PstS1-LEP).

It can be seen from the above examples that the compound adjuvant of the present invention can enhance the induction of humoral and cellular immunity by Mycobacterium tuberculosis protein, and can also enhance the induction of humoral and cellular immunity by viral protein (CMV virus). A single water-oil microsphere mainly enhances the Th2-type immune response, while a compound adjuvant combined with water-oil microspheres and heat-inactivated Bacillus Calmette-Guerin (hBCG) induces the immune response to drift to Th1-type cellular immune response; enhances the body's ability to clear Mycobacterium tuberculosis , is an important candidate adjuvant for tuberculosis preventive and therapeutic vaccines.

Although the foregoing invention has been described in detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. Accordingly, all suitable modifications or equivalents may be considered to fall within the scope of the present invention as defined in the claims. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

sequence listing

<110> No. 309 Hospital of the Chinese People's Liberation Army

<120> Composite adjuvant and vaccine containing the same

<130> PBK00886

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1050

<212>DNA

<213> Artificial Sequence

<220>

<223> The coding sequence of amino acid 25-amino acid 374 of PstS1 antigen

<400> 1

ggctcgaaac caccgagcgg ttcgcctgaa acgggcgccg gcgccggtac tgtcgcgact 60

accccccgcgt cgtcgccggt gacgttggcg gagaccggta gcacgctgct ctacccgctg 120

ttcaacctgt ggggtccggc ctttcacgag aggtatccga acgtcacgat caccgctcag 180

ggcaccggtt ctggtgccgg gatcgcgcag gccgccgccg ggacggtcaa cattggggcc 240

tccgacgcct atctgtcgga aggtgatatg gccgcgcaca aggggctgat gaacatcgcg 300

ctagccatct ccgctcagca ggtcaactac aacctgcccg gagtgagcga gcacctcaag 360

ctgaacggaa aagtcctggc ggccatgtac cagggcacca tcaaaacctg ggacgacccg 420

cagatcgctg cgctcaaccc cggcgtgaac ctgcccggca ccgcggtagt tccgctgcac 480

cgctccgacg ggtccggtga caccttcttg ttcacccagt acctgtccaa gcaagatccc 540

gagggctggg gcaagtcgcc cggcttcggc accaccgtcg acttcccggc ggtgccgggt 600

gcgctgggtg agaacggcaa cggcggcatg gtgaccggtt gcgccgagac accgggctgc 660

gtggcctata tcggcatcag cttcctcgac caggccagtc aacggggact cggcgaggcc 720

caactaggca atagctctgg caatttcttg ttgcccgacg cgcaaagcat tcaggccgcg 780

gcggctggct tcgcatcgaa aaccccggcg aaccaggcga tttcgatgat cgacgggccc 840

gccccggacg gctacccgat catcaactac gagtacgcca tcgtcaacaa ccggcaaaag 900

gacgccgcca ccgcgcagac cttgcaggca tttctgcact gggcgatcac cgacggcaac 960

aaggcctcgt tcctcgacca ggttcatttc cagccgctgc cgcccgcggt ggtgaagttg 1020

tctgacgcgt tgatcgcgac gatttccagc 1050

<210> 2

<211> 540

<212>DNA

<213> Artificial Sequence

<220>

<223> Coding sequence of polyepitopic antigen LEP amino acid

<400> 2

gtgccgctct atggacagct gggagccaaa ggggtagtcg gtaccggcgc cgaattgctc 60

gacgacatta gggcattctt gcggcggttc cggacccagt tcgaggatgc gtggtcccgg 120

tatctctctg ccgacaccac ccgggtgacg atcctgaccg gcagacggat gaccgatttg 180

gtggtcatct tcgtgacact gggcgccgcg gccatcccgg cgtggccgat cgcgctggct 240

tacttcgtgg cgttgtcccg acagcggtgg cgagagtttt tgacaaagct cactggcgca 300

ggcgcagcgg cattccccgc cacgttcacc gaagacgtcc gccaggcgtt gtacgcctcc 360

aagggccgct acttccggct gttgaccggg tgggtcgggg gcggaacctg gccgtacccc 420

gaaatcgcta ccgcagccat gatttcgcca ttcaaggact actttggcct ggcgcacgac 480

ctgccgaagt gggcgtggta cgcggtcgac gtcttttcca ctcttttggt agtccctggg 540

<210> 3

<211> 1596

<212>DNA

<213> Artificial Sequence

<220>

<223> amino acid coding sequence of PstS1-LEP fusion protein (without PstS1 N-terminal 24 amino acids)

<400> 3

ggctcgaaac caccgagcgg ttcgcctgaa acgggcgccg gcgccggtac tgtcgcgact 60

accccccgcgt cgtcgccggt gacgttggcg gagaccggta gcacgctgct ctacccgctg 120

ttcaacctgt ggggtccggc ctttcacgag aggtatccga acgtcacgat caccgctcag 180

ggcaccggtt ctggtgccgg gatcgcgcag gccgccgccg ggacggtcaa cattggggcc 240

tccgacgcct atctgtcgga aggtgatatg gccgcgcaca aggggctgat gaacatcgcg 300

ctagccatct ccgctcagca ggtcaactac aacctgcccg gagtgagcga gcacctcaag 360

ctgaacggaa aagtcctggc ggccatgtac cagggcacca tcaaaacctg ggacgacccg 420

cagatcgctg cgctcaaccc cggcgtgaac ctgcccggca ccgcggtagt tccgctgcac 480

cgctccgacg ggtccggtga caccttcttg ttcacccagt acctgtccaa gcaagatccc 540

gagggctggg gcaagtcgcc cggcttcggc accaccgtcg acttcccggc ggtgccgggt 600

gcgctgggtg agaacggcaa cggcggcatg gtgaccggtt gcgccgagac accgggctgc 660

gtggcctata tcggcatcag cttcctcgac caggccagtc aacggggact cggcgaggcc 720

caactaggca atagctctgg caatttcttg ttgcccgacg cgcaaagcat tcaggccgcg 780

gcggctggct tcgcatcgaa aaccccggcg aaccaggcga tttcgatgat cgacgggccc 840

gccccggacg gctacccgat catcaactac gagtacgcca tcgtcaacaa ccggcaaaag 900

gacgccgcca ccgcgcagac cttgcaggca tttctgcact gggcgatcac cgacggcaac 960

aaggcctcgt tcctcgacca ggttcatttc cagccgctgc cgcccgcggt ggtgaagttg 1020

tctgacgcgt tgatcgcgac gatttccagc ggatccgtgc cgctctatgg acagctggga 1080

gccaaagggg tagtcggtac cggcgccgaa ttgctcgacg acattagggc attcttgcgg 1140

cggttccgga cccagttcga ggatgcgtgg tcccggtatc tctctgccga caccacccgg 1200

gtgacgatcc tgaccggcag acggatgacc gatttggtgg tcatcttcgt gacactggggc 1260

gccgcggcca tcccggcgtg gccgatcgcg ctggcttact tcgtggcgtt gtcccgacag 1320

cggtggcgag agtttttgac aaagctcact ggcgcaggcg cagcggcatt ccccgccacg 1380

ttcaccgaag acgtccgcca ggcgttgtac gcctccaagg gccgctactt ccggctgttg 1440

accgggtggg tcgggggcgg aacctggccg taccccgaaa tcgctaccgc agccatgatt 1500

tcgccattca aggactactt tggcctggcg cacgacctgc cgaagtgggc gtggtacgcg 1560

gtcgacgtct tttccactct tttggtagtc cctggg 1596

<210> 4

<211> 532

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of PstS1-LEP fusion protein (without 24 amino acids at the N-terminal of PstS1)

<400> 4

Gly Ser Lys Pro Pro Ser Gly Ser Pro Glu Thr Gly Ala Gly Ala Gly

1 5 10 15

Thr Val Ala Thr Thr Pro Ala Ser Ser Pro Val Thr Leu Ala Glu Thr

20 25 30

Gly Ser Thr Leu Leu Tyr Pro Leu Phe Asn Leu Trp Gly Pro Ala Phe

35 40 45

His Glu Arg Tyr Pro Asn Val Thr Ile Thr Ala Gln Gly Thr Gly Ser

50 55 60

Gly Ala Gly Ile Ala Gln Ala Ala Ala Gly Thr Val Asn Ile Gly Ala

65 70 75 80

Ser Asp Ala Tyr Leu Ser Glu Gly Asp Met Ala Ala His Lys Gly Leu

85 90 95

Met Asn Ile Ala Leu Ala Ile Ser Ala Gln Gln Val Asn Tyr Asn Leu

100 105 110

Pro Gly Val Ser Glu His Leu Lys Leu Asn Gly Lys Val Leu Ala Ala

115 120 125

Met Tyr Gln Gly Thr Ile Lys Thr Trp Asp Asp Pro Gln Ile Ala Ala

130 135 140

Leu Asn Pro Gly Val Asn Leu Pro Gly Thr Ala Val Val Pro Leu His

145 150 155 160

Arg Ser Asp Gly Ser Gly Asp Thr Phe Leu Phe Thr Gln Tyr Leu Ser

165 170 175

Lys Gln Asp Pro Glu Gly Trp Gly Lys Ser Pro Gly Phe Gly Thr Thr

180 185 190

Val Asp Phe Pro Ala Val Pro Gly Ala Leu Gly Glu Asn Gly Asn Gly

195 200 205

Gly Met Val Thr Gly Cys Ala Glu Thr Pro Gly Cys Val Ala Tyr Ile

210 215 220

Gly Ile Ser Phe Leu Asp Gln Ala Ser Gln Arg Gly Leu Gly Glu Ala

225 230 235 240

Gln Leu Gly Asn Ser Ser Gly Asn Phe Leu Leu Pro Asp Ala Gln Ser

245 250 255

Ile Gln Ala Ala Ala Ala Ala Gly Phe Ala Ser Lys Thr Pro Ala Asn Gln

260 265 270

Ala Ile Ser Met Ile Asp Gly Pro Ala Pro Asp Gly Tyr Pro Ile Ile

275 280 285

Asn Tyr Glu Tyr Ala Ile Val Asn Asn Arg Gln Lys Asp Ala Ala Thr

290 295 300

Ala Gln Thr Leu Gln Ala Phe Leu His Trp Ala Ile Thr Asp Gly Asn

305 310 315 320

Lys Ala Ser Phe Leu Asp Gln Val His Phe Gln Pro Leu Pro Pro Ala

325 330 335

Val Val Lys Leu Ser Asp Ala Leu Ile Ala Thr Ile Ser Ser Ser Gly Ser

340 345 350

Val Pro Leu Tyr Gly Gln Leu Gly Ala Lys Gly Val Val Gly Thr Gly

355 360 365

Ala Glu Leu Leu Asp Asp Ile Arg Ala Phe Leu Arg Arg Phe Arg Thr

370 375 380

Gln Phe Glu Asp Ala Trp Ser Arg Tyr Leu Ser Ala Asp Thr Thr Arg

385 390 395 400

Val Thr Ile Leu Thr Gly Arg Arg Met Thr Asp Leu Val Val Ile Phe

405 410 415

Val Thr Leu Gly Ala Ala Ala Ile Pro Ala Trp Pro Ile Ala Leu Ala

420 425 430

Tyr Phe Val Ala Leu Ser Arg Gln Arg Trp Arg Glu Phe Leu Thr Lys

435 440 445

Leu Thr Gly Ala Gly Ala Ala Ala Phe Pro Ala Thr Phe Thr Glu Asp

450 455 460

Val Arg Gln Ala Leu Tyr Ala Ser Lys Gly Arg Tyr Phe Arg Leu Leu

465 470 475 480

Thr Gly Trp Val Gly Gly Gly Thr Trp Pro Tyr Pro Glu Ile Ala Thr

485 490 495

Ala Ala Met Ile Ser Pro Phe Lys Asp Tyr Phe Gly Leu Ala His Asp

500 505 510

Leu Pro Lys Trp Ala Trp Tyr Ala Val Asp Val Phe Ser Thr Leu Leu

515 520 525

Val Val Pro Gly

530

Claims (10)

1. An immune complex adjuvant comprising sorbitan trioleate, squalene, surfactant and heat-inactivated BCG (hBCG) or high-pressure homogeneously broken BCG. 2. The composite adjuvant of claim 1, wherein the amount of said sorbitan trioleate is 0.1% to 1% (w/v). 3. The composite adjuvant according to claim 1 or 2, wherein the squalene is 1-10% (v/v). 4. The composite adjuvant according to any one of claims 1-3, wherein the surfactant is a nonionic surfactant; preferably Tween; more preferably Tween 80. 5. The compound adjuvant according to any one of claims 1-4, which also comprises a buffer; preferably the buffer is a sodium citrate buffer; preferably the buffer has a concentration of 5-10 mM and a pH of 6-7. 6. The composite adjuvant according to any one of claims 1-5, wherein the amount of said sorbitan trioleate is 0.3% to 0.7%; preferably 0.4-0.6%, more preferably 0.5%. 7. The composite adjuvant according to any one of claims 1-6, wherein said squalene is 3-7%; preferably 4-6%; more preferably 5%. 8. The composite adjuvant according to any one of claims 1-7, wherein the concentration of the surfactant is 0.3-0.7% (w/v); preferably 0.4-0.6%; more preferably 0.5%. 9. The composite adjuvant according to any one of claims 1-8, wherein the amount of heat-inactivated BCG (hBCG) or high-pressure homogeneously broken BCG is 5-500 μg, more preferably 25-500 μg; more preferably 50-500 μg; More preferably 50-450 μg; more preferably 50-400 μg; more preferably 50-400 μg; more preferably 50-300 μg; most preferably 50-250 μg. 10. The method for preparing the composite adjuvant of any one of claims 1-9, comprising 1) Add a surfactant to the buffer, stir evenly, and use it as the water phase; 2) Weighing sorbitan trioleate, adding squalene and fully mixing, as the oil phase; 3) Add the oil phase to the water phase and mix well; 4) Add heat-inactivated Bacillus Calmette-Guerin (hBCG) or high-pressure homogeneously crushed BCG after fully emulsifying and degerming.