WO2020211857A1 - 用于免疫增强的锰组合物 - Google Patents

用于免疫增强的锰组合物 Download PDF

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WO2020211857A1
WO2020211857A1 PCT/CN2020/085507 CN2020085507W WO2020211857A1 WO 2020211857 A1 WO2020211857 A1 WO 2020211857A1 CN 2020085507 W CN2020085507 W CN 2020085507W WO 2020211857 A1 WO2020211857 A1 WO 2020211857A1
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virus
manganese
colloidal
cancer
precipitated
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PCT/CN2020/085507
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English (en)
French (fr)
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蒋争凡
王晨光
张睿
吕梦泽
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北京大学
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Priority to US17/594,468 priority Critical patent/US20220193125A1/en
Priority to JP2021561978A priority patent/JP7308562B2/ja
Priority to KR1020217037373A priority patent/KR20220002968A/ko
Priority to EP20791542.2A priority patent/EP3957312A4/en
Priority to CN202080029675.2A priority patent/CN113692280A/zh
Publication of WO2020211857A1 publication Critical patent/WO2020211857A1/zh

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Definitions

  • the present invention provides divalent manganese colloid or divalent manganese neoprecipitate for enhancing immunity, and its use for enhancing immunity, which can be used as an immune adjuvant, antiviral or antitumor, for example.
  • the body protects the body from foreign pathogens through the natural immune system and adaptive immune system. Innate immunity can promote adaptive immunity.
  • Innate immunity can promote adaptive immunity.
  • the body When the body is infected by a pathogen, it first initiates a natural immune response, recognizes the pathogen's pathogen-related molecular patterns through pattern recognition receptors, and activates a variety of signaling pathways, such as TLR pathway (1), RLR pathway (2), cGAS-STING Pathway (3), inflammasome activation (4), etc.
  • the activation of these pathways leads to the production of many downstream cytokines, including type I interferon, IL-1 ⁇ , IL-18 and so on.
  • Type I interferon can activate the JAK-STAT pathway through autocrine and paracrine pathways, induce the expression of a large number of antiviral genes, and achieve the effect of resisting viral infections (5,6).
  • type I interferon can promote the maturation of antigen-presenting cells (7).
  • the antigen-presenting cells present pathogenic microorganisms or tumor antigens to T cells, and activate antigen-specific CD4 + T cells and CD8 + T cells; it can also Promote the activation of B cells to produce antigen-specific antibodies; and promote the production of memory B cells and immune memory.
  • the pro-inflammatory factor IL-1 ⁇ can directly act on CD4 + T cells and promote the proliferation of T cells (8); IL-18 can effectively enhance Th1 immune response, and can also promote the proliferation and cytotoxicity of T cells and NK cells (9) ).
  • Aluminum adjuvants have been widely used since 1920, and are used in hepatitis A (HAV) vaccine, hepatitis B (HBV) vaccine, DTP vaccine, human papillomavirus (HPV) vaccine, Haemophilus influenzae (HiB) ) Vaccines, etc.
  • HAV hepatitis A
  • HBV hepatitis B
  • HPV human papillomavirus
  • HiB Haemophilus influenzae
  • AS04 is an adjuvant containing MPL and aluminum salts and is used in HPV vaccines and HBV vaccines.
  • the adjuvant can activate NF-kB, produce pro-inflammatory factors, and has the ability to activate Th1 response that the aluminum adjuvant does not originally have.
  • MF59 is a water-in-oil adjuvant made with squalene as the oil phase. It contains degradable squalene, Tween 80 and Span 85 and is used in influenza vaccines. The mechanism is still unclear. Its half-life in the body is 42 hours, and it can activate both Th1 and Th2 responses.
  • AS03 is an oil-in-water adjuvant containing alpha tocopherol, squalene, and Tween 80, which is used in influenza vaccines, but the H1N1 influenza virus vaccine Pandemrix TM containing AS03 may cause narcolepsy (11). AS03 can induce the production of pro-inflammatory factors by activating the NF-kB pathway, recruit immune cells, and induce antibody production.
  • ligands that activate the cGAS-STING pathway can be used as adjuvants, such as DMXAA (12), c-di-GMP (13), cGAMP (14), chitosan (15).
  • CN107412260A discloses that divalent manganese is a cGAS-STING pathway activator, which has the function of enhancing immunity, for example, it can be used as an immune adjuvant.
  • divalent manganese itself still has some defects, and an improved solution is needed to eliminate at least one of the defects.
  • the inventors further studied the immuno-enhancing effect of divalent manganese and found that although the divalent manganese solution can produce an immune-enhancing effect in the body, this immune-enhancing effect is not high enough. More importantly, during the research process, it was also found that when the concentration of the divalent manganese solution is increased to improve the immune enhancement effect, it is easy to produce manganese precipitation, which makes it impossible to obtain a uniform and stable high-concentration manganese solution, which makes the experiment difficult. Repeatability cannot be effectively guaranteed. This manganese precipitation gradually agglomerates and grows with increasing storage time.
  • the inventors had to use divalent manganese precipitation to continue the research on its immune enhancement effect. Due to the negligence of the experimental operation, the inventor inadvertently used divalent manganese precipitates left for different periods of time. The inventors unexpectedly discovered that the divalent manganese precipitates with different storage time have different immune enhancement effects, and the immuno-enhancing effects of the newborn precipitates are significantly better than the manganese precipitation with longer storage time. The inventor further compared the divalent manganese solution with the divalent manganese precipitation stored for different time, and unexpectedly found that the immunoenhancing effect of the new manganese salt precipitation was even significantly better than that of the divalent manganese solution.
  • divalent manganese compounds can form colloidal solutions. What is even more surprising is that this manganese colloid shows an immune-enhancing effect comparable to or even stronger than that of the new manganese precipitation.
  • the present invention provides an immune-enhancing composition comprising nascent precipitated manganese, colloidal manganese, and/or a source capable of forming nascent precipitated manganese and/or colloidal manganese.
  • the newly precipitated manganese and/or colloidal manganese are selected from the following forms: manganese phosphate, manganese carbonate, manganese hydroxide and any mixtures thereof.
  • the time for the nascent precipitated manganese to be converted from a non-precipitated form to a precipitated form does not exceed 1 day, or does not exceed 24, 22, 20, 18, 16, 15, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour, or no more than 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute.
  • the present invention provides a vaccine composition comprising
  • Newly precipitated manganese, colloidal manganese and/or can form a source of new precipitated manganese and or colloidal manganese
  • components A and B may be in the same and/or separate containers.
  • the vaccine immunogen is derived from viruses, bacteria and/or parasites,
  • the virus is selected from: DNA virus and RNA virus
  • the virus is selected from: Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Mini RNAviridae, hepatotropic DNAviridae, flaviviridae, papillomaviridae, poxviridae, and retroviridae
  • the virus is selected from: herpes simplex virus, vesicular stomatitis virus, vaccinia virus , HIV and HBV;
  • the bacteria are selected from Gram-negative bacteria and Gram-positive bacteria, preferably the bacteria are selected from Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, and meningitis. Meningococcus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca ), Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa and Acinetobacter baumanni;
  • the vaccine immunogen is derived from influenza virus, hepatitis virus (such as hepatitis A virus, hepatitis B virus), polio virus, rabies virus, HPV virus, encephalitis virus (such as Japanese encephalitis virus), mumps Virus, rubella virus, tetanus bacillus, bacillus pertussis, diphtheria bacillus, leprosy bacillus, tuberculosis bacillus, meningococcus, pneumococcus and any combination thereof.
  • hepatitis virus such as hepatitis A virus, hepatitis B virus
  • polio virus rabies virus
  • HPV virus encephalitis virus
  • mumps Virus rubella virus
  • tetanus bacillus bacillus pertussis
  • diphtheria bacillus diphtheria bacillus
  • leprosy bacillus tuberculosis bacill
  • the present invention provides a method of manufacturing an immune enhancing composition, which comprises
  • the newly precipitated manganese and/or colloidal manganese are selected from manganese phosphate, manganese carbonate, manganese hydroxide and any mixtures thereof.
  • the time for the nascent precipitated manganese to be converted from a non-precipitated form to a precipitated form does not exceed 1 day, or does not exceed 24, 22, 20, 18, 16, 15, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 hour, or no more than 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute.
  • the present invention provides a method of manufacturing a vaccine composition, which comprises
  • the vaccine immunogen is derived from viruses, bacteria and/or parasites, as described above, and will not be repeated here.
  • the present invention provides a source of nascent precipitated manganese, colloidal manganese and/or capable of forming nascent precipitated manganese and or colloidal manganese in the preparation of an immune enhancement composition or vaccine composition for improving innate immunity and/or adaptive immunity Use in.
  • the nascent precipitated manganese, colloidal manganese and/or a source capable of forming nascent precipitated manganese and or colloidal manganese improves innate immunity and/or adaptive immunity by increasing the expression of type I interferon.
  • the newly-precipitated manganese, colloidal manganese and/or the source capable of forming newly-precipitated manganese and or colloidal manganese improve innate immunity and/or adaptive immunity by inducing cleavage of inflammatory factors to produce active forms.
  • the nascent precipitated manganese, colloidal manganese and/or a source capable of forming nascent precipitated manganese and or colloidal manganese improve innate immunity and/or adaptive immunity by promoting antibody production.
  • the present invention provides a method of enhancing immunity, which comprises administering the immune enhancing composition to a subject in need thereof.
  • the immune enhancement is, for example, A) improving innate immunity and/or adaptive immunity, B) increasing the expression of type I interferon, C) inducing the production of inflammatory factors in active form, D) promoting antibody production, E) promoting T Cell proliferation, and/or F) promote the maturation of dendritic cells.
  • the administration is selected from intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal administration, and any combination thereof.
  • the enhanced immunity is used to prevent and/or treat diseases, such as bacterial infections, fungal infections, viral infections, parasitic infections, tumors, and autoimmune diseases.
  • said virus is selected from: DNA virus and RNA virus, preferably said virus is selected from: Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Mini RNAviridae, hepatotropic DNAviridae, flaviviridae, papillomaviridae, poxviridae, and retroviridae, specifically the virus is selected from: herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, influenza virus, hepatitis virus (such as hepatitis A virus, hepatitis B virus), polio virus, rabies virus, HPV virus, encephalitis virus (such as Japanese encephalitis virus), mumps virus, rubella virus and any combination thereof.
  • Herpesviridae Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyx
  • said bacteria are selected from Gram-negative bacteria and Gram-positive bacteria, preferably said bacteria are selected from Streptococcus pneumoniae, Haemophilus influenzae (Haemophilus influenzae), Salmonella (Salmonella), meningitis double Meningococcus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca ), Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter baumanni, Tetanus, Pertussis, Diphtheria , Leprosy Bacillus, Mycobacterium tuberculosis, Meningococcus, Pneumococcus and any combination thereof.
  • the parasite is an intracellular parasite, preferably selected from Plasmodium, Toxoplasma, Trypanosoma, Schistosoma, Filaria, and Leishmania.
  • the autoimmune disease is selected from type I diabetes, psoriasis, rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis.
  • the tumor is selected from ovarian cancer, lung cancer, gastric cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, cholangiocarcinoma, bladder cancer, kidney cancer, colon cancer, placental choriocarcinoma, Cervical cancer, testicular cancer, uterine cancer and leukemia.
  • the immune enhancing composition is administered to a subject in need thereof together with another preventive/therapeutic agent.
  • the present invention provides an immunization method, which comprises
  • component A and component B can be administered at the same time or at different times when they are in different containers;
  • the immune enhancing composition of the present invention and optionally the vaccine immunogen are administered to a subject in need thereof.
  • the administration is selected from intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal administration, and any combination thereof.
  • the immunization is used to prevent diseases such as bacterial infections, fungal infections, viral infections, parasitic infections, tumors and autoimmune diseases.
  • diseases such as bacterial infections, fungal infections, viral infections, parasitic infections, tumors and autoimmune diseases.
  • the disease is the same as that described above, and will not be repeated here.
  • the immune enhancing composition is administered to a subject in need thereof together with another preventive/therapeutic agent.
  • the present invention provides the use of newly precipitated manganese, colloidal manganese and/or a source capable of forming newly precipitated manganese and/or colloidal manganese in the preparation of drugs for enhancing type I interferon.
  • the present invention provides the use of newly precipitated manganese, colloidal manganese and/or a source capable of forming newly precipitated manganese and/or colloidal manganese in the preparation of a medicament for inducing the cleavage of inflammatory factors to produce an active form.
  • the present invention provides the use of nascent precipitated manganese, colloidal manganese and/or a source capable of forming nascent precipitated manganese and/or colloidal manganese in the preparation of a drug for stimulating antibody production.
  • the present invention provides the use of nascent precipitated manganese, colloidal manganese and/or a source capable of forming nascent precipitated manganese and/or colloidal manganese as an immune adjuvant.
  • the immune adjuvant activates T cell activation and/or antibody production.
  • the immune adjuvant is used in a vaccine composition for the treatment of diseases selected from bacterial infections, viral infections, parasites, autoimmune diseases and cancer.
  • the present invention provides a kit for immunization, which comprises: a first container containing one or more antigens therein; and a second container containing newly precipitated manganese, colloidal manganese, and /Or can form a source of newly precipitated manganese and/or colloidal manganese.
  • the first container and/or the second container further comprise a pharmaceutically acceptable carrier.
  • the kit is used for one or more purposes of the present invention.
  • the antigen and the nascent precipitated manganese, colloidal manganese and/or the source capable of forming nascent precipitated manganese and/or colloidal manganese are located in the same container.
  • the antigen used in the present invention is selected from virus or bacterial or parasite antigens, for example, hepatitis A, B, C, D and E-3 hepatitis virus, HIV, herpes virus 1, Types 2, 6 and 7, cytomegalovirus, varicella-zoster virus, papilloma virus, Epstein-Barr virus, influenza virus, parainfluenza virus, adenovirus, Bunya virus (Hantavirus), Coxsackie virus, Picornavirus, rotavirus, respiratory syncytial virus, poxvirus, rhinovirus, rubella virus, papilloma virus, mumps virus and measles virus, mycobacteria that cause tuberculosis and leprosy, pneumococcus, aerobic leather Collins-negative bacilli, mycoplasma, staphylococcal infections, streptococcal infections, salmonella and chlamydia, Helicobacter pylori, malaria,
  • the nascent precipitated manganese, colloidal manganese and/or the source and/or antigen capable of forming nascent precipitated manganese and/or colloidal manganese in the present invention are effective amounts.
  • the present invention provides a method for increasing the expression of type I interferon in a subject, which comprises administering to the subject nascent precipitated manganese, colloidal manganese and/or capable of forming nascent precipitated manganese and/or colloidal manganese source.
  • the present invention also provides a method for enhancing the activity of type I interferon in cells in vitro, which comprises applying newly precipitated manganese, colloidal manganese and/or a source capable of forming newly precipitated manganese and/or colloidal manganese to the cells, Preferably, the method is for non-therapeutic purposes.
  • the present invention provides a method for inducing the cleavage of inflammatory factors to produce an active form in a subject, which comprises administering to the subject nascent precipitated manganese, colloidal manganese and/or capable of forming nascent precipitated manganese and/or colloidal manganese The source.
  • the present invention also provides a method for inducing cleavage of inflammatory factors in cells to produce active forms in vitro, which comprises applying to the cells a source of newly precipitated manganese, colloidal manganese and/or capable of forming newly precipitated manganese and/or colloidal manganese.
  • the method is for non-therapeutic purposes.
  • the present invention provides a method for stimulating antibody production in a subject, which comprises administering to the subject nascent precipitated manganese, colloidal manganese, and/or a source capable of forming nascent precipitated manganese and/or colloidal manganese.
  • the present invention also provides a method for stimulating cells to produce antibodies in vitro, which comprises applying newly precipitated manganese, colloidal manganese and/or a source capable of forming newly precipitated manganese and/or colloidal manganese to the cells, preferably, The method described is for non-therapeutic purposes.
  • Figure 1 illustrates that newly precipitated manganese and colloidal manganese (Mn 2 OHPO 4 ) have good adjuvant effects.
  • Figure 1A shows the different states of divalent manganese in the solution;
  • Figure 1B shows the transmission electron micrograph of colloidal manganese (Mn2OHPO4);
  • Figure 1C shows the level of OVA antibody IgG1 after immunization;
  • Figure 1D shows the MnCl 2 solution and colloidal manganese (Mn 2 OHPO) 4 ) Comparison of residence time after intramuscular injection.
  • Figure 2 illustrates the different existence states and properties of MnCl 2 solution mixed with other anions in physiological saline. And determine the ability of these precipitates or colloids to activate type I interferon or inflammatory factors, and the adjuvant effect.
  • Figure 2A shows that MnCl 2 solution and different concentrations of carbonate ion, bicarbonate ion, phosphate ion, monohydrogen phosphate ion, and hydroxide ion form different states;
  • Figure 2B shows that different states of manganese ion activate Type I interference It can induce the production of IL-1 ⁇ , an inflammatory factor, and the ability to produce antibodies as an adjuvant mixed with OVA protein.
  • Figure 2C shows the products formed by laser pointer irradiation of manganese ions and different anions, and observe the Tyndall effect.
  • Figure 3 illustrates the colloidal manganese (Mn 2 OHPO 4 ) and aluminum adjuvant mixed with chicken ovalbumin (OVA) to immunize mice by intramuscular injection for 10 days (A), 17 days (B), 24 days (C) and After 31 days (D), the amount of anti-OVA antibody produced.
  • Mn 2 OHPO 4 colloidal manganese
  • OVA chicken ovalbumin
  • Figure 4 illustrates the colloidal manganese (Mn 2 OHPO 4 ) and cholera toxin B (CTB) adjuvants respectively mixed with OVA after nasal drip to immunize mice with alveolar lavage (A), oral lavage (B), and serum IgA (C) and serum IgG1 (D) anti-OVA antibody amount.
  • Mn 2 OHPO 4 colloidal manganese
  • C serum IgA
  • D serum IgG1
  • Figure 5 illustrates the use of colloidal manganese (Mn 2 OHPO 4 ) as an adjuvant and mixed OVA intramuscular injection (A) or intranasal immunization (B) of mice according to the method of Figure 1 and Figure 2 and the determination of mice for 1 to 6 months The amount of anti-OVA antibodies in serum, oral lavage fluid, and alveolar lavage fluid.
  • Mn 2 OHPO 4 colloidal manganese
  • Figure 6 illustrates MnCl 2 activation of type I interferon pathway and inflammasome activation.
  • Figure 6A-B shows the expression levels of type I interferon, downstream inducible factors ISG54 and viperin after MnCl 2 treatment of mouse peritoneal macrophages, with aluminum adjuvant as a control.
  • Figure 6C-D shows that MnCl 2 can activate inflammasome activation, and aluminum adjuvant is used as a control.
  • Figure 6E-F shows that the activation of inflammasomes activated by MnCl 2 is dependent on NLRP3 and ASC.
  • Figure 7 illustrates that colloidal manganese (Mn 2 OHPO 4 ) promotes the proliferation of CD4 + T cells, with aluminum adjuvant as a control.
  • Figure 8 illustrates that the amount of antibodies produced by mice during intramuscular injection of colloidal manganese (Mn 2 OHPO 4 ) and mucosal immunization depends on the cGAS-STING pathway and the inflammasome pathway.
  • Figure 9 illustrates that the ability of colloidal manganese (Mn 2 OHPO 4 ) to promote the maturation of dendritic cells and the activation of T cells depends on the cGAS-STING pathway and the inflammasome pathway.
  • Figure 9A shows that colloidal manganese (MnOHPO 4 ) induces the maturation of antigen-presenting cells BMDC.
  • Figure 9B shows that colloidal manganese (MnOHPO 4 ) plus antigen OVA stimulates the proliferation of CD4 + T cells.
  • Figure 9C shows that colloidal manganese (MnOHPO 4 ) was used as an adjuvant to stimulate CD8 + T cell activation after immunizing mice.
  • FIG 10 illustrates that colloidal manganese (Mn 2 OHPO 4 ) as an adjuvant can significantly enhance the immune protection effect of inactivated vesicular stomatitis virus (VSV) and herpes simplex virus (HSV-1).
  • Figure 10A shows the experimental flow.
  • Figure 10B shows that the inactivated VSV virus was diluted into doses of 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , and 10 -6 to immunize mice, and the survival rate of the mice was observed for 14 days.
  • Fig. 10C shows the death curve of mice in 14 days after immunization with VSV inactivated virus diluted to a dose of 10 -3 .
  • Figure 10D shows the virus titers in the brains of mice on the 4th day of infection with the VSV virus.
  • Figures 10E-G show that adding colloidal manganese to the inactivated HSV-1 virus has the same protective effect.
  • Figure 11 illustrates that colloidal manganese (Mn 2 OHPO 4 ) as an adjuvant can significantly enhance the immune protective effect of inactivated influenza virus PR8.
  • Figure 11A-B shows the survival rate and body weight of mice after the inactivated influenza virus was diluted to 1 , 10 -1 , 10 -2 , and 10 -3 doses after intranasal immunization, 7 days later Variety.
  • Figure 11C-D shows the survival rate and body weight changes of mice infected with influenza virus after immunizing mice twice with intranasal drops.
  • FIG. 12 illustrates that colloidal manganese (Mn 2 OHPO 4 ) as an adjuvant can significantly enhance the immunoprotective effect of influenza subunit HA vaccine.
  • Fig. 12A shows the content of anti-HA protein IgG1 antibody in mouse serum at 14, 21, and 28 days after intramuscular injection of mice.
  • Fig. 12B shows the anti-HA protein IgA antibody content in the mouse serum at 14, 21, and 28 days after intranasal immunization of mice.
  • Figure 12C shows the weight change of mice after infection with influenza virus.
  • Figure 12D shows the pathological changes of mouse lung tissue on day 5 of infection.
  • Figure 13 illustrates the use of colloidal manganese Mn 2 OHPO 4 as an adjuvant to enhance the protective effect of inactivated influenza virus and subunit vaccines against homeotype influenza virus (WSN) and heterologous influenza virus (H3N2).
  • Figure 13A shows the weight change of WSN virus-infected mice after immunization;
  • Figure 13B shows the weight change of H3N2 virus-infected mice after immunization.
  • Figure 14 illustrates that colloidal manganese (Mn 2 OHPO 4 ) can significantly inhibit the growth of tumors in situ and metastases.
  • Figure 14A shows the size of the subcutaneous tumor taken with the imager at different times.
  • Figure 14B shows the tumor volume change after tumor inoculation.
  • Figure 14C shows the survival curve of mice after tumor inoculation.
  • Figure 14D shows that the tumor metastasized lung tissue was photographed 21 days after different immunization methods.
  • Figure 14E shows the statistics of the number of tumor cells in Figure 14D.
  • Figure 15 illustrates the effect of colloidal manganese (Mn 2 OHPO 4 ) and the chemotherapy drug cyclophosphamide (CTX) in the treatment of subcutaneous tumor model-melanoma B16-F10.
  • Figure 15A shows pictures of tumors 14 days after different treatments.
  • Figure 15B shows the growth curve of mouse subcutaneous tumor B16F10.
  • Figure 15C shows the tumor weight of the mice in the corresponding group on day 14 in Figure 15B.
  • Figure 16 illustrates the effect of colloidal manganese (Mn 2 OHPO 4 ) combined with PD1 antibody drugs on the subcutaneous tumor model-melanoma B16-F10.
  • Figure 16A shows pictures of tumors after different treatments.
  • Figure 16B shows the growth curve of the tumor.
  • Figure 16C shows the tumor weights of the mice in the B panel.
  • Figure 16D-E shows the number of CD8 + T cells infiltrated in the tumor analyzed by flow cytometry.
  • Figure 17 illustrates the effect of colloidal manganese (Mn 2 OHPO 4 ) combined with PD1 antibody drugs in the treatment of subcutaneous tumor model-colon cancer tumor MC38.
  • Figure 17A shows pictures of tumors after different treatments.
  • Figure 17B shows the growth curve of the tumor.
  • Figure 17C shows the tumor weight of the corresponding group in Figure 17B.
  • Figure 17D shows a tissue section of the tumor in the corresponding group in Figure A, with immunofluorescence staining of DAPI and CD8 + T cells.
  • innate immunity refers to the natural immune defense function formed by the body during germline development and evolution, that is, the non-specific defense function already possessed after birth, also known as non-specific immunity (non-specific immunity).
  • Innate immunity involves a variety of cells and molecules, such as macrophages, natural killer cells, complement, cytokines (IL, CSF, IFN, TNF, TGF- ⁇ ), chemokines (including CC chemokines, such as CCL1, CCL2) , CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, etc.
  • CXC chemokines such as CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, etc., C Factor, CX3C chemokine), lysozyme and so on.
  • adaptive immunity refers to the specific immunity of the body against the antigen formed after the stimulation of the antigen molecule, which involves cellular immunity and humoral immunity.
  • adjuvant refers to an agent that does not constitute a specific antigen, but enhances the strength and duration of the immune response to the co-administered antigen.
  • divalent manganese or “divalent manganese compound” as used herein can be hydrochloride, carbonate, hydrobromide, sulfate, nitrate, phosphate, tartrate, fumarate , Maleate, lactate, benzenesulfonate, pantothenate, ascorbate, hydroxide, etc., or any combination thereof.
  • the divalent manganese compound is pharmaceutically acceptable.
  • colloidal manganese refers to the state in which divalent manganese exists in the form of colloid (colloid, also known as colloidal solution) formed with anions.
  • colloid has the meaning understood by those of ordinary skill in the art.
  • the particle size of colloidal particles is generally about 1 to 100 nm, especially 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 nm, or a value between any two of the above values, such as 1 to 20, 5 to 20 nm, 10 nm.
  • colloidal solutions exhibit the Tyndall effect when exposed to light.
  • the particle size range of colloidal particles is on the order of nanometers
  • “colloidal manganese” in this article can also be referred to as “nano manganese” according to its particle size range.
  • cent precipitated manganese refers to the state of existence of the divalent manganese compound in a short period of time after the non-precipitated form (for example, the solution form) is converted to the precipitated form.
  • the short period of time generally does not exceed 1 day, and typically does not exceed 24, 22, 20, 18, 16, 15, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1. Hours, such as not exceeding 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute.
  • derived from viruses, bacteria and/or parasites means to include inactivated viruses, bacteria and/or parasites, or proteins and/or nucleic acids extracted from the above pathogenic substances (including cutting , Engineering and other processed substances), or purified recombinant protein substances, or chemically synthesized peptide substances.
  • pharmaceutically acceptable carrier can be selected from: water, buffered aqueous solution, isotonic salt solution such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, stearin Magnesium acid, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc.
  • the type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • composition according to the present invention may contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring substances, flavoring substances and/or aromatic substances and the like as additives.
  • composition according to the present invention can be administered by any suitable route, such as oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.
  • administration means to provide a substance to a subject in a pharmacologically usable manner.
  • pharmaceutical effective amount and “effective amount” refer to a dose sufficient to show its benefit to the administered subject.
  • the actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the individual being treated.
  • the prescription of treatment (such as the decision on dosage, etc.) is ultimately the responsibility of the general practitioner and other doctors and rely on them to make decisions, usually taking into account the disease to be treated, the individual patient’s condition, the delivery site, the method of application, and what is already known to the doctor. Know other factors.
  • subject means animals, including warm-blooded mammals such as humans and primates; birds; domesticated domestic or farm animals such as cats, dogs, sheep, goats, cows, horses, and pigs ; Laboratory animals, such as mice, rats and guinea pigs; fish; reptiles; zoo animals and wild animals.
  • any component, element, attribute or step disclosed in an embodiment of the method and product can be applied to any other method and product disclosed herein.
  • anti-GAPDH antibody (sc-25778) was purchased from Santa Cruz.
  • Anti-Viperin antibodies, anti-ISG54 antibodies, anti-Casp1/p20 antibodies, anti-IL1 ⁇ /p17 antibodies and anti-ASC antibodies are all prepared and used by published methods (16).
  • the cDNA of the antigen fragment is inserted into the pET-21b vector (Novagen) and expressed in E. coli BL21(DE3), the recombinant protein is purified by Ni-NTA affinity column, and then injected into mice or rabbits , To obtain antiserum that can recognize the corresponding antigen.
  • L929-ISRE (Jiang Zhengfan’s laboratory self-made cell line, stably transfected pGL3 ISRE-Luciferase plasmid into L929 cells, CCL-1), BHK21( CCL-10), B16-OVA( CRL-6322) and B16F10( CRL-6475) cells were cultured in DMEM (Gibco) medium supplemented with 10% FBS (Gibco), 5 ⁇ g/ml penicillin and 10 ⁇ g/ml streptomycin.
  • BMDCs Bone marrow derived macrophages
  • RPMI-1640 (Gibco) medium containing 10ng/mL GM-CSF, 10ng/mL IL-4, and 10% FBS (Gibco)
  • FBS FBS
  • Peritoneal macrophages were harvested from mice 5 days after the induction of thioglycolate (BD, Sparks, MD) injection, and cultured in DMEM medium supplemented with 5% FBS.
  • Tmem173 -/- mice were established by injecting Cas9 mRNA (100ng/ ⁇ l) and gRNA (50ng/ ⁇ l) cytoplasm into C57BL/6J mouse fertilized eggs using the CRISPR-cas9 method.
  • Cas9 mRNA and single guide RNA (gRNA) were transcribed in vitro by mMESSAGE mMACHINE T7 Ultra (Ambion, am1345).
  • Nlrp3 -/- , Nlrc4 -/- , Pycard -/- and Aim2 -/- gene knockout mice were donated by Vishva Dixit (Genentech Inc, USA).
  • Tmem173 -/- Pycard -/- double knockout mice were identified by mating Tmem173 -/- mice and Pycard -/- .
  • mice All mice were fed in the Laboratory Animal Center of Peking University under aseptic conditions in accordance with the NIH National Institute of Health Guide for Care and Use of Laboratory Animals.
  • Type I IFN Type I-IFN bioassay
  • IFN-stimulated response element IFN-stimulated response element
  • pGL3-Basic vector Promega
  • IFN-sensitive luciferase vector IFN-sensitive luciferase vector
  • L929-ISRE cells were seeded into a 96-well plate and incubated with the cell culture supernatant. Recombinant human and mouse IFN- ⁇ (R&D Systems) were used as standards. After 4 hours, L929-ISRE cells were lysed and assayed by Luciferase Reporter Assay System (Promega).
  • H1N1 influenza virus PR8 strain from Yonghui Zhang, Tsinghua University, VR-95
  • H1N1 influenza virus WSN strain, A/WSN/33(H1N1) from Wenjun Liu, Institute of Microbiology, CAS
  • H3N2 influenza virus strain, H3N2 subtype A/Jiangxi/2005 from Min Fang, Institute of Microbiology, CAS
  • BHK21 cells were used to determine the titers of HSV-1 and VSV viruses.
  • MDCK cells were used to determine influenza virus titer.
  • HSV-1 and VSV viruses are amplified with vero cells.
  • PR8, WSN, and H3N2 influenza viruses are amplified with chicken embryos.
  • the amplified virus was purified with PEG8000.
  • the purified virus was inactivated with 0.2% formaldehyde at 37 degrees Celsius for 24 hours. The plaque method was used to measure whether the virus was completely inactivated, and then the inactivated virus was used to immunize mice.
  • Mouse survival experiment Infect 8-12 weeks of mice intravenously with HSV-1 (1.4 x 10 7 pfu/mouse) and VSV (8 x 10 8 pfu/mouse), or with PR8 (1 x 10 5 pfu/mouse), WSN (1 x 10 6 pfu/mouse), H3N2 (1 x 10 6 pfu/mouse) intranasally infected mice for 8-12 weeks.
  • Plaque assay BHK21 cells were incubated with homogenate from infected mouse organs (a series of dilutions in serum-free DMEM) for 2 hours. Then, the medium was replaced with serum-free DMEM containing 0.5% methylcellulose. After 60 hours, the cells were fixed with 0.5% (vol/vol) glutaraldehyde and stained with 1% (wt/vol) crystal violet (dissolved in 70% ethanol). The plaques are counted to calculate the virus titer in plaque forming units.
  • the purified plasmid of influenza virus HA1 protein was donated by Zhang Yonghui's laboratory of Tsinghua University (18).
  • the plasmid expresses the 11-324 amino acids of HA protein (Gene ID: 956529).
  • the bacterial solution is shaken to an OD 600 of 0.6, it is induced by adding 0.5 mM IPTG and incubated at 37°C for 5 hours.
  • the inclusion bodies are collected by ultrasound, and the HA1 protein is obtained after denaturation and renaturation. Then the HA1 protein was purified by gel filtration chromatography.
  • Ovalbumin (OVA) as an immunogen to immunize mice
  • the method of immunization by intramuscular injection of MnCl 2 solution is to add 10 ⁇ g MnCl 2 and 10 ⁇ g OVA protein to 100 ⁇ L PBS, mix and leave for 5 minutes, and then immunize mice by intramuscular injection.
  • Intranasal immunization method of MnCl 2 solution is to add 5 ⁇ g MnCl 2 and 10 ⁇ g OVA protein to 20 ⁇ L PBS, mix and place for 5 minutes, and then immunize mice by intranasal drip.
  • Colloidal manganese (take Mn 2 OHPO 4 as an example) intramuscular injection immunization method is to add 10 ⁇ g colloidal manganese and 10 ⁇ g OVA protein to 100 ⁇ L of normal saline, mix well and intramuscularly immunize mice.
  • Colloidal manganese nasal drip immunization method is to add 5 ⁇ g colloidal manganese and 10 ⁇ g OVA protein to 20 ⁇ L of normal saline, mix well and immunize mice by nasal drip.
  • Aluminum adjuvant intramuscular injection immunization method is to add 10 ⁇ g/20 ⁇ L aluminum adjuvant and 10 ⁇ g OVA protein to 80 ⁇ L normal saline, emulsify with an emulsifier, and then intramuscularly immunize mice.
  • Cholera toxin B (CTB) intranasal immunization method is to add 5 ⁇ g CTB and 10 ⁇ g OVA protein to 20 ⁇ L of normal saline, mix well and immunize mice by intrana
  • the ELISA method measures OVA-specific IgG1, IgG2c, total IgG, and IgA antibodies: the diluted serum from the immunized mice is incubated on an ELISA plate coated with 100 ⁇ g/ml OVA. After washing, use HRP-conjugated antibody mouse IgG1 (eBioscience, #18-4015-82), IgG2c (GeneTex, GTX77297), IgG total (Invitrogen, G21040), IgA (GeneTex, GTX77223) to detect the bound corresponding antibodies . Then, the plate was incubated with the substrate TMB (eBioscience), the reaction was stopped with 1M H 3 PO 4 , and then the absorbance at 450 nm was measured.
  • HRP-conjugated antibody mouse IgG1 eBioscience, #18-4015-82
  • IgG2c GeneTex, GTX77297
  • IgG total Invitrogen, G21040
  • IgA GeneTex,
  • Mantel-Cox test was used to compare survival curves.
  • Example 1 The state of manganese compound in solution and its effect as an adjuvant
  • mice were randomly divided into 6 groups according to their body weight, namely: 1) PBS control group, 2) antigen OVA (10 ⁇ g), 3) MnCl 2 reacted in saline for 2 days (10 ⁇ g MnCl 2 +OVA), 4) MnCl 2 Reaction in PBS for 5 minutes group (10 ⁇ g MnCl 2 +OVA), 5) MnCl 2 reaction in PBS for 2 days group (10 ⁇ g MnCl 2 +OVA), 6) MnCl 2 reaction to generate colloidal manganese for 30 days group (10 ⁇ g Mn 2 OHPO 4 +OVA). 10 ⁇ g here refers to the mass of manganese.
  • mice were randomly divided into 2 groups according to their body weight, namely: intramuscular injection of MnCl 2 solution group (20 ⁇ g MnCl 2 dissolved in 100 ⁇ l of saline), intramuscular injection of colloidal manganese (20 ⁇ g of colloidal manganese suspended in 100 ⁇ l of saline), where 20 ⁇ g were both Refers to the quality of manganese.
  • MnCl 2 solution group (20 ⁇ g MnCl 2 dissolved in 100 ⁇ l of saline
  • colloidal manganese 20 ⁇ g of colloidal manganese suspended in 100 ⁇ l of saline
  • the content of manganese was determined by ICP-MS (Thermo X SERIES II).
  • colloidal manganese can stay at the injection site longer, which can activate the body to produce antibodies for a longer time, while the solution manganese is quickly metabolized.
  • Mn 3 (PO 4 ) 2 is produced at low concentration, and Mn 2 OHPO 4 is produced at high concentration; divalent free manganese reacts with HPO 4 2- to produce white MnHPO 4 ; reacts with OH - to produce slightly yellow Precipitated Mn(OH) 2 .
  • P-IRF3 is the main transcription factor regulating the expression of type I interferon.
  • Mn 2 OHPO 4 and MnHPO 4 can also significantly induce the production of IL-1 ⁇ .
  • non-colloid Mn(OH) 2 can only activate the production of type I interferon, but cannot induce the production of inflammatory factor IL-1 ⁇ .
  • the mouse serum was taken to measure the anti-OVA IgG1 antibody content. The effect of Mn 2 OHPO 4 and MnHPO 4 in stimulating the body to produce antibodies is significantly better than that of manganese compounds in the precipitated state.
  • the leftmost MnCl 2 in the antibody test is the adjuvant effect of the precipitation of MnCl 2 reacted with PBS for 5 minutes. Other solid precipitates placed overnight also have a partial adjuvant effect.
  • the reaction product was resuspended in physiological saline and then irradiated with a laser pointer. It can be seen that the Tyndall effect of Mn 2 OHPO 4 is the most obvious, and MnHPO 4 has a part of Tyndall effect, probably because its diameter is slightly larger than 100 nm. The diameter of Mn 2 OHPO 4 is about 10 nm, which meets the definition of colloid.
  • Example 3 Colloidal manganese Mn 2 OHPO 4 as an adjuvant for intramuscular injection and mucosal immunity
  • mice were randomly divided into 6 groups according to their body weight, namely: PBS control group, antigen OVA (10 ⁇ g), colloidal manganese Mn 2 OHPO 4 group (10 ⁇ g Mn 2 OHPO 4 +OVA), low-dose aluminum adjuvant group (440 ⁇ g aluminum adjuvant)
  • the medium-dose aluminum adjuvant group 800 ⁇ g aluminum adjuvant+OVA
  • the high-dose aluminum adjuvant group (1220 ⁇ g aluminum adjuvant+OVA).
  • Each group was immunized by intramuscular injection according to the experimental design amount, and immunized once on the 0th day and the 10th day.
  • colloidal manganese Mn 2 OHPO 4 at a dose of 10 ⁇ g/mouse is more effective than a high-dose (1220 ⁇ g/mouse) aluminum adjuvant. This indicates that colloidal manganese can achieve a better immune enhancement effect at a dose 100 times lower than that of aluminum adjuvants, which can greatly reduce the side effects of aluminum adjuvants currently widely used.
  • Example 4 Colloidal manganese Mn 2 OHPO 4 can be used as an adjuvant for intramuscular injection and mucosal immunity to make the body persistently produce specific antibodies
  • mice were immunized with colloidal manganese Mn 2 OHPO 4 as an adjuvant mixed with OVA by intramuscular injection, and the anti-OVA antibody content in the serum of the mice was determined 1 week, 1 month, 3 months, and 6 months after immunization. .
  • the anti-OVA antibody in the serum remained at a high level by the sixth month, which indicates that the colloidal manganese Mn 2 OHPO 4 as an adjuvant can produce a lasting protective effect.
  • the life span of mice is generally 2 years. It is expected that colloidal manganese Mn 2 OHPO 4 can produce protective antibodies throughout the life cycle of the body.
  • Example 3 Refer to the method in Example 3 after mucosal immunization of mice, the blood, alveolar lavage fluid, and oral lavage fluid of the mice were collected at different times to determine the antibody content. As shown in Figure 5B, the concentration of specific antibodies remained at a high level until 6 months after immunization, indicating that colloidal manganese Mn 2 OHPO 4 can also produce a lasting protective effect when used for mucosal immunity.
  • Example 5 Manganese (II) activates the production of inflammatory factors
  • the inflammatory factor IL-1 ⁇ can directly act on CD4 + T cells and promote the proliferation of T cells (8).
  • IL-1 ⁇ and IL-1 ⁇ can promote the infiltration of neutrophils (19); the inflammatory factor IL-18 can be effective Enhancing Th1 immune response can also promote the proliferation and cytotoxicity of T cells and NK cells (9). It has been reported that the adjuvant effect of aluminum adjuvant is achieved by activating NLRP3 inflammasome and releasing inflammatory factors (20-22), so we tested and compared MnCl 2 /colloidal manganese Mn 2 OHPO 4 with aluminum adjuvant to activate inflammatory factors. Ability.
  • MnCl 2 and colloidal manganese Mn 2 OHPO 4 have the same ability to activate inflammatory factors (see Figure 2B) and mechanism.
  • MnCl 2 is used as an example.
  • the primary culture of mouse peritoneal macrophages cultured in vitro were treated with 10, 20, 50, 100 ⁇ g/mL manganese chloride or aluminum adjuvants, and the interferon-inducing genes ISG54 and Viperin were detected by Western blotting, and the supernatant was detected by Bioassay The total type I interferon content (the sum of the concentrations of interferon alpha and interferon beta). The results are shown in Figures 6A and 6B.
  • the peritoneal macrophages pretreated with LPS were treated with MnCl 2 , ATP (classical NLRP3 inflammasome activator), and VacV (classical Aim2 inflammasome activator).
  • Western blotting was used to detect the cleavage of caspase1 and IL-1 ⁇ in the supernatant Cut (the upper band is the precursor, the lower band is the active form of caspase1 and IL-1 ⁇ ), and the production of inflammatory factors IL-1 ⁇ and IL-18 in the supernatant is detected by ELISA.
  • the results are shown in Figures 6E and 6F.
  • NLRP3 knockout cells the ability of MnCl 2 to activate inflammatory factors was significantly weakened, so NLRP3 inflammasome was mainly activated by MnCl 2 .
  • OT-II cells labeled with CFSE dye into the tail vein of mice.
  • the usage amount described here is the amount of one mouse immunization. Lymph nodes were collected three days after the immunized mice, and the proliferation of OT-II cells in the lymph nodes was analyzed by flow cytometry.
  • the rightmost peak in the figure is the OT-II cells originally injected into the mouse body.
  • the CFSE in the cell will be evenly distributed between the two cells, so the brightness of the CFSE in the cell will decrease, and the cells with decreased CFSE on the left are all dividing cells.
  • the results show that when the same amount of antigen is used, the ability of colloidal manganese to promote the proliferation of T cells is stronger than that of aluminum adjuvant, and the amount of colloidal manganese only needs 1/100 of that of aluminum adjuvant to achieve similar effects.
  • Example 7 Colloidal manganese Mn 2 OHPO 4 activated antibody production depends on the STING pathway and the inflammasome pathway
  • Figure 8A shows the intramuscular injection of WT, Pycard knockout (ASC protein deletion), Sting knockout (STING protein deletion), Pycard and Sting double knockout with 10 ⁇ g of colloidal manganese per mouse plus 10 ⁇ g of OVA protein.
  • the mouse serum was taken to measure the IgG1, IgG2c, and total IgG contents of anti-OVA antibodies in the serum by ELISA.
  • the results show that knockout alone will only partially lose the adjuvant effect of colloidal manganese, while double knockout will significantly reduce the adjuvant effect.
  • Fig. 8B shows that mice were immunized three times with 5 ⁇ g colloidal manganese per mouse and 10 ⁇ g OVA protein mucosa.
  • the serum, alveolar lavage fluid, and oral lavage fluid were collected by ELISA to measure the secreted anti-OVA IgA antibody content. The results showed that the adjuvant effect was significantly reduced in double knocked mice.
  • Example 8 Colloidal manganese Mn 2 OHPO 4 promotes the maturation of dendritic cells
  • Figure 9A shows the use of 100, 200, and 400 ⁇ M colloidal manganese to treat bone marrow-derived dendritic cells (BMDC) cells for 20 hours, and use a flow cytometer to detect the CD86 protein, a marker of dendritic cell maturation.
  • BMDC bone marrow-derived dendritic cells
  • FIG 9B the method described in Example 6 was used in four mice to test the efficiency of colloidal manganese in promoting T cell proliferation.
  • Example 9 Colloidal manganese Mn 2 OHPO 4 as an adjuvant enhances the immune protective effect of inactivated vesicular stomatitis virus (VSV) and herpes simplex virus (HSV-1)
  • the experimental procedure is shown in Figure 10A.
  • the mice were injected intramuscularly with the vaccine on day 0, and the mice were infected with virus on day 10.
  • the virus titer in mouse tissues was measured on day 14.
  • the mice were observed and the death curve was recorded to day 14. .
  • the PEG-purified VSV virus was inactivated and used as a vaccine, and diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 dilutions to immunize mice by intramuscular injection.
  • the virus concentration is high enough, it can produce enough immune effect to protect mice when the virus is used alone. After the virus is diluted by 10 times several gradients, the mice are immunized.
  • the survival rate of mice is only 8.3%.
  • the concentration was 10 -4 , 10 -5 , and 10 -6 , all mice died; but when Mn 2 OHPO 4 colloid was added as an adjuvant to immunize mice with 10 -3 concentration of inactivated virus, the survival rate of mice Increase to 75%, as shown in Figures 10B and 10C. It shows that colloidal manganese as an adjuvant can enhance the immune effect of low-concentration immunogens, using a small amount of immunogen can play a protective effect, and the active virus titer in mice is also reduced, as shown in Figure 10D.
  • the PEG-purified HSV-1 virus was inactivated and used as a vaccine to immunize mice by intramuscular injection.
  • the virus dilution is 10 -1 , 10 -2 , it can produce sufficient immune effect to protect mice when the virus is used alone.
  • the survival rate of mice was only 16.7%, and all the mice died when the dilution was lower; while adding Mn 2 OHPO 4 colloid to the virus at the 10 -4 dilution, the survival rate of mice increased to 100%, as shown in Figures 10E and 10F.
  • the active virus titer in mice was reduced, as shown in Figure 10G. This indicates that colloidal manganese as an adjuvant can achieve the same protective effect while reducing the dose of inactivated HSV-1 virus by 10 times.
  • the PEG-purified influenza PR8 virus was inactivated and diluted to 1 , 10 -1 , 10 -2 , and 10 -3 dilutions, and the mice were immunized once by nasal drip. Seven days later, the mice were infected with influenza virus, and the survival rate and weight change of the mice were recorded. All doses have no protective effect when no adjuvant is added, and the weight of the mouse has been reduced until death; and after adding Mn 2 OHPO 4 colloid as an adjuvant, the virus diluted to a dose of 10 -1 can make the weight of the mouse drop to The increase appeared after the 4th and 5th days, the body weight returned to the level before the virus infection after the 10th day, and the condition improved, as shown in Figures 11A and 11B. This result indicates that colloidal manganese can be used as an adjuvant to protect an immunogen that is originally non-immunogenic or weakly immunogenic after immunizing a subject once.
  • the PEG-purified influenza PR8 virus was inactivated and diluted to dilutions of 1 , 10 -1 , 10 -2 , and 10 -3 .
  • the mice were immunized twice (day 0 and day 7). immunity). After 14 days, the mice were infected with influenza virus, and the survival rate and weight change of the mice were recorded. All doses have no protective effect without adjuvant, and the weight of the mice has been reduced until death; and after adding Mn 2 OHPO 4 colloid as an adjuvant, the mice were immunized twice with the virus diluted to a dose of 10 -1 .
  • Example 10 Colloidal manganese Mn 2 OHPO 4 is used as an adjuvant to enhance the protective effect of influenza subunit vaccine
  • mice were randomly divided into 4 groups according to their body weight, namely: blank control group (ctrl), HA antigen group (HA), HA antigen and colloidal manganese group (HA+Mn) and HA antigen and aluminum adjuvant group (HA+Al ).
  • ctrl blank control group
  • HA HA antigen group
  • H+Mn HA antigen and colloidal manganese group
  • HA+Al HA antigen and aluminum adjuvant group
  • Example 11 Colloidal manganese Mn 2 OHPO 4 is used as an adjuvant to enhance inactivated influenza virus and subunit vaccines to have protective effects on homeotype influenza virus (WSN) and heterologous influenza virus (H3N2) bacteria
  • mice were randomly divided into 5 groups according to their body weight, namely: blank control group (con), inactivated virus PR8 group, inactivated virus PR8+ colloidal manganese group (PR8+MnJ), PR8-HA1 protein group (HA1) and PR8- HA1 protein + colloidal manganese group (HA1+MnJ).
  • blank control group con
  • inactivated virus PR8 group inactivated virus PR8+ colloidal manganese group
  • PR8+MnJ PR8-HA1 protein group
  • HA1+MnJ PR8-HA1 protein + colloidal manganese group
  • mice were randomly divided into 5 groups according to their body weight, namely: blank control group (con), inactivated virus PR8 group, inactivated virus PR8+ colloidal manganese group (PR8+MnJ), PR8-HA1 protein group (HA1) and PR8- HA1 protein + colloidal manganese group (HA1+MnJ).
  • Blank control group con
  • inactivated virus PR8 group inactivated virus PR8+ colloidal manganese group
  • PR8+MnJ PR8-HA1 protein group
  • HA1+MnJ PR8-HA1 protein + colloidal manganese group
  • the weight of the mice will drop to 80% before recovery, and the HA1 group alone will reduce to 90% recovery.
  • the HA1 group with colloidal manganese basically did not lose weight, and the inactivated virus group with colloidal manganese added only 5% of the weight to recover.
  • Example 12 Colloidal manganese Mn 2 OHPO 4 as an immune enhancer enhances the body's anti-tumor effect
  • mice were randomly divided into 4 groups according to their body weight, namely: blank control group (con), OVA control group, OVA and colloidal manganese group (OVA+Mn), and OVA and aluminum adjuvant group (OVA+Al). After immunizing the mice three times according to the experimental design (once every 7 days), the melanoma B16-OVA was subcutaneously inoculated to establish an orthotopic tumor model.
  • the use of aluminum adjuvants in advance cannot inhibit tumor growth and improve the survival rate of mice; while the use of colloidal manganese in advance can significantly inhibit tumor growth and increase the survival rate of mice; 21 days after tumor inoculation, the control group is small the average tumor size of mice is 1906mm 3, the average tumor size of mice was OVA 925.5mm 3, manganese colloid tumors in mice immunized with an average size of 222.9mm 3, the average tumor size of mice aluminum adjuvant is 1946mm 3, As shown in Figure 14B; the survival time of control mice was 26 days, and the survival time of mice immunized with colloidal manganese was 49 days, which was significantly longer than that of mice immunized with Lu adjuvant at 27 days, as shown in Figure 14C. The results indicate that colloidal manganese can be used as an immune enhancer to enhance the body's epitopic tumor effect.
  • Mn 2 OHPO 4 colloid and aluminum adjuvant were mixed with antigen OVA to immunize mice, immunized every 7 days, immunized 3 times to activate the immune response in the body; B16-OVA tumor was injected into the tail vein to establish a tumor metastasis model. The mice were sacrificed 21 days after the tumor injection, and the lung tissues were taken out. It can be seen that the lungs of the mice in the blank control group were covered with melanomas, and a certain number of melanomas appeared on the lungs of the mice immunized with OVA, while those immunized with OVA + colloidal manganese There were almost no tumors on the lungs of the mice, as shown in Figure 14D.
  • mice with melanoma B16F10 After subcutaneously inoculating mice with melanoma B16F10, they were randomly divided into 4 groups: control group (con), Mn 2 OHPO 4 colloid group (5 mg/kg), Cyclophosphamide Monohydrate (CTX) group (140 mg/kg), and Combination medication group (CTX+Mn). Cyclophosphamide (CTX) is a widely used anti-tumor chemotherapy drug in clinical practice. Mn 2 OHPO 4 colloidal intramuscular injection (treatment every two days after tumor inoculation), intraperitoneal injection of CTX (treatment each on the 6, 9 and 12 days after tumor inoculation), and the control group was given an equal volume of normal saline.
  • CTX Cyclophosphamide
  • the combination of colloidal manganese and CTX can significantly inhibit tumor growth. 14 days after tumor inoculation, the mice were sacrificed, the tumors were dissected and their volumes were measured. The average tumor size of the control group was 960.1mm 3 , the tumor size of mice treated with colloidal manganese alone was 659.5mm 3 , and the size of mice treated with CTX alone was 393.9 mm 3 , the tumor size of the combined mice was 238 mm 3 , as shown in Figure 15B; weighing the weight of each tumor at the same time, the results showed that the combined use of colloidal manganese and CTX inhibited tumor growth, as shown in Figure 15C. This indicates that colloidal manganese as an immune enhancer enhances traditional treatment methods, such as chemotherapy, and its combined use has a significant inhibitory effect on tumor growth.
  • mice with melanoma B16F10 After subcutaneously inoculating mice with melanoma B16F10, they were randomly divided into 4 groups: control group (con), Mn 2 OHPO 4 colloid group (5 mg/kg), anti-PD1 antibody group (200 ⁇ g/mouse), and combined Medication group (anti-PD1+Mn).
  • PD1 antibody is a new drug used in immunotherapy in recent years.
  • PD1 antibody (Clone 29 F.1A12, BioXCell) was treated once each on the 3rd, 7th, and 11th days after tumor inoculation. Each time 200ug of antibody was dissolved in 200ul PBS and injected intraperitoneally.
  • mice 15 days after tumor inoculation, the mice were sacrificed, and the tumors were dissected and measured.
  • the average tumor size of mice in the control group was 1847 mm 3
  • the average tumor size of mice in the colloidal manganese group alone was 797.3 mm 3 .
  • tumor bearing mice PD1 antibody alone group average size of 687.3mm 3, while tumors in mice treated with a mean size of 342mm 3, PD1 antibodies significantly lower than used alone.
  • Flow cytometry was used to analyze the proportion of CD8 + T cells infiltrating the tumor.
  • Colloidal manganese increased the effect of PD1 antibody drug from 27.7% to 45.4%. This indicates that colloidal manganese can cooperate with PD1 antibody drugs to treat melanoma.
  • mice were subcutaneously inoculated with colon cancer tumor MC38 and randomly divided into 4 groups, namely: control antibody group (Isotype, 200 ⁇ g/mouse), Mn 2 OHPO 4 colloid and control antibody group (Isotype+Mn), anti-PD1 antibody In the group (200 ⁇ g/mouse), and the combination group (anti-PD1+Mn), the use of PD-1 is the same as in experiment (d). Twenty days after tumor inoculation, the mice were sacrificed and the tumors were dissected and measured.
  • mice in the control group were 319.8 mm 3
  • the size of mice treated with manganese alone was 126.9 mm 3
  • the size of mice treated with PD1 antibody drug alone was 143.1 mm 3
  • the tumor size of the mice treated with the combination treatment was 66.74 mm 3 , as shown in Figure 17B.
  • the tumor tissue sections were made, and immunofluorescence staining of DAPI and CD8 + T cells was performed. It can be seen that the proportion of CD8 + T cells infiltrating into the tumor of mice treated with PD1 antibody and colloidal manganese was significantly increased, as shown in Figure 17D. This indicates that colloidal manganese can cooperate with PD1 antibody drugs to treat colon cancer tumors.

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Abstract

包含新生沉淀锰和／或胶体锰的免疫增强组合物和疫苗组合物，其制备方法，以及它们用于增强免疫和或疫苗接种的用途。

Description

用于免疫增强的锰组合物
本申请要求于2019年04月19日提交中国专利局、申请号为201910319344.1的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明提供了用于增强免疫的二价锰胶体或二价锰新生沉淀，以及其用于增强免疫的用途，这种免疫增强用途例如可用于作为免疫佐剂、抗病毒或抗肿瘤。
背景技术
机体通过天然免疫系统和适应性免疫系统保护机体免于外来病原体的侵染。天然免疫对于适应性免疫有着促进作用。当机体被病源体感染时，首先启动了天然免疫反应，通过模式识别受体识别病原体的病源相关分子模式，激活多种信号通路，如TLR通路(1)、RLR通路(2)、cGAS-STING通路(3)、炎症小体活化(4)等，这些通路的激活导致下游众多细胞因子的产生，包括I型干扰素、IL-1β、IL-18等。
I型干扰素通过自分泌和旁分泌途径可激活JAK-STAT通路，诱导大量抗病毒基因的表达，达到抵抗病毒感染的效果(5,6)。同时I型干扰素可促进抗原递呈细胞的成熟(7)，抗原递呈细胞将病源微生物或肿瘤抗原递呈给T细胞，激活抗原特异性CD4 +T细胞和CD8 +T细胞；同时也可促进B细胞活化产生抗原特异性抗体；及促进产生记忆性B细胞及免疫记忆。
促炎因子IL-1β能直接作用于CD4 +T细胞，促进T细胞的增殖(8)；IL-18可以有效增强Th1免疫反应，也可以促进T细胞和NK细胞的增殖和细胞毒作用(9)。
目前已经被FDA批准在人身上使用的佐剂有四种：铝佐剂、MF59、AS03、AS04。铝佐剂从1920年以来一直被广泛使用，被用于甲肝(HAV)疫苗、乙肝(HBV)疫苗、百白破(DTP)疫苗、人乳头瘤病毒(HPV)疫苗、流感嗜血杆菌(HiB)疫苗等。铝佐剂只能激活Th2反应(以抗体产生为主的体液免 疫)，不能激活Th1反应(以CD8 +T细胞为主的细胞免疫)(10)。AS04是包含MPL和铝盐的佐剂，被用于HPV疫苗和HBV疫苗。该佐剂能激活NF-kB，产生促炎因子，具备了铝佐剂原来不具备的激活Th1反应的能力。MF59是一种以角鲨烯为油相制成的油包水类型佐剂，含有可降解鲨烯、Tween 80和Span 85，被用于流感疫苗。其机制目前还不清楚，它在体内的半衰期是42小时，能同时激活Th1反应和Th2反应。AS03是含有α生育酚、角鲨烯、Tween 80的水包油类型佐剂，被用于流感疫苗，但是含有AS03的H1N1流感病毒疫苗Pandemrix TM可能会引起嗜睡症(11)。AS03可通过激活NF-kB通路诱导促炎因子的产生，招募免疫细胞，诱导抗体产生。
目前未批准临床应用，但在实验室中可使用的佐剂有很多，其中很多效果很好，但是由于其副作用限制了在临床上的应用，如弗氏佐剂的矿物油成分代谢能力差，在注射部位会形成结节。还有细胞因子类型的佐剂，如白细胞介素-2、干扰素，它们的缺点是成本昂贵。
目前有报道证明激活cGAS-STING通路的配体能够作为佐剂，如DMXAA(12)、c-di-GMP(13)、cGAMP(14)、chitosan(15)。
CN107412260A公开了二价锰是一种cGAS-STING通路激活剂，具有增强免疫的作用，例如可用作免疫佐剂。然而，在进一步研究中，发明人意外发现二价锰本身还存在一些缺陷，还需要改进的方案以消除至少一种所述缺陷。
发明内容
发明人在进一步研究二价锰免疫增强作用中发现，尽管二价锰溶液可以在机体中产生免疫增强作用，然而这种免疫增强作用还不够高。更重要的是，研究过程中还发现，当希望提高二价锰溶液的浓度，以便提高免疫增强作用时，很容易产生锰沉淀，导致无法获得均匀稳定的高浓度二价锰溶液，使得实验的可重复性无法得到有效保证。这种锰沉淀随着放置时间增加而逐渐团聚化增长。
在无法实现期望的高浓度二价锰溶液的情况下，发明人不得已使用二价锰沉淀继续其免疫增强作用的研究。由于实验操作的疏忽，发明人无意中使用了放置不同时间的二价锰沉淀。发明人意外发现，不同放置时间的二价锰沉淀具有不同的免疫增强作用，而且新生沉淀的免疫增强作用显著优于放置时间更 长的锰沉淀。发明人进一步比较了二价锰溶液和放置不同时间的二价锰沉淀，又意外地发现，新生锰盐沉淀的免疫增强作用竟然还显著优于二价锰溶液。
在尝试不同二价锰体系过程中，发明人还意外发现，二价锰化合物可以形成胶体溶液。更加令人惊奇的是，这种锰胶体显示出与新生锰沉淀相当、甚至更强的免疫增强作用。
发明人在此基础上完成了本发明，并提供以下技术方案。
在一个方面，本发明提供一种免疫增强组合物，其包含新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。
在一个实施方案中，所述新生沉淀锰和/或胶体锰是选自以下物质的存在形式：磷酸锰盐、碳酸锰盐和氢氧化锰及其任意混合物。
优选地，所述新生沉淀锰从非沉淀形式转化成沉淀形式的时间不超过1天，或者不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，或者不超过60、50、45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
在另一个方面，本发明提供一种疫苗组合物，其包含
A)疫苗免疫原，和
B)新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和或胶体锰的源，
任选地，组分A和B可以处于相同和/或分开的容器中。
优选地，所述疫苗免疫原源自病毒、细菌和/或寄生虫，
例如所述病毒选自：DNA病毒和RNA病毒，优选地所述病毒选自：疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、冠状病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，更优选地所述病毒选自：单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV和HBV；
例如所述细菌选自革兰氏阴性菌和革兰氏阳性菌，优选地所述细菌选自肺炎链球菌(Streptococcus pneumoniae)、流感嗜血杆菌(Haemophilus influenzae)、沙门氏菌(Salmonella)、脑膜炎双球菌(Meningococcus)、表皮葡萄球菌(Staphylococcus epidermidis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、肺炎克雷伯氏菌(Klebsiella  pneumoniae)、产酸克雷伯氏菌(Klebsiella oxytoca)、阴沟肠杆菌(Enterobacter cloacae)、弗氏柠檬酸杆菌(Citrobacter freundii)、绿脓假单胞菌(Pseudomonas aeruginosa)和波美不动杆菌(Acinetobacter baumanni)；
具体地，例如所述疫苗免疫原源自流感病毒、肝炎病毒(如甲肝病毒、乙肝病毒)、脊髓灰质炎病毒、狂犬病毒、HPV病毒、脑炎病毒(如乙型脑炎病毒)、腮腺炎病毒、风疹病毒、破伤风杆菌、百日咳杆菌、白喉杆菌、麻风杆菌、结核杆菌、脑膜炎双球菌、肺炎双球菌及其任意组合。
在另一个方面，本发明提供一种制造免疫增强组合物的方法，其包括
1)提供能够形成新生沉淀锰和/或胶体锰的源；和
2)任选地使所述能够形成新生沉淀锰和/或胶体锰的源转化为新生沉淀锰和/或胶体锰。
优选地，所述新生沉淀锰和/或胶体锰选自磷酸锰盐、碳酸锰盐和氢氧化锰及其任意混合物。
优选地，所述新生沉淀锰从非沉淀形式转化成沉淀形式的时间不超过1天，或者不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，或者不超过60、50、45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
在另一个方面，本发明提供一种制造疫苗组合物的方法，其包括
1)提供疫苗免疫原；和
2)提供新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。
优选地，所述疫苗免疫原源自病毒、细菌和/或寄生虫，同上所述，在此不再赘述。
在一个方面，本发明提供了新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和或胶体锰的源在制备用于改善固有免疫和/或适应性免疫的免疫增强组合物或疫苗组合物中的用途。在一些实施方案中，所述新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和或胶体锰的源通过增加I型干扰素表达改善固有免疫和/或适应性免疫。在另一些实施方案中，所述新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和或胶体锰的源通过诱导炎症因子切割产生活性形式改善固有免疫和/或适应性免疫。在又一些实施方案中，所述新生沉淀锰、胶体锰和/ 或能够形成新生沉淀锰和或胶体锰的源通过促进抗体产生来改善固有免疫和/或适应性免疫。
在一个方面，本发明提供一种增强免疫的方法，其包括向有此需要的对象施用所述免疫增强组合物。
具体地，所述免疫增强例如是A)改善固有免疫和/或适应性免疫，B)增加I型干扰素表达，C)诱导产生活性形式的炎症因子，D)促进抗体产生，E)促进T细胞增殖，和/或F)促进树突细胞成熟。
优选地，所述施用选自肌肉注射、皮内注射、皮下注射、静脉注射、粘膜施用及其任意组合。
特别地，所述增强免疫是用于预防和/或治疗疾病，例如细菌感染、真菌感染、病毒感染、寄生虫感染、肿瘤、自身免疫病。
其中所述病毒选自：DNA病毒和RNA病毒，优选地所述病毒选自：疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、冠状病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，具体地所述病毒选自：单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV、流感病毒、肝炎病毒(如甲肝病毒、乙肝病毒)、脊髓灰质炎病毒、狂犬病毒、HPV病毒、脑炎病毒(如乙型脑炎病毒)、腮腺炎病毒、风疹病毒及其任意组合。
其中所述细菌选自革兰氏阴性菌和革兰氏阳性菌，优选地所述细菌选自肺炎链球菌(Streptococcus pneumoniae)、流感嗜血杆菌(Haemophilus influenzae)、沙门氏菌(Salmonella)、脑膜炎双球菌(Meningococcus)、表皮葡萄球菌(Staphylococcus epidermidis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、产酸克雷伯氏菌(Klebsiella oxytoca)、阴沟肠杆菌(Enterobacter cloacae)、弗氏柠檬酸杆菌(Citrobacter freundii)、绿脓假单胞菌(Pseudomonas aeruginosa)、波美不动杆菌(Acinetobacter baumanni)、破伤风杆菌、百日咳杆菌、白喉杆菌、麻风杆菌、结核杆菌、脑膜炎双球菌、肺炎双球菌及其任意组合。
其中所述寄生虫是细胞内寄生虫，优选地选自疟原虫、弓形虫、锥虫、血吸虫、丝虫和利什曼原虫。
其中所述自身免疫性疾病选自I型糖尿病、银屑病、类风湿性关节炎、系统性红斑狼疮和多发性硬化。
其中所述肿瘤选自卵巢癌、肺癌、胃癌、乳腺癌、肝癌、胰腺癌、皮肤癌、恶性黑色素瘤、头颈癌、肉瘤、胆管癌、膀胱癌、肾癌、结肠癌、胎盘绒毛膜癌、子宫颈癌、睾丸癌、子宫癌和白血病。
在一些实施方案中，所述免疫增强组合物与另外的预防/治疗剂一起施用于有此需要的对象。
在另一方面，本发明提供一种免疫接种方法，其包括
向有此需要的对象施用本发明的疫苗组合物，其中当组分A和组分B处于不同容器中时可以同时或不同时施用；和/或
向有此需要的对象施用本发明的免疫增强组合物和任选地疫苗免疫原。
优选地，所述施用选自肌肉注射、皮内注射、皮下注射、静脉注射、粘膜施用及其任意组合。
特别地，所述免疫接种用于预防疾病，例如细菌感染、真菌感染、病毒感染、寄生虫感染、肿瘤和自身免疫病。所述疾病同上所述，在此不再赘述。
在一些实施方案中，所述免疫增强组合物与另外的预防/治疗剂一起施用于有此需要的对象。
在又一个方面，本发明提供了新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源在制备用于增强I型干扰素的药物中的用途。
在又一个方面，本发明提供了新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源在制备用于诱导炎症因子切割产生活性形式的药物中的用途。
在又一个方面，本发明提供了新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源在制备用于刺激抗体产生的药物中的用途。
在又一个方面，本发明提供了新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源作为免疫佐剂的用途。在一些实施方案中，所述免疫佐剂激活T细胞活化和/或抗体生产。优选地，所述免疫佐剂用于治疗选自细菌感染、病毒感染、寄生虫、自身免疫性疾病和癌症之疾病的疫苗组合物中。
在又一个方面，本发明提供了一种用于免疫接种的试剂盒，其包含：第一容器，其中容纳一种或更多种抗原；和第二容器，其中容纳新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。优选地，所述第一容器和/或第二容器还包含药学上可接受的载体。特别地，所述试剂盒用于本发明的一种或更多种目的。在一些实施方案中，抗原与新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源位于同一容器中。
在一些实施方案中，本发明中所使用的抗原选自：病毒或细菌或寄生物抗原，例如，甲型、乙型、丙型、丁型和戊3型肝炎病毒、HIV、疱疹病毒1、2、6和7型、巨细胞病毒、水痘带状疱疹病毒、乳头状瘤病毒、EB病毒、流感病毒、副流感病毒、腺病毒、布尼亚病毒(汉坦病毒)、柯萨奇病毒、小RNA病毒、轮状病毒、呼吸道合胞病毒、痘病毒、鼻病毒、风疹病毒、乳多泡病毒、腮腺炎病毒和麻疹病毒、导致结核和麻风病的分枝杆菌、肺炎球菌、需氧革兰氏阴性杆菌、支原体、葡萄球菌感染、链球菌感染、沙门氏菌和衣原体、幽门螺杆菌、疟疾、利什曼病、锥虫病、弓形虫病、血吸虫病和丝虫病。
在一些实施方案中，本发明中的新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源和/或抗原是有效量的。
在又一个方面，本发明提供了用于在对象中增加I型干扰素表达的方法，其包括向所述对象施用新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。此外，本发明还提供了用于体外增强细胞中I型干扰素活性的方法，其包括向所述细胞施加新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源，优选地，所述方法是非治疗目的的。
在又一个方面，本发明提供了用于在对象中诱导炎症因子切割产生活性形式的方法，其包括向所述对象施用新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。此外，本发明还提供了用于体外诱导细胞中炎症因子切割产生活性形式的方法，其包括向所述细胞施加新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源，优选地，所述方法是非治疗目的的。
在又一个方面，本发明提供了用于在对象中刺激抗体产生的方法，其包括向所述对象施用新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。此外，本发明还提供了用于体外刺激细胞产生抗体的方法，其包括向所述细胞施加新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源，优选地，所述方法是非治疗目的的。
附图说明
下面通过对本发明的详细描述以及附图来清楚地说明本发明前面叙述的方面以及其他方面。为了举例说明本发明，在附图中的实施方案是目前优选的，然而，可以理解，本发明并不限于所公开的特定实施方案。
图1图示了新生沉淀锰和胶体锰(Mn 2OHPO 4)具有良好的佐剂效果。图1A示二价锰在溶液中的不同存在状态；图1B示胶体锰(Mn2OHPO4)的透射电镜图；图1C示免疫后OVA抗体IgG1水平；图1D示MnCl 2溶液与胶体锰(Mn 2OHPO 4)肌肉注射后驻留时间对比。
图2图示了将MnCl 2溶液与其他阴离子在生理盐水中混合后的不同存在状态及性质。且测定这些沉淀或胶体激活I型干扰素或炎症因子产生能力，佐剂效果。图2A示MnCl 2溶液与不同浓度的碳酸根离子、碳酸氢根离子、磷酸根离子、磷酸一氢根离子、氢氧根离子形成不同的状态；图2B示不同状态的锰离子激活I型干扰素、诱导炎症因子IL-1β产生、及作为佐剂混合OVA蛋白免疫产生抗体的能力；图2C示激光笔照射锰离子与不同阴离子形成的产物，观察丁达尔效应。
图3图示了胶体锰(Mn 2OHPO 4)和铝佐剂分别混合鸡卵清蛋白(OVA)通过肌注免疫小鼠10天(A)、17天(B)、24天(C)和31天(D)后产生抗OVA的抗体量。
图4图示了胶体锰(Mn 2OHPO 4)和霍乱毒素B(CTB)佐剂分别混合OVA通过滴鼻免疫小鼠后肺泡灌洗液(A)、口腔灌洗液(B)、血清IgA(C)和血清IgG1(D)抗OVA的抗体量。
图5图示了按照图1和图2的方法用胶体锰(Mn 2OHPO 4)作为佐剂混合OVA肌注(A)或滴鼻免疫(B)小鼠后测定1至6个月小鼠血清、口腔灌洗液、肺泡灌洗液中的抗OVA的抗体量。
图6图示了MnCl 2激活I型干扰素通路和炎症小体活化。图6A-B示MnCl 2处理小鼠腹腔巨噬细胞后，I型干扰素、下游诱导因子ISG54和viperin的表达水平，铝佐剂作为对照。图6C-D示MnCl 2可以激活炎症小体活化，铝佐剂作为对照。图6E-F示MnCl 2激活的炎症小体活化依赖于NLRP3和ASC。
图7图示了胶体锰(Mn 2OHPO 4)促进CD4 +T细胞增殖，铝佐剂作为对照。
图8图示了胶体锰(Mn 2OHPO 4)肌注和粘膜免疫时小鼠产生抗体量依赖于cGAS-STING通路和炎症小体通路。
图9图示了胶体锰(Mn 2OHPO 4)促进树突状细胞成熟、T细胞活化的能力依赖于cGAS-STING通路和炎症小体通路。图9A示胶体锰(MnOHPO 4)诱导抗原递呈细胞BMDC的成熟。图9B示胶体锰(MnOHPO 4)加抗原OVA刺激CD4 +T细胞的增殖能力。图9C示胶体锰(MnOHPO 4)作为佐剂免疫小鼠后刺激CD8 +T细胞活化。
图10图示了胶体锰(Mn 2OHPO 4)作为佐剂能显著增强灭活水疱性口炎病毒(VSV)和单纯疱疹病毒(HSV-1)的免疫保护效果。图10A示实验流程。图10B示将灭活VSV病毒稀释成10 -1、10 -2、10 -3、10 -4、10 -5、10 -6的剂量免疫小鼠，在观察14天后小鼠的存活率。图10C显示用稀释成10 -3的剂量的VSV灭活病毒免疫小鼠后，观察14天中小鼠的死亡曲线。图10D显示感染VSV病毒第4天时小鼠大脑中的病毒滴度。图10E-G显示在灭活HSV-1病毒中加入胶体锰也有同样的保护效果。
图11图示了胶体锰(Mn 2OHPO 4)作为佐剂能显著增强灭活流感病毒PR8的免疫保护效果。图11A-B显示将灭活流感病毒稀释成1、10 -1、10 -2、10 -3的剂量滴鼻免疫小鼠一次，7天后用流感病毒感染小鼠后小鼠的存活率及体重变化。图11C-D显示滴鼻免疫小鼠两次后用流感病毒感染小鼠的存活率及体重变化。
图12图示了胶体锰(Mn 2OHPO 4)作为佐剂能显著增强流感亚单位HA疫苗的免疫保护效果。图12A显示肌注小鼠后14天、21天、28天时小鼠血清中抗HA蛋白IgG1抗体含量。图12B显示滴鼻免疫小鼠后14天、21天、28天时小鼠血清中抗HA蛋白IgA抗体含量。图12C显示感染流感病毒后小鼠的体重变化。图12D显示感染第5天时小鼠肺部组织的病变情况。
图13图示了胶体锰Mn 2OHPO 4作为佐剂增强灭活流感病毒及亚单位疫苗对于同源异型流感病毒(WSN)及异源流感病毒(H3N2)的保护效果。图13A显示免疫后WSN病毒感染小鼠的体重变化；图13B显示免疫后H3N2病毒感染小鼠的体重变化。
图14图示了胶体锰(Mn 2OHPO 4)能显著抑制原位瘤和转移瘤的生长。图 14A显示用成像仪在不同时间拍摄皮下肿瘤大小。图14B显示接种肿瘤后肿瘤体积变化。图14C显示接种肿瘤后小鼠生存曲线。图14D显示不同免疫方法21天后肿瘤转移肺组织拍照。图14E显示为图14D中肿瘤细胞的数量统计。
图15图示了胶体锰(Mn 2OHPO 4)与化疗药环磷酰胺(CTX)联合治疗皮下肿瘤模型-黑色素瘤B16-F10效果。图15A显示不同治疗方法14天后肿瘤图片。图15B显示小鼠皮下肿瘤B16F10的生长曲线。图15C图显示15B图中的小鼠第14天对应组的肿瘤重量。
图16图示了胶体锰(Mn 2OHPO 4)与PD1抗体药联合治疗皮下肿瘤模型-黑色素瘤B16-F10效果。图16A显示不同治疗方法后肿瘤图片。图16B显示肿瘤的生长曲线。图16C图显示B图中的小鼠对应组的肿瘤重量。图16D-E图显示肿瘤中用流式细胞仪分析肿瘤中浸润的CD8 +T细胞数量。
图17图示了胶体锰(Mn 2OHPO 4)与PD1抗体药联合治疗皮下肿瘤模型-结肠癌肿瘤MC38效果。图17A显示不同治疗方法后肿瘤图片。图17B显示肿瘤的生长曲线。图17C显示17B图中对应组的肿瘤重量。图17D显示A图中对应组肿瘤的组织切片，做了DAPI和CD8 +T细胞的免疫荧光染色。
具体实施方式
定义
本文中所使用的术语“固有免疫”是指机体在种系发育和进化过程中形成的天然免疫防御功能，即出生后就已具备的非特异性防御功能，也称为非特异性免疫(non-specific immunity)。固有免疫涉及多种细胞和分子，例如巨噬细胞、自然杀伤细胞、补体、细胞因子(IL、CSF、IFN、TNF、TGF-β)、趋化因子(包括CC趋化因子，例如CCL1、CCL2、CCL3、CCL4、CCL5、CCL6、CCL7、CCL8、CCL9、CCL10、CCL11、CCL12等，CXC趋化因子，例如CXCL1、CXCL2、CXCL3、CXCL4、CXCL5、CXCL6、CXCL7、CXCL8、CXCL9等，C趋化因子，CX3C趋化因子)、溶菌酶等等。
本文中所使用的术语“适应性免疫”又称获得性免疫或特异性免疫，是指机体在抗原分子刺激之后形成的针对该抗原的特异性免疫，其涉及细胞免疫和体液免疫。
本文中所使用的术语“佐剂”是指并不构成特异性抗原，但是增强对共同给药的抗原的免疫应答的强度和持续时间的试剂。
本文中所使用的术语“二价锰”或“二价锰化合物”，可以是盐酸盐、碳酸盐、氢溴酸盐、硫酸盐、硝酸盐、磷酸盐、酒石酸盐、富马酸盐、马来酸盐、乳酸盐、苯磺酸盐、泛酸盐、抗坏血酸盐、氢氧化物等，或者其任意组合。优选地，所述二价锰化合物是药学上可接受的。
本文中所使用的术语“胶体锰”，是指二价锰以与阴离子形成的胶体(colloid，亦称胶体溶液)形式存在的状态，术语“胶体”具有本领域普通技术人员所理解的含义。例如，胶体颗粒的粒径一般为约1至100nm，尤其是1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、60、70、80、90、100nm，或者以上任意两数值之间的值，例如1至20、5至20nm、10nm。典型地，胶体溶液在光照时表现出丁达尔效应。考虑到胶体颗粒的粒径范围在纳米量级，本文中“胶体锰”也可根据其粒径范围而称为“纳米锰”。
本文所用术语“新生沉淀锰”，是指二价锰化合物从非沉淀形式(例如溶液形式)转化为沉淀形式后一个短时间期间的存在状态。所述短时间期间一般不超过1天，典型地不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，例如不超过60、50、45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
本文使用的术语例如“包含”、“含”、“含有”和“包括”不意在限制。此外，除非另有说明，“或”、“或者”意指“和/或”。
本文所述“源自病毒、细菌和/或寄生虫”，意指包括灭活病毒、细菌和/或寄生虫，或从上述致病物中提取的蛋白和/或核酸类物质(亦包括切割、工程化等处理后的上述物质)，或纯化的重组蛋白质类物质，或者化学合成的肽段类物质。
另外应注意，如本说明书中所使用的，单数形式包括其所指对象的复数形式，除非清楚且明确的限于一个所指对象。而且如果提到一个特定的数值，至少会包括该数值，除非文章清楚的表明了其另有所指。
当数值表示近似值，应理解为特定的数值形成了另一个实施方案。就如所使用的，“约X”(其中X是一个数值)是指±10％(包含)所列出值。如果存在，所有范围都是包括和可以组合的。
本文使用的术语“药学上可接受的载体”可选自：水、缓冲水溶液、、等渗盐溶液如PBS(磷酸盐缓冲液)、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇或聚亚烷基二醇如聚丙二醇、甘油三酯等。所用可药用载体的类型尤其依赖于根据本发明的组合物是否配制为用于经口、鼻、皮内、皮下、肌内或静脉施用。根据本发明的组合物可包含润滑剂、防腐剂、稳定剂、湿润剂、乳化剂、影响渗透压的盐、缓冲剂、着色物质、矫味物质和/或芳香物质等作为添加剂。
根据本发明的药物组合物可通过任何适宜的途径施用，例如可经口、鼻、皮内、皮下、肌内或静脉内施用。
本文使用的术语“施用”意指以在药理学上可用的方式向对象提供物质。
本文使用的“药物有效量”、“有效量”是指足以显示其对于所施用对象益处的剂量。施用的实际量，以及施用的速率和时间过程会取决于所治疗者的自身情况和严重程度。治疗的处方(例如对剂量的决定等)最终是全科医生及其它医生的责任并依赖其做决定，通常考虑所治疗的疾病、患者个体的情况、递送部位、施用方法以及对于医生来说已知的其它因素。
本文所使用的术语“对象”意指动物，包括温血哺乳动物，例如人和灵长类动物；鸟类；驯养的家养或农场动物，例如猫、狗、绵羊、山羊、牛、马和猪；实验室动物，例如小鼠、大鼠和豚鼠；鱼；爬行动物；动物园动物和野生动物等。
除非另有定义，本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。
除非另有说明，任何关于本方法和产品一种实施方案公开的组分、元素、属性或者步骤可以应用于任何其他在此公开的方法和产品。
本公开的每项专利、专利申请、引用的出版物或者本文件中的描述以参考的方式整体并入文本中。
本发明在下面的实施例中进一步的定义。应了解这些实施例仅仅以举例的方式来说明，并不旨在限制本发明的范围。从上面的讨论以及这些例子中，本领域技术人员可以确定本发明的本质特征，而且在不脱离其本质和范围的情况下，能够对本发明做出各方面的改变和修改来使之适应各种各样的用法和条件。
实施例
材料和方法
抗体和试剂
抗体来源如下：抗GAPDH抗体(sc-25778)购自Santa Cruz。抗蝰蛇毒素(Viperin)抗体、抗ISG54抗体、抗Casp1/p20抗体、抗IL1β/p17抗体和抗ASC抗体均通过已公开方法制得和使用(16)。简言之，抗原片段的cDNA插入到pET-21b载体(Novagen)并表达于大肠杆菌E.coli BL21(DE3)，通过Ni-NTA亲和柱纯化重组蛋白，然后将其注射进小鼠或兔子，获得能够识别对应抗原的抗血清。
所有化学品购自Sigma-Aldrich(St.Louis,MO)，另有说明除外。铝佐剂(Imject Alum,Thermo,77161)、小鼠IL-1beta ELISA Kit(MULTI SCIENCES,EK201B2/2)、小鼠IL-18 ELISA Kit(MULTI SCIENCES,EK2181)、脂多糖(Sigma,L4130)、卵清蛋白(InvivoGen,#vac-pova)均为商业化产品。
细胞
L929-ISRE(蒋争凡实验室自制细胞系，将pGL3 ISRE-Luciferase质粒稳定转染至L929细胞，
Figure PCTCN2020085507-appb-000001
CCL-1)、BHK21(
Figure PCTCN2020085507-appb-000002
CCL-10)、B16-OVA(
Figure PCTCN2020085507-appb-000003
CRL-6322)和B16F10(
Figure PCTCN2020085507-appb-000004
CRL-6475)细胞，培养于添加有10％FBS(Gibco)、5μg/ml青霉素和10μg/ml链霉素的DMEM(Gibco)培养基中。骨髓来源的树突细胞(bone marrow derived macrophages,BMDCs)在含有10ng/mL GM-CSF、10ng/mL IL-4、10％FBS(Gibco)的RPMI-1640(Gibco)培养基中诱导，第3天半量换液，第7天进行实验。腹膜巨噬细胞收获自巯基乙醇酸盐(BD,Sparks,MD)注射诱导后5天的小鼠，培养于添加有5％FBS的DMEM培养基中。
小鼠
Tmem173 -/-小鼠是用CRISPR-cas9方法，通过将Cas9 mRNA(100ng/μl)和gRNA(50ng/μl)胞质注射到C57BL/6J小鼠受精卵而建立。通过mMESSAGE mMACHINE T7 Ultra(Ambion,am1345)体外转录Cas9 mRNA和单引导(single guide)RNA(gRNA)。Nlrp3 -/-、Nlrc4 -/-、Pycard -/-和Aim2 -/- 基因敲除小鼠由Vishva Dixit(Genentech Inc,USA)惠赠。Tmem173 -/-Pycard -/-双敲除小鼠通过将Tmem173 -/-小鼠和Pycard -/-交配后鉴定获得。
所有小鼠均按照NIH实验动物管理使用指南(National Institute of Health Guide for Care and Use of Laboratory Animals)在无菌条件下喂养于北京大学实验动物中心。
I型IFN(Type I-IFN)生物测定
按照公开方法测定I型IFN的浓度(17)。简言之，通过将IFN-刺激应答元件(IFN-stimulated response element，ISRE)克隆进pGL3-Basic载体(Promega)来构建IFN敏感萤光素酶载体，然后将其稳定转染进L929细胞。将L929-ISRE细胞接种到96孔板，分别与细胞培养上清一起孵育。使用重组人和小鼠IFN-β(R&D Systems)作为标准品。4小时后，裂解L929-ISRE细胞，通过Luciferase Reporter Assay System(Promega)进行测定。
病毒感染
单纯疱疹病毒1(HSV-1,来自Hongbing Shu,Wuhan University，
Figure PCTCN2020085507-appb-000005
VR-1544)和水疱性口炎病毒(VSV,Indiana strain，
Figure PCTCN2020085507-appb-000006
VR-1238)由上述同仁惠赠。H1N1流感病毒PR8株(来自Yonghui Zhang,Tsinghua University，
Figure PCTCN2020085507-appb-000007
VR-95)。H1N1流感病毒WSN株，A/WSN/33(H1N1)(来自Wenjun Liu,Institute of Microbiology,CAS)。H3N2流感病毒株，H3N2 subtype A/Jiangxi/2005(来自Min Fang,Institute of Microbiology,CAS)。使用BHK21细胞测定HSV-1和VSV病毒滴度。使用MDCK细胞测定流感病毒滴度。
病毒扩增和纯化：HSV-1、VSV病毒用vero细胞扩增。PR8、WSN、H3N2流感病毒用鸡胚扩增。扩增出来的病毒用PEG8000纯化，纯化的病毒用0.2％的甲醛于37摄氏度灭活24小时，用空斑法测量病毒是否被完全灭活，然后将灭活病毒用于免疫小鼠。
小鼠存活实验：用HSV-1(1.4 x10 7pfu/只小鼠)和VSV(8 x 10 8pfu/只小鼠)静脉内感染8-12周小鼠，或者用PR8(1 x 10 5pfu/只小鼠)、WSN(1 x 10 6pfu/只小鼠)、H3N2(1 x 10 6pfu/只小鼠)鼻内感染8-12周小鼠。
空斑测定：将BHK21细胞与来自被感染小鼠器官的匀浆(在无血清DMEM中的一系列稀释液)一起孵育2小时。然后，用无血清DMEM含有0.5％甲基纤维素替换培养基。60小时后，用0.5％(vol/vol)戊二醛固定细胞， 用1％(wt/vol)结晶紫(溶解于70％乙醇)染色。对空斑进行计数，从而计算以空斑形成单位计的病毒滴度。
蛋白质表达和纯化
流感病毒HA1蛋白纯化质粒由清华大学张永辉实验室馈赠(18)。质粒表达HA蛋白(Gene ID:956529)的11-324氨基酸，当菌液摇至OD 600为0.6时，加入0.5mM IPTG诱导，置于37℃培养5小时。收菌后，超声收集包涵体，经过变性复性得到HA1蛋白。再将HA1蛋白用凝胶过滤层析的方法纯化。
卵清蛋白(Ovalbumin,OVA)作为免疫原免疫小鼠
MnCl 2溶液肌注免疫方法是100μL PBS中加入10μg MnCl 2和10μg OVA蛋白，混匀放置5分钟后肌注免疫小鼠。MnCl 2溶液滴鼻免疫方法是20μL PBS中加入5μg MnCl 2和10μg OVA蛋白，混匀放置5分钟后滴鼻免疫小鼠。胶体锰(以Mn 2OHPO 4为例)肌注免疫方法是100μL生理盐水中加入10μg胶体锰和10μg OVA蛋白，混匀后肌注免疫小鼠。胶体锰滴鼻免疫方法是20μL生理盐水中加入5μg胶体锰和10μg OVA蛋白，混匀后滴鼻免疫小鼠。铝佐剂肌注免疫方法是80μL生理盐水中加入10μg/20μL铝佐剂和10μg OVA蛋白，用乳化仪乳化后肌注免疫小鼠。霍乱毒素B(CTB)滴鼻免疫方法是20μL生理盐水中加入5μg CTB和10μg OVA蛋白，混匀后滴鼻免疫小鼠。
ELISA方法测定OVA特异性IgG1、IgG2c、总IgG、IgA抗体：将来自免疫小鼠的稀释血清孵育在涂覆了100μg/ml OVA的ELISA板上。清洗后，使用HRP-缀合的抗体小鼠IgG1(eBioscience,#18-4015-82)、IgG2c(GeneTex,GTX77297)、IgG total(Invitrogen,G21040)、IgA(GeneTex,GTX77223)检测结合的对应抗体。然后，将板与底物TMB(eBioscience)一起孵育，用1M H 3PO 4停止反应，然后测定450nm吸收波长的吸光度。
统计分析
使用t检验分析数据。使用Mantel-Cox检验比较存活曲线。
实施例1.锰化合物在溶液中的状态及其作为佐剂的效果
实验(a)MnCl 2与PBS反应产生沉淀，如下所述制备胶体锰Mn 2OHPO 4
如图1A所示，实验中生理盐水(0.9％NaCl)自己配制，PBS(pH 7.4), 购买自Gibco。共有3组反应体系，分别是：100μl MnCl 2(0.2M)加入900μl生理盐水；100μl MnCl 2(0.2M)加入900μl PBS；50μl Na 3PO 4加入850μl生理盐水，再加入100μl MnCl 2(0.2M)制成胶体锰Mn 2OHPO 4。该胶体锰的分子式是通过X射线光电子能谱分析X-ray photoelectron spectroscopy(XPS)测定。MnCl 2在生理盐水中没有沉淀产生，在PBS中反应过夜后有白色沉淀，第三个反应生成的胶体不会沉淀。光照第三个反应产物有丁达尔效应。用透射电镜拍摄胶体锰的精细结构，如图1B所示，是直径约10nm左右的颗粒。
实验(b)比较PBS中锰化合物沉淀和胶体锰的佐剂效果
小鼠按体重随机分为6组，分别是：1)PBS对照组，2)抗原OVA(10μg)，3)MnCl 2在生理盐水中反应2天组(10μg MnCl 2+OVA)，4)MnCl 2在PBS中反应5分钟组(10μg MnCl 2+OVA)，5)MnCl 2在PBS中反应2天组(10μg MnCl 2+OVA)，6)MnCl 2反应生成胶体锰30天组(10μg Mn 2OHPO 4+OVA)。这里的10μg均指锰元素的质量。各组按实验设计量进行肌注免疫，在第0天，7天，14天各免疫一次。在第21天取血，分离血清，用ELISA方法测抗OVA的IgG1抗体含量。如图1C所示，MnCl 2在PBS中反应短时间的颗粒具有佐剂效果，但是反应长时间后，由于聚集成大颗粒效果减弱，而胶体锰(图中缩写为MnJ)Mn 2OHPO 4放置一个月效果仍然很好。这表明胶体锰Mn 2OHPO 4是一种效果好且稳定的佐剂。
实验(c)比较MnCl 2溶液和胶体锰Mn 2OHPO 4在注射部位的停留时间
小鼠按体重随机分为2组，分别是：肌肉注射MnCl 2溶液组(20μg MnCl 2溶于100μl生理盐水)，肌肉注射胶体锰组(20μg胶体锰悬于100μl生理盐水)，这里的20μg均指锰元素的质量。在注射后不同时间，取注射部位肌肉0.1g，用微波消解仪消解，然后用ICP-MS(Thermo X SERIES II)测定锰元素的含量。如图1D所示，胶体锰能停留在注射部位更久，可以更加持久地激活机体产生抗体，而溶液锰很快就代谢掉。
实施例2.锰离子(II)与其他阴离子反应生成的沉淀性质与细胞效应
实验(a)固定浓度锰离子(II)与不同浓度的阴离子反应
在生理盐水反应体系中，用20mM的MnCl 2与其他不同浓度(2.5,5,10,20,30,40mM)的来自钠盐的阴离子(Na 2CO 3,NaHCO 3,Na 3PO 4,Na 2HPO 4, NaOH)混合。如图2A所示，二价游离锰与CO 3 2-反应生成黑色沉淀状MnCO 3；与HCO 3 -反应生成白色沉淀状Mn(HCO 3) 2；与PO 4 3-反应生成两种状态的产物，低浓度时生成白色沉淀状物质，高浓度时生成微黄色胶体物质。经测定，低浓度时生成的是Mn 3(PO 4) 2,高浓度时生成的是Mn 2OHPO 4；二价游离锰与HPO 4 2-反应生成白色MnHPO 4；与OH -反应生成微黄色沉淀状Mn(OH) 2.
实验(b)比较不同锰离子(II)产物激活I型干扰素和炎症因子切割产生IL-1β的能力及佐剂效果
如图2B所示，按从左向右的顺序，在Eppendorf管中用100μL 0.2M的MnCl 2加900μL生理盐水放置过夜(MnCl 2)，加100μL 0.5M NaHCO 3+800μL生理盐水放置过夜(Mn(HCO 3) 2)，加100μL 0.5M Na 2CO 3+800μL生理盐水放置过夜(MnCO 3)，加20μL 0.5M Na 2CO 3+20μL 0.5M NaOH+860μL生理盐水放置过夜(Mn 2OHCO 3)，加100μL 0.5M NaH 2PO 4+800μL生理盐水放置过夜(Mn(H 2PO 4) 2)，加100μL 0.5M Na 2HPO 4+800μL生理盐水放置过夜(MnHPO 4)，加26.6μL 0.5M Na 3PO 4+873.4μL生理盐水放置过夜(Mn 3(PO 4) 2)，加50μL 0.5M Na 3PO 4+850μL生理盐水放置过夜(Mn 2(OH)PO 4)，以及加100μL 0.5M NaOH+800μL生理盐水放置过夜(Mn(OH) 2)，反应生成如图所示的胶体或固体沉淀。用生理盐水将沉淀洗一遍后用于后续实验(MnCl 2没有洗，因为在生理盐水中没有沉淀，使用溶液进行实验)。使用等摩尔量(含有500μM的Mn 2+)的各种产物(Mn(H 2PO 4) 2产物易溶于水，沉淀少，不足500μM，其他阴离子都能使Mn 2+完全沉淀)刺激2×10 6THP1细胞18小时，测量上清中I型干扰素的水平。结果如图2B所示，Mn 2OHPO 4和MnHPO 4能显著激活I型干扰素产生，图中所示P-IRF3是调控I型干扰素表达的主要转录因子。Mn 2OHPO 4和MnHPO 4还能显著诱导症因子IL-1β的产生。而非胶体的Mn(OH) 2只能激活I型干扰素产生，不能诱导炎症因子IL-1β的产生。每次用10μg各种产物(10μg指锰元素的质量)分别混合10μg OVA蛋白肌肉免疫小鼠3次后，取小鼠血清测量其中抗OVA的IgG1抗体含量。Mn 2OHPO 4和MnHPO 4刺激机体产生抗体的效果明显好于沉淀状态的锰化合物。抗体试验中最左侧的MnCl 2为MnCl 2与PBS反应5分钟的沉淀的佐剂效果。其他的放置过夜固体沉淀也有部分佐剂效果。我们选取效果最好的胶体锰Mn 2OHPO 4进行后续的实验证明。
实验(c)比较不同锰离子(II)产物的丁达尔效应
如图2C所示，将反应生成物重悬在生理盐水中，然后用激光笔照射。可以看到Mn 2OHPO 4的丁达尔效应最明显，而MnHPO 4有部分丁达尔效应，可能因为其直径稍大于100nm。而Mn 2OHPO 4的直径约10nm，符合胶体的定义。
实施例3.胶体锰Mn 2OHPO 4作为肌注和粘膜免疫佐剂
实验(a)胶体锰Mn 2OHPO 4作为肌注佐剂
小鼠按体重随机分为6组，分别是：PBS对照组，抗原OVA(10μg)，胶体锰Mn 2OHPO 4组(10μg Mn 2OHPO 4+OVA)，低剂量铝佐剂组(440μg铝佐剂+OVA)，中剂量铝佐剂组(800μg铝佐剂+OVA)和高剂量铝佐剂组(1220μg铝佐剂+OVA)。各组按实验设计量进行肌注免疫，在第0天和第10天各免疫一次。在第10天、第17天、第24天和第31天取血，分离血清，用ELISA方法测抗OVA的IgG1抗体含量。如图3所示，胶体锰Mn 2OHPO 4在10μg/小鼠的剂量下，效果强于高剂量(1220μg/小鼠)的铝佐剂。这表明胶体锰可以在低于铝佐剂100倍的剂量下达到更好的增强免疫的效果，这可以极大降低目前广泛使用的铝佐剂的副作用。
实验(b)胶体锰Mn 2OHPO 4作为粘膜佐剂
如图4所示，用等量(5μg/小鼠)胶体锰Mn 2OHPO 4与经典粘膜免疫佐剂霍乱毒素B(CTB)分别混合抗原OVA(10μg)，滴鼻免疫小鼠。在第0天、第7天和第14天各免疫一次，第21天时取血分离血清，收集口腔灌洗液、肺泡灌洗液，用ELISA方法测抗OVA的IgA抗体和IgG1抗体含量。如图4所示，胶体锰Mn 2OHPO 4作为粘膜佐剂免疫小鼠时，在肺泡灌洗液中的抗体含量高于霍乱毒素B作为佐剂。在口腔灌洗液和血清中，Mn 2OHPO 4与等量霍乱毒素B效果相当。这表明胶体锰Mn 2OHPO 4也是一种很好的粘膜免疫佐剂。
实施例4.胶体锰Mn 2OHPO 4作为肌注和粘膜免疫佐剂可使机体持久产生特异性抗体
实验(a)胶体锰Mn 2OHPO 4作为肌注佐剂效果持久
采用参见实施例3中的方法，用胶体锰Mn 2OHPO 4作为佐剂混合OVA肌 注免疫小鼠，测定免疫后1周、1月、3月、6月小鼠血清中抗OVA的抗体含量。如图5A所示，至第6个月，血清中抗OVA抗体仍维持在较高水平，这表明胶体锰Mn 2OHPO 4作为佐剂可以产生持久的保护效果。小鼠寿命一般2年，预计胶体锰Mn 2OHPO 4可以使机体整个生命周期中产生保护性抗体。
实验(b)胶体锰Mn 2OHPO 4作为粘膜佐剂效果持久
参见实施例3中的方法粘膜免疫小鼠后，在不同时间收取小鼠血液、肺泡灌洗液、口腔灌洗液测定抗体含量。如图5B所示，直至免疫后6个月，特异性抗体浓度依然维持在较高水平，这表明胶体锰Mn 2OHPO 4用于粘膜免疫时也能产生持久的保护效果。
实施例5.锰离子(II)激活炎症因子产生
炎症因子IL-1β能直接作用于CD4 +T细胞，促进T细胞的增殖(8)，IL-1α和IL-1β能促进嗜中性粒细胞的浸润(19)；炎症因子IL-18可以有效增强Th1免疫反应，也可以促进T细胞和NK细胞的增殖和细胞毒作用(9)。有报道称铝佐剂的佐剂效果是通过激活NLRP3炎症小体，释放炎症因子实现的(20-22)，所以我们检测并比较MnCl 2/胶体锰Mn 2OHPO 4与铝佐剂激活炎症因子的能力。
MnCl 2和胶体锰Mn 2OHPO 4具有相同的激活炎症因子的能力(见图2B)和机制，这里只用MnCl 2作示例。分别用10、20、50、100μg/mL的氯化锰或者铝佐剂处理体外培养的小鼠腹腔巨噬细胞原代培养，用免疫印迹检测干扰素诱导基因ISG54和Viperin，用Bioassay检测上清中总的I型干扰素含量(干扰素α和干扰素β的浓度总和)。结果如图6A和6B所示，只有MnCl 2能激活I型干扰素的表达，铝佐剂则不能。用同样浓度的氯化锰和铝佐剂处理LPS预处理的腹腔巨噬细胞，用免疫印迹检测上清中caspase1的切割和IL-1β的切割(上面的条带是前体，下面的条带是活性形式的caspase1和IL-1β)，用ELISA检测上清中炎症因子IL-1β和IL-18的产生。结果如图6C和6D所示，MnCl 2激活炎症因子的能力显著强于铝佐剂。用MnCl 2、ATP(经典NLRP3炎症小体激活剂)、VacV(经典Aim2炎症小体激活剂)处理LPS预处理的腹腔巨噬细胞，用免疫印迹检测上清中caspase1的切割和IL-1β的切割(上面的条带是前体，下面的条带是活性形式的caspase1和IL-1β)，用ELISA检测上清中炎症因子IL-1β和IL-18的产生。结果如图6E和6F所示，在NLRP3敲除的细 胞中，MnCl 2激活炎症因子的能力显著削弱，因此MnCl 2激活的主要是NLRP3炎症小体。
实施例6.胶体锰Mn 2OHPO 4促进T细胞增殖
将CFSE染料标记的1×10 6OT-II细胞尾静脉注射入小鼠，一天后用OVA蛋白进行免疫，分别为：PBS对照组，不同量的OVA对照组(1μg、2μg和5μg)，10μg胶体锰分别与1μg、2μg和5μg OVA蛋白混合组，以及1220μg铝佐剂分别与1μg、2μg和5μg OVA蛋白混合组，此处描述的使用量均为一只小鼠一次免疫的量。免疫小鼠三天后取淋巴结，用流式细胞仪分析淋巴结中OT-II细胞的增殖情况。
如图7所示，图中最右侧的峰是原始注射到小鼠体中的OT-II细胞。当细胞分裂后，细胞中的CFSE会平均分配到两个细胞中，所以细胞中的CFSE亮度会下降，左侧CFSE下降的细胞都是分裂的细胞。结果显示在使用相同量抗原的情况下，胶体锰促进T细胞增殖的能力强于铝佐剂，胶体锰的量只需要铝佐剂的1/100即可达到类似效果。
实施例7.胶体锰Mn 2OHPO 4激活抗体产生依赖于STING通路和炎症小体通路
如图8所示，图8A为用10μg/只的胶体锰加10μg/只的OVA蛋白肌肉注射WT、Pycard敲除(ASC蛋白缺失)、Sting敲除(STING蛋白缺失)、Pycard和Sting双敲小鼠三针后，取小鼠血清用ELISA测量血清中抗OVA抗体的IgG1、IgG2c、总IgG的含量。结果显示单独敲除只会使胶体锰的佐剂效果部分丧失，而双敲会使佐剂效果显著降低。图8B为用5μg/只的胶体锰加10μg/只的OVA蛋白粘膜免疫小鼠三次后取血清、肺泡灌洗液、口腔灌洗液用ELISA测量分泌型抗OVA的IgA抗体含量。结果显示在双敲的小鼠中佐剂效果显著降低。
实施例8.胶体锰Mn 2OHPO 4促进树突状细胞成熟
如图9所示，图9A为使用100、200、400μM的胶体锰处理骨髓来源树 突状细胞(BMDC)细胞20小时，用流式细胞仪检测树突状细胞成熟标志CD86蛋白。结果显示在野生型小鼠中随着浓度增加，越能促进树突状细胞的成熟。而在双敲细胞中则不能促进细胞成熟。图9B中在四种小鼠中采用参见实施例6中的方法，测试胶体锰促进T细胞增殖的效率。结果显示在双敲的小鼠中，胶体锰佐剂促进T细胞增殖的能力明显下降，这也与树突状细胞的成熟是一致的。图9C中使用10μg/只的胶体锰加10μg/只的OVA蛋白肌肉注射野生型小鼠和双敲小鼠，用流式细胞仪分析CD8 +T细胞表面的OVA tetramer蛋白含量(表征佐剂激活的OVA特异性的CD8 +T细胞)。结果表明胶体锰能激活CD8 +T细胞的免疫反应，且依赖于STING和ASC两条通路。
实施例9.胶体锰Mn 2OHPO 4作为佐剂增强灭活水疱性口炎病毒(VSV)和单纯疱疹病毒(HSV-1)的免疫保护效果
实验(a)VSV灭活病毒疫苗
实验流程如图10A所示，第0天时用疫苗肌注小鼠，第10天时用病毒感染小鼠，第14天测定小鼠组织中病毒滴度，观察小鼠并记录死亡曲线至第14天。将经过PEG纯化的VSV病毒灭活后作为疫苗，稀释成10 -1、10 -2、10 -3、10 -4、10 -5和10 -6稀释度肌注免疫小鼠。当病毒浓度足够高时，单独使用就能够产生足够的免疫效果保护小鼠；将病毒10倍稀释几个梯度后免疫小鼠，当浓度为10 -3时，小鼠存活率仅为8.3％，当浓度为10 -4、10 -5、10 -6时，所有小鼠都死亡；但是当在10 -3浓度灭活病毒中加入Mn 2OHPO 4胶体作为佐剂免疫小鼠，小鼠存活率提高到75％，如图10B和10C所示。说明胶体锰作为佐剂可以增强低浓度免疫原的免疫效果，使用少量免疫原就能起到保护作用，且小鼠中的活动病毒滴度也下降了，如图10D所示。
实验(b)HSV-1灭活病毒疫苗
将经过PEG纯化的HSV-1病毒灭活后作为疫苗肌注免疫小鼠，当病毒稀释度为10 -1、10 -2时，单独使用就能产生足够的免疫效果保护小鼠，但是当病毒稀释度降为10 -4时，小鼠的存活率只有16.7％，更低时小鼠全部死亡；而在10 -4稀释度的病毒中加入Mn 2OHPO 4胶体，小鼠的存活率上升到100％，如图10E和10F所示。此外，小鼠中的活动病毒滴度降低，如图10G所示。这表明胶体锰作为佐剂可以在降低灭活HSV-1病毒10倍剂量的同时达到同样的保 护效果。
实验(c)PR8灭活病毒疫苗粘膜免疫一次
将经过PEG纯化的流感PR8病毒灭活后稀释成1、10 -1、10 -2、10 -3稀释度，滴鼻免疫小鼠一次。7天后用流感病毒感染小鼠，记录小鼠的存活率及体重变化。所有的剂量在不加佐剂时都没有保护效果，小鼠体重一直下降直至死亡；而加入Mn 2OHPO 4胶体作为佐剂后，稀释成10 -1剂量的病毒就能使小鼠体重在下降至第4天和第5天后出现上升，第10天后体重恢复到病毒感染前水平，病情好转，如图11A和11B所示。该结果表明胶体锰作为佐剂能使原本没有免疫原性或免疫原性弱的免疫原在免疫对象一次后就产生保护效果。
实验(d)PR8灭活病毒疫苗粘膜免疫两次
如图11C和11D所示，将经过PEG纯化的流感PR8病毒灭活后稀释成1、10 -1、10 -2、10 -3稀释度，滴鼻免疫小鼠两次(0天和7天免疫)。14天后用流感病毒感染小鼠，记录小鼠的存活率及体重变化。所有的剂量在不加佐剂时都没有保护效果，小鼠体重一直下降直至死亡；而加入Mn 2OHPO 4胶体作为佐剂后，稀释成10 -1剂量的病毒免疫小鼠两次后，小鼠体重在感染病毒后只是下降10％就很快恢复；而不稀释的病毒免疫后小鼠体重没有下降，也没有出现感染症状。该结果表明胶体锰作为佐剂加入原本没有免疫效果的疫苗后，免疫两次就可以达到显著的预防效果。
实施例10.胶体锰Mn 2OHPO 4作为佐剂增强流感亚单位疫苗的保护效果
小鼠按体重随机分为4组，分别为：空白对照组(ctrl)，HA抗原组(HA)，HA抗原与胶体锰组(HA+Mn)和HA抗原与铝佐剂组(HA+Al)。肌注小鼠后，第14天、第21天和第28天检测小鼠血清中抗HA蛋白IgG1抗体含量。如图12A所示，单独使用HA蛋白(5μg/小鼠)时，没有产生可检测到的特异性抗体；当加入Mn 2OHPO 4胶体(50μg/小鼠)作为佐剂后，肌注免疫产生的抗HA IgG1抗体量显著高多铝佐剂(800μg/只)的效果。对比胶体锰与CTB作为佐剂滴鼻免疫小鼠的效果，当加入Mn 2OHPO 4胶体(20μg/小鼠)作为佐剂后，滴鼻免疫产生的抗HA IgA抗体在免疫4周后显著高于CTB(5μg/小鼠)作为佐剂的效果。且以胶体锰为佐剂免疫小鼠后，小鼠体重没有变化，肺部也没有炎症产生；而以铝佐剂和CTB为佐剂免疫小鼠后，小鼠体重略下降后恢 复，肺部有少量炎症，如图12C和12D所示。该结果表明，胶体锰可以作为亚单位疫苗佐剂，比传统佐剂的安全性更高，效果更好。
实施例11.胶体锰Mn 2OHPO 4作为佐剂增强灭活流感病毒及亚单位疫苗对于同源异型流感病毒(WSN)及异源流感病毒(H3N2)菌均有保护效果
实验(a)胶体锰Mn 2OHPO 4混合PR8灭活病毒或者PR8-HA1蛋白制作的疫苗能预防WSN病毒感染
小鼠按体重随机分为5组，分别为：空白对照组(con)，灭活病毒PR8组，灭活病毒PR8+胶体锰组(PR8+MnJ)，PR8-HA1蛋白组(HA1)及PR8-HA1蛋白+胶体锰组(HA1+MnJ)。在第0天，7天，14天各免疫小鼠一次，第21天用WSN病毒感染小鼠，连续14天记录小鼠体重。如图13A所示，在加了胶体锰的实验组中，小鼠体重都不会下降，表明对病毒的保护效果极好，而不加佐剂和空白对照组的小鼠体重都很快下降。
实验(b)胶体锰Mn 2OHPO 4混合PR8灭活病毒或者PR8-HA1蛋白制作的疫苗能预防H3N2病毒感染
小鼠按体重随机分为5组，分别为：空白对照组(con)，灭活病毒PR8组，灭活病毒PR8+胶体锰组(PR8+MnJ)，PR8-HA1蛋白组(HA1)及PR8-HA1蛋白+胶体锰组(HA1+MnJ)。在第0天，7天，14天各免疫小鼠一次，第21天用H3N2病毒感染小鼠，连续14天记录小鼠体重。如图13B所示，H3N2病毒毒性不会导致小鼠死亡，在空白对照组和单独灭活病毒组中，小鼠体重都会下降到80％才恢复，单独HA1组下降到90％恢复，而加了胶体锰的HA1组体重基本不降，加了胶体锰的灭活病毒组，体重只降5％就恢复。
实施例12.胶体锰Mn 2OHPO 4作为免疫增强剂增强机体的抗肿瘤效果
实验(a)胶体锰Mn 2OHPO 4疫苗抑制原位瘤生长
小鼠按体重随机分为4组，分别为：空白对照组(con)，OVA对照组，OVA与胶体锰组(OVA+Mn)，及OVA与铝佐剂组(OVA+Al)。根据实验设计免疫小鼠三次后(每隔7天免疫一次)，皮下接种黑色素瘤B16-OVA，建立原位瘤模型。如图14A至14C所示，预先使用铝佐剂不能抑制肿瘤生长及提 高小鼠存活率；而预先使用胶体锰能显著抑制肿瘤生长并提高小鼠存活率；在接种肿瘤21天后，对照组小鼠的肿瘤平均大小为1906mm 3，OVA组小鼠的肿瘤平均大小为925.5mm 3，胶体锰免疫的小鼠肿瘤平均大小为222.9mm 3，铝佐剂组小鼠的肿瘤平均大小为1946mm 3，如图14B所示；对照小鼠存活时间为26天，免疫胶体锰的小鼠存活时间为49天，比免疫吕佐剂小鼠的27天显著延长，如图14C所示。该结果表明，胶体锰可作为免疫增强剂增强机体的抗原位瘤效果。
实验(b)胶体锰Mn 2OHPO 4疫苗抑制肿瘤转移
Mn 2OHPO 4胶体和铝佐剂分别与抗原OVA混合后免疫小鼠,每隔7天免疫一次，免疫3次，激活体内免疫反应；尾静脉注射B16-OVA肿瘤，建立肿瘤转移模型。在注射肿瘤21天后处死小鼠，取出肺组织，可见空白对照组小鼠肺上长满了黑色素瘤，仅免疫了OVA的小鼠肺上出现一定数量的黑色素瘤，而免疫了OVA+胶体锰的小鼠肺上几乎没有肿瘤，如图14D所示。将肿瘤组织研磨后用流式细胞仪统计肿瘤细胞数量，显示免疫了OVA+胶体锰的小鼠肺中肿瘤细胞数量明显少于其他两组，如图14E所示。该结果表明，胶体锰可作为免疫增强剂增强机体的抗转移瘤效果。
实验(c)胶体锰Mn 2OHPO 4与环磷酰胺联用抗肿瘤
在小鼠皮下接种黑色素瘤B16F10后，随机分为4组，分别为：对照组(con)，Mn 2OHPO 4胶体组(5mg/kg)，Cyclophosphamide Monohydrate(CTX)组(140mg/kg)，以及联合用药组(CTX+Mn)。环磷酰胺(CTX)是临床上广泛使用的抗肿瘤化疗药物。Mn 2OHPO 4胶体肌肉注射(肿瘤接种后每两天治疗一次)，CTX腹腔注射(肿瘤接种后的第6，9，12天各治疗一次)，对照组施以等体积的生理盐水。如图15所示，胶体锰与CTX联用能显著抑制肿瘤的生长。在接种肿瘤14天后，处死小鼠，解剖取出肿瘤并测量体积，对照组肿瘤平均大小为960.1mm 3，单独使用胶体锰治疗小鼠肿瘤大小为659.5mm 3，单独CTX治疗小鼠肿瘤大小为393.9mm 3，联用小鼠肿瘤大小为238mm 3，如图15B所示；同时称量各肿瘤重量，结果显示胶体锰与CTX联用抑制肿瘤的生长，如图15C所示。这表明胶体锰作为免疫增强剂增强传统治疗方法，如化疗，联合使用，对肿瘤生长具有显著的抑制作用。
实验(d)胶体锰Mn 2OHPO 4与PD-1抗体联用抗黑色素瘤
在小鼠皮下接种黑色素瘤B16F10后，随机分为4组，分别为：对照组(con)，Mn 2OHPO 4胶体组(5mg/kg)，anti-PD1抗体组(200μg/只)，以及联合用药组(anti-PD1+Mn)。PD1抗体是近年用于免疫治疗的新药。PD1抗体(Clone 29 F.1A12,BioXCell)在肿瘤接种后的第3，7，11天各治疗一次，每次200ug抗体溶解在200ul PBS中腹腔注射。图16A-C所示，接种肿瘤15天后，处死小鼠，解剖取出肿瘤并进行测量，对照组小鼠肿瘤平均大小为1847mm 3，单独使用胶体锰组的小鼠肿瘤平均大小为797.3mm 3，单独PD1抗体组的小鼠肿瘤平均大小为687.3mm 3，而联用治疗组小鼠肿瘤平均大小为342mm 3，显著低于单独使用PD1抗体。用流式细胞仪分析肿瘤中浸润的CD8 +T细胞比例，胶体锰将PD1抗体药的效果从27.7％提高到45.4％。这表明胶体锰能协同PD1抗体药治疗黑色素瘤。
实验(e)胶体锰Mn 2OHPO 4与PD-1抗体联用抗结肠癌肿瘤
在小鼠皮下接种结肠癌肿瘤MC38，随机分为4组，分别为：对照抗体组(Isotype，200μg/小鼠)，Mn 2OHPO 4胶体和对照抗体组(Isotype+Mn)，anti-PD1抗体组(200μg/小鼠)，以及联合用药组(anti-PD1+Mn)，PD-1使用与实验(d)一样。接种肿瘤20天后，处死小鼠，解剖取出肿瘤并进行测量，对照组小鼠肿瘤平均大小为319.8mm 3，单独锰治疗小鼠大小为126.9mm 3，单独PD1抗体药治疗小鼠肿瘤大小为143.1mm 3，联用治疗小鼠肿瘤大小为66.74mm 3，如图17B所示。制作肿瘤组织切片，进行DAPI和CD8 +T细胞的免疫荧光染色，可以看出PD1抗体和胶体锰联用治疗的小鼠肿瘤内部浸润的CD8 +T细胞比例显著增加，如图17D所示。这表明胶体锰能协同PD1抗体药治疗结肠癌肿瘤。
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Claims (41)

  1. 一种免疫增强组合物，其包含新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。
  2. 根据权利要求1所述的免疫增强组合物，其用作免疫佐剂。
  3. 根据权利要求1或2所述的免疫增强组合物，其中所述新生沉淀锰和/或胶体锰选自磷酸锰盐、碳酸锰盐、氢氧化锰及其任意混合物。
  4. 根据权利要求1至3任一项所述的免疫增强组合物，其中所述新生沉淀锰从非沉淀形式转化成沉淀形式的时间不超过1天，或者不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，或者不超过60、50、45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
  5. 一种疫苗组合物，其包含
    A)疫苗免疫原，和
    B)新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源，任选地，组分A和B可以处于相同和/或分开的容器中。
  6. 根据权利要求5的疫苗组合物，其中所述疫苗免疫原源自病毒、细菌和/或寄生虫，
    例如所述病毒选自：DNA病毒和RNA病毒，优选地所述病毒选自：疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、冠状病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，更优选地所述病毒选自：单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV和HBV；
    例如所述细菌选自革兰氏阴性菌和革兰氏阳性菌，优选地所述细菌选自肺炎链球菌(Streptococcus pneumoniae)、流感嗜血杆菌(Haemophilus influenzae)、沙门氏菌(Salmonella)、脑膜炎双球菌(Meningococcus)、表皮葡萄球菌(Staphylococcus epidermidis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、产酸克雷伯氏菌(Klebsiella oxytoca)、阴沟肠杆菌(Enterobacter cloacae)、弗氏柠檬酸杆菌(Citrobacter freundii)、绿脓假单胞菌(Pseudomonas aeruginosa)和 波美不动杆菌(Acinetobacter baumanni)；
    具体地，例如所述疫苗免疫原源自流感病毒、肝炎病毒(如甲肝病毒、乙肝病毒)、脊髓灰质炎病毒、狂犬病毒、HPV病毒、脑炎病毒(如乙型脑炎病毒)、腮腺炎病毒、风疹病毒、破伤风杆菌、百日咳杆菌、白喉杆菌、麻风杆菌、结核杆菌、脑膜炎双球菌、肺炎双球菌及其任意组合。
  7. 根据权利要求5或6的疫苗组合物，其中所述新生沉淀锰和/或胶体锰选自磷酸锰盐、碳酸锰盐、氢氧化锰及其任意混合物。
  8. 根据权利要求5至7任一项所述的疫苗组合物，其中所述新生沉淀锰从非沉淀形式转化成沉淀形式的时间不超过1天，或者不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，或者不超过60、50、45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
  9. 一种制造免疫增强组合物的方法，其包括
    1)提供能够形成新生沉淀锰和/或胶体锰的源；和
    2)任选地使所述能够形成新生沉淀锰和/或胶体锰的源转化为新生沉淀锰和/或胶体锰。
  10. 根据权利要求9所述的方法，其中所述新生沉淀锰和/或胶体锰选自磷酸锰盐、碳酸锰盐、氢氧化锰及其任意混合物。
  11. 根据权利要求9或10所述的方法，其中所述新生沉淀锰从非沉淀形式转化成沉淀形式的时间不超过1天，或者不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，或者不超过60、50、45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
  12. 一种制造疫苗组合物的方法，其包括
    1)提供疫苗免疫原；和
    2)提供新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和/或胶体锰的源。
  13. 根据权利要求12的方法，其中所述疫苗免疫原源自病毒、细菌和/或寄生虫，
    例如所述病毒选自：DNA病毒和RNA病毒，优选地所述病毒选自：疱疹 病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、冠状病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，更优选地所述病毒选自：单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV和HBV；
    例如所述细菌选自革兰氏阴性菌和革兰氏阳性菌，优选地所述细菌选自肺炎链球菌(Streptococcus pneumoniae)、流感嗜血杆菌(Haemophilus influenzae)、沙门氏菌(Salmonella)、脑膜炎双球菌(Meningococcus)、表皮葡萄球菌(Staphylococcus epidermidis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、产酸克雷伯氏菌(Klebsiella oxytoca)、阴沟肠杆菌(Enterobacter cloacae)、弗氏柠檬酸杆菌(Citrobacter freundii)、绿脓假单胞菌(Pseudomonas aeruginosa)和波美不动杆菌(Acinetobacter baumanni)；
    具体地，例如所述疫苗免疫原源自流感病毒、肝炎病毒(如甲肝病毒、乙肝病毒)、脊髓灰质炎病毒、狂犬病毒、HPV病毒、脑炎病毒(如乙型脑炎病毒)、腮腺炎病毒、风疹病毒、破伤风杆菌、百日咳杆菌、白喉杆菌、麻风杆菌、结核杆菌、脑膜炎双球菌、肺炎双球菌及其任意组合。
  14. 根据权利要求12或13所述的方法，其中所述新生沉淀锰和/或胶体锰选自磷酸锰盐、碳酸锰盐、氢氧化锰及其任意混合物。
  15. 根据权利要求12至14任一项所述的方法，其中所述新生沉淀锰从非沉淀形式转化成沉淀形式的时间不超过1天，或者不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，或者不超过60、50、45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
  16. 新生沉淀锰、胶体锰和/或能够形成新生沉淀锰和或胶体锰的源在制备免疫增强组合物或疫苗组合物中的用途。
  17. 根据权利要求16的用途，其中所述免疫增强组合物是免疫佐剂。
  18. 根据权利要求16或17的用途，其中所述新生沉淀锰和/或胶体锰选自磷酸锰盐、碳酸锰盐、氢氧化锰及其任意混合物。
  19. 根据权利要求16至18任一项所述的用途，其中所述新生沉淀锰从非沉淀形式转化成沉淀形式的时间不超过1天，不超过24、22、20、18、16、15、14、12、10、9、8、7、6、5、4、3、2或1小时，或者不超过60、50、 45、40、35、30、25、20、15、10、9、8、7、6、5、4、3、2或1分钟。
  20. 根据权利要求16至19任一项所述的用途，其中所述免疫增强是改善固有免疫和/或适应性免疫。
  21. 根据权利要求16至20任一项所述的用途，其中所述免疫增强是增加I型干扰素表达。
  22. 根据权利要求16至20任一项所述的用途，其中所述免疫增强是诱导炎症因子切割产生活性形式。
  23. 根据权利要求16至20任一项所述的用途，其中所述免疫增强是促进抗体产生。
  24. 一种增强免疫的方法，其包括，向有此需要的对象施用根据权利要求1至4任一项所述的或者根据权利要求9至11任一项所述方法获得的免疫增强组合物，
    具体地，所述免疫增强例如是
    A)改善固有免疫和/或适应性免疫，
    B)增加I型干扰素表达，
    C)诱导产生活性形式的炎症因子，
    D)促进抗体产生，
    E)促进T细胞增殖，和/或
    F)促进树突细胞成熟。
  25. 根据权利要求24的方法，其中所述施用选自肌肉注射、皮内注射、皮下注射、静脉注射、粘膜施用及其任意组合。
  26. 根据权利要求24或25的方法，其中所述增强免疫是用于预防和/或治疗疾病，例如细菌感染、真菌感染、病毒感染、寄生虫感染、肿瘤、自身免疫病。
  27. 根据权利要求24至26任一项所述的方法，其中所述病毒选自：DNA病毒和RNA病毒，优选地所述病毒选自：疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、冠状病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，具体地所述 病毒选自：单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV、流感病毒、肝炎病毒(如甲肝病毒、乙肝病毒)、脊髓灰质炎病毒、狂犬病毒、HPV病毒、脑炎病毒(如乙型脑炎病毒)、腮腺炎病毒、风疹病毒及其任意组合。
  28. 根据权利要求24至26任一项所述的方法，其中所述细菌选自革兰氏阴性菌和革兰氏阳性菌，优选地所述细菌选自肺炎链球菌(Streptococcus pneumoniae)、流感嗜血杆菌(Haemophilus influenzae)、沙门氏菌(Salmonella)、脑膜炎双球菌(Meningococcus)、表皮葡萄球菌(Staphylococcus epidermidis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、产酸克雷伯氏菌(Klebsiella oxytoca)、阴沟肠杆菌(Enterobacter cloacae)、弗氏柠檬酸杆菌(Citrobacter freundii)、绿脓假单胞菌(Pseudomonas aeruginosa)、波美不动杆菌(Acinetobacter baumanni)、破伤风杆菌、百日咳杆菌、白喉杆菌、麻风杆菌、结核杆菌、脑膜炎双球菌、肺炎双球菌及其任意组合。
  29. 根据权利要求24至26任一项所述的方法，其中所述自身免疫性疾病选自I型糖尿病、银屑病、类风湿性关节炎、系统性红斑狼疮和多发性硬化。
  30. 根据权利要求24至26任一项所述的方法，其中所述肿瘤选自卵巢癌、肺癌、胃癌、乳腺癌、肝癌、胰腺癌、皮肤癌、恶性黑色素瘤、头颈癌、肉瘤、胆管癌、膀胱癌、肾癌、结肠癌、胎盘绒毛膜癌、子宫颈癌、睾丸癌、子宫癌和白血病。
  31. 根据权利要求24至26任一项所述的方法，其中所述寄生虫是细胞内寄生虫，优选地选自疟原虫、弓形虫、锥虫、血吸虫、丝虫和利什曼原虫。
  32. 根据权利要求24至26任一项所述的方法，其还包括向所述对象施用另外的预防/治疗剂。
  33. 一种免疫接种方法，其包括
    向有此需要的对象施用根据权利要求5至8任一项所述的疫苗组合物，其中当组分A和组分B处于不同容器中时可以同时或不同时施用；和/或
    向有此需要的对象施用根据权利要求1至4任一项所述的免疫增强组合物和任选地疫苗免疫原。
  34. 根据权利要求33的方法，其中所述施用选自肌肉注射、皮内注射、 皮下注射、静脉注射、粘膜施用及其任意组合。
  35. 根据权利要求33或34的方法，其中所述免疫接种用于预防疾病，例如细菌感染、真菌感染、病毒感染、寄生虫感染、肿瘤和自身免疫病。
  36. 根据权利要求33至35任一项所述的方法，其中所述病毒选自：DNA病毒和RNA病毒，优选地所述病毒选自：疱疹病毒科、弹状病毒科、丝状病毒科、正粘病毒科、副粘病毒科、冠状病毒科、小RNA病毒科、嗜肝DNA病毒科、黄病毒科、乳头瘤病毒科、痘病毒科、和逆转录病毒科，具体地所述病毒选自：单纯疱疹病毒、水疱性口炎病毒、牛痘病毒、HIV、流感病毒、肝炎病毒(如甲肝病毒、乙肝病毒)、脊髓灰质炎病毒、狂犬病毒、HPV病毒、脑炎病毒(如乙型脑炎病毒)、腮腺炎病毒、风疹病毒及其任意组合。
  37. 根据权利要求33至35任一项所述的方法，其中所述细菌选自革兰氏阴性菌和革兰氏阳性菌，优选地所述细菌选自肺炎链球菌(Streptococcus pneumoniae)、流感嗜血杆菌(Haemophilus influenzae)、沙门氏菌(Salmonella)、脑膜炎双球菌(Meningococcus)、表皮葡萄球菌(Staphylococcus epidermidis)、金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichia coli)、肺炎克雷伯氏菌(Klebsiella pneumoniae)、产酸克雷伯氏菌(Klebsiella oxytoca)、阴沟肠杆菌(Enterobacter cloacae)、弗氏柠檬酸杆菌(Citrobacter freundii)、绿脓假单胞菌(Pseudomonas aeruginosa)、波美不动杆菌(Acinetobacter baumanni)、破伤风杆菌、百日咳杆菌、白喉杆菌、麻风杆菌、结核杆菌、脑膜炎双球菌、肺炎双球菌及其任意组合。
  38. 根据权利要求33至35任一项所述的方法，其中所述自身免疫性疾病选自I型糖尿病、银屑病、类风湿性关节炎、系统性红斑狼疮和多发性硬化。
  39. 根据权利要求33至35任一项所述的方法，其中所述肿瘤选自卵巢癌、肺癌、胃癌、乳腺癌、肝癌、胰腺癌、皮肤癌、恶性黑色素瘤、头颈癌、肉瘤、胆管癌、膀胱癌、肾癌、结肠癌、胎盘绒毛膜癌、子宫颈癌、睾丸癌、子宫癌和白血病。
  40. 根据权利要求33至35任一项所述的方法，其中所述寄生虫是细胞内寄生虫，优选地选自疟原虫、弓形虫、锥虫、血吸虫、丝虫和利什曼原虫。
  41. 根据权利要求33至35任一项所述的方法，其还包括向所述对象施用另外的预防剂。
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CN113274550A (zh) * 2021-05-31 2021-08-20 福州大学 一种具有抗炎作用的血管化骨仿生多功能组织工程支架及其制备方法
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CN113797329A (zh) * 2021-10-19 2021-12-17 启锰生物科技(江苏)有限公司 一种二价锰佐剂和CpG佐剂的疫苗佐剂组合物及其制作方法
WO2023123959A1 (zh) * 2021-12-28 2023-07-06 广东粤港澳大湾区国家纳米科技创新研究院 一种铝锰复合纳米晶及其制备方法和应用

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