Manganese compositions for immune enhancement

Abstract

The present invention provides immunopotentiating compositions and vaccine compositions comprising neo-natal precipitated manganese and / or colloidal manganese, methods of making them, and their use for enhancing immunization and or vaccination.

Description

[0001] This application claims priority to Chinese Patent Application No. 201910319344.1, filed with the Chinese Patent Office on April 19, 2019, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present invention provides divalent manganese colloids or divalent manganese neoprecipitates for enhancing immunity, and their use for enhancing immunity, such as as an immune adjuvant, antiviral or antitumor agent. Background Technology

[0003] The body protects itself from infection by foreign pathogens through the innate immune system and the adaptive immune system. Innate immunity promotes adaptive immunity. When the body is infected by pathogens, the innate immune response is initiated first. Through pattern recognition receptors, the pathogen-associated molecular patterns of pathogens are recognized, activating multiple signaling pathways, such as the TLR pathway (1), RLR pathway (2), cGAS-STING pathway (3), and inflammasome activation (4). The activation of these pathways leads to the production of many downstream cytokines, including type I interferon, IL-1β, and IL-18.

[0004] Type I interferon can activate the JAK-STAT pathway through autocrine and paracrine pathways, inducing the expression of a large number of antiviral genes to resist viral infection (5,6). At the same time, type I interferon can promote the maturation of antigen-presenting cells (7). Antigen-presenting cells present pathogenic microorganisms or tumor antigens to T cells, activating antigen-specific CD4+. + T cells and CD8 + T cells; they can also promote B cell activation to produce antigen-specific antibodies; and promote the production of memory B cells and immune memory.

[0005] The pro-inflammatory factor IL-1β can directly act on CD4. + T cells, promote T cell proliferation (8); IL-18 can effectively enhance Th1 immune response, and can also promote the proliferation and cytotoxic effects of T cells and NK cells (9).

[0006] Currently, four adjuvants have been approved by the FDA for use in humans: aluminum adjuvant, MF59, AS03, and AS04. Aluminum adjuvant has been widely used since 1920 for vaccines such as Hepatitis A (HAV), Hepatitis B (HBV), DTP, HPV, and Haemophilus influenzae (HiB). Aluminum adjuvant can only activate Th2 responses (humoral immunity primarily driven by antibody production) and cannot activate Th1 responses (those driven by CD8+). +T-cell-based cellular immunity (10). AS04 is an adjuvant containing MPL and aluminum salts, used in HPV and HBV vaccines. This adjuvant can activate NF-κB, producing pro-inflammatory factors, and possesses the ability to activate Th1 responses, which aluminum adjuvants did not originally possess. MF59 is a water-in-oil adjuvant made with squalene as the oil phase, containing degradable squalene, Tween 80, and Span 85, used in influenza vaccines. Its mechanism is currently unclear; its half-life in vivo is 42 hours, and it can simultaneously activate Th1 and Th2 responses. AS03 is an oil-in-water adjuvant containing α-tocopherol, squalene, and Tween 80, used in influenza vaccines, but the H1N1 influenza virus vaccine Pandemrix, which contains AS03, is also mentioned. TM It may cause narcolepsy (11). AS03 can induce the production of pro-inflammatory factors, recruit immune cells, and induce antibody production by activating the NF-κB pathway.

[0007] Currently, there are many adjuvants that are not approved for clinical use but can be used in laboratories, many of which are very effective. However, their clinical application is limited by their side effects. For example, Freund's adjuvant has poor mineral oil metabolism and can form nodules at the injection site. There are also cytokine-type adjuvants, such as interleukin-2 and interferon, but their main drawback is their high cost.

[0008] There are reports demonstrating that ligands that activate the cGAS-STING pathway can act as adjuvants, such as DMXAA (12), c-di-GMP (13), cGAMP (14), and chitosan (15).

[0009] CN107412260A discloses that divalent manganese is a cGAS-STING pathway activator with immune-enhancing effects, such as its potential use as an immune adjuvant. However, in further research, the inventors unexpectedly discovered that divalent manganese itself has some defects, and improved solutions are needed to eliminate at least one of these defects. Summary of the Invention

[0010] In further research on the immunomodulatory effects of divalent manganese, the inventors discovered that although divalent manganese solution could produce an immunomodulatory effect in the body, this effect was not strong enough. More importantly, the research also revealed that when the concentration of the divalent manganese solution was increased to enhance the immunomodulatory effect, manganese precipitation easily occurred, making it impossible to obtain a uniform and stable high-concentration divalent manganese solution, thus compromising the reproducibility of the experiment. This manganese precipitate gradually aggregated and grew with increasing storage time.

[0011] Unable to achieve the desired high concentration of divalent manganese solution, the inventors were forced to continue their research on the immunomodulatory effects of divalent manganese precipitate. Due to an oversight in the experimental procedure, the inventors inadvertently used divalent manganese precipitate stored for different periods. They unexpectedly discovered that divalent manganese precipitate stored for different times exhibited different immunomodulatory effects, and that the immunomodulatory effect of newly formed precipitate was significantly superior to that of manganese precipitate stored for longer periods. Further comparing divalent manganese solution and divalent manganese precipitate stored for different times, the inventors unexpectedly discovered that the immunomodulatory effect of newly formed manganese salt precipitate was also significantly superior to that of divalent manganese solution.

[0012] In experimenting with different divalent manganese systems, the inventors unexpectedly discovered that divalent manganese compounds can form colloidal solutions. Even more surprisingly, these manganese colloids exhibited immune-enhancing effects comparable to, or even stronger than, newly formed manganese precipitates.

[0013] Based on this, the inventor completed the present invention and provides the following technical solution.

[0014] In one aspect, the present invention provides an immune-enhancing composition comprising newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese.

[0015] In one embodiment, the newly precipitated manganese and / or colloidal manganese is in the form of manganese phosphates, manganese carbonates, and manganese hydroxides, and any mixture thereof.

[0016] Preferably, the time for the newly formed precipitated manganese to transform 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 does not exceed 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute.

[0017] In another aspect, the present invention provides a vaccine composition comprising

[0018] A) Vaccine immunogen, and

[0019] B) Sources of newly formed precipitated manganese, colloidal manganese, and / or manganese capable of forming newly formed precipitated manganese and / or colloidal manganese.

[0020] Optionally, components A and B may be in the same and / or separate containers.

[0021] Preferably, the vaccine immunogenicity originates from viruses, bacteria, and / or parasites.

[0022] For example, the virus is selected from DNA viruses and RNA viruses, preferably from the Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Picornaviridae, Hepatoviridae, Flaviviridae, Papillomavirusidae, Poxviridae, and Retroviridae families, and more preferably from the Herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV;

[0023] For example, the bacteria are selected from Gram-negative and Gram-positive bacteria, preferably selected from Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Meningococcus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumanni.

[0024] Specifically, the vaccine's immunogenicity may be derived from influenza virus, hepatitis virus (such as hepatitis A virus, hepatitis B virus), poliovirus, rabies virus, HPV virus, encephalitis virus (such as Japanese encephalitis virus), mumps virus, rubella virus, tetanus bacillus, pertussis bacillus, diphtheria bacillus, leprosy bacillus, tuberculosis bacillus, meningococcus, pneumococcus, or any combination thereof.

[0025] In another aspect, the present invention provides a method for manufacturing an immune-enhancing composition, comprising:

[0026] 1) Provide a source capable of forming newly formed precipitated manganese and / or colloidal manganese; and

[0027] 2) Optionally, the source capable of forming newly formed precipitated manganese and / or colloidal manganese is converted into newly formed precipitated manganese and / or colloidal manganese.

[0028] Preferably, the newly precipitated manganese and / or colloidal manganese is selected from manganese phosphates, manganese carbonates, and manganese hydroxides, and any mixture thereof.

[0029] Preferably, the time for the newly formed precipitated manganese to transform 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 does not exceed 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute.

[0030] In another aspect, the present invention provides a method for manufacturing a vaccine composition, comprising:

[0031] 1) Provide vaccine immunogen; and

[0032] 2) Provide a source of newly formed precipitated manganese, colloidal manganese and / or manganese capable of forming newly formed precipitated manganese and / or colloidal manganese.

[0033] Preferably, the vaccine immunogenicity is derived from viruses, bacteria and / or parasites, as described above, and will not be repeated here.

[0034] In one aspect, the present invention provides the use of neoprecipitated manganese, colloidal manganese, and / or sources capable of forming neoprecipitated manganese and / or colloidal manganese in the preparation of immune-enhancing compositions or vaccine compositions for improving innate and / or adaptive immunity. In some embodiments, the neoprecipitated manganese, colloidal manganese, and / or sources capable of forming neoprecipitated manganese and / or colloidal manganese improve innate and / or adaptive immunity by increasing type I interferon expression. In other embodiments, the neoprecipitated manganese, colloidal manganese, and / or sources capable of forming neoprecipitated manganese and / or colloidal manganese improve innate and / or adaptive immunity by inducing the cleavage of inflammatory factors to produce active forms. In still other embodiments, the neoprecipitated manganese, colloidal manganese, and / or sources capable of forming neoprecipitated manganese and / or colloidal manganese improve innate and / or adaptive immunity by promoting antibody production.

[0035] In one aspect, the present invention provides a method for enhancing immunity, comprising administering the immune-enhancing composition to a subject in need of it.

[0036] Specifically, the immune enhancement includes, for example, A) improving innate and / or adaptive immunity, B) increasing type I interferon expression, C) inducing the production of active forms of inflammatory factors, D) promoting antibody production, E) promoting T cell proliferation, and / or F) promoting dendritic cell maturation.

[0037] Preferably, the administration is selected from intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal application, and any combination thereof.

[0038] Specifically, the immune enhancement is used to prevent and / or treat diseases such as bacterial infections, fungal infections, viral infections, parasitic infections, tumors, and autoimmune diseases.

[0039] The viruses mentioned herein are selected from DNA viruses and RNA viruses. Preferably, the viruses are selected from the following families: Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Picornaviridae, Hepatoviridae, Flaviviridae, Papillomavirusidae, Poxviridae, and Retroviridae. Specifically, the viruses are selected from the following families: Herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, influenza virus, hepatitis virus (such as hepatitis A virus, hepatitis B virus), poliovirus, rabies virus, HPV virus, encephalitis virus (such as Japanese encephalitis virus), mumps virus, rubella virus, and any combination thereof.

[0040] The bacteria are selected from Gram-negative and Gram-positive bacteria, preferably from Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Neisseria meningitidis, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, Acinetobacter baumanni, Clostridium tetani, Bordetella pertussis, Corynebacterium diphtheriae, Mycobacterium leprae, Mycobacterium tuberculosis, Neisseria meningitidis, Neisseria pneumoniae, and any combination thereof.

[0041] The parasites mentioned therein are intracellular parasites, preferably selected from Plasmodium, Toxoplasma gondii, Trypanosoma, Schistosoma, filariasis, and Leishmania.

[0042] The autoimmune diseases mentioned are selected from type 1 diabetes, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

[0043] The tumors mentioned are selected from ovarian cancer, lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, malignant melanoma, head and neck cancer, sarcoma, bile duct cancer, bladder cancer, kidney cancer, colon cancer, placental choriocarcinoma, cervical cancer, testicular cancer, uterine cancer, and leukemia.

[0044] In some embodiments, the immune-enhancing composition is administered to the recipient in combination with additional preventative / therapeutic agents.

[0045] In another aspect, the present invention provides an immunization method, comprising:

[0046] Administer the vaccine composition of the present invention to an object in need, wherein components A and B may be administered simultaneously or at different times when they are in different containers; and / or

[0047] The immune-enhancing composition of the present invention and, optionally, the vaccine immunogen, are administered to those who require it.

[0048] Preferably, the administration is selected from intramuscular injection, intradermal injection, subcutaneous injection, intravenous injection, mucosal application, and any combination thereof.

[0049] Specifically, the immunization is used to prevent diseases such as bacterial infections, fungal infections, viral infections, parasitic infections, tumors, and autoimmune diseases. These diseases are as described above and will not be repeated here.

[0050] In some embodiments, the immune-enhancing composition is administered to the recipient in combination with additional preventative / therapeutic agents.

[0051] In another aspect, the present invention provides the use of newly precipitated manganese, colloidal manganese and / or sources capable of forming newly precipitated manganese and / or colloidal manganese in the preparation of a medicament for enhancing type I interferon.

[0052] In another aspect, the present invention provides the use of newly precipitated manganese, colloidal manganese and / or sources 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.

[0053] In another aspect, the present invention provides the use of newly precipitated manganese, colloidal manganese and / or sources capable of forming newly precipitated manganese and / or colloidal manganese in the preparation of a medicament for stimulating antibody production.

[0054] In another aspect, the present invention provides the use of newly precipitated manganese, colloidal manganese, and / or sources capable of forming newly precipitated manganese and / or colloidal manganese as an immune adjuvant. In some embodiments, the immune adjuvant activates T cell activation and / or antibody production. Preferably, the immune adjuvant is used in vaccine compositions for treating diseases selected from bacterial infections, viral infections, parasitic diseases, autoimmune diseases, and cancer.

[0055] In another aspect, the present invention provides a kit for immunization comprising: a first container containing one or more antigens; and a second container containing newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese. Preferably, the first and / or second containers further comprise pharmaceutically acceptable carriers. In particular, the kit is used for one or more purposes of the present invention. In some embodiments, the antigen is located in the same container as the newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese.

[0056] In some embodiments, the antigens used in this invention are selected from: viral or bacterial or parasitic antigens, such as hepatitis A, B, C, D and E3 viruses, HIV, herpesviruses 1, 2, 6 and 7, cytomegalovirus, varicella-zoster virus, papillomavirus, Epstein-Barr virus, influenza virus, parainfluenza virus, adenovirus, Bunyavirus (Hantavirus), Coxsackievirus, ciliary RNA virus, rotavirus, respiratory syncytial virus, poxvirus, rhinovirus, rubella virus, polyvesicular papillomavirus, mumps virus and measles virus, mycobacteria that cause tuberculosis and leprosy, pneumococcus, aerobic Gram-negative bacilli, mycoplasma, staphylococcal infection, streptococcal infection, Salmonella and Chlamydia, Helicobacter pylori, malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis and filariasis.

[0057] In some embodiments, the newly precipitated manganese, colloidal manganese, and / or sources and / or antigens capable of forming newly precipitated manganese and / or colloidal manganese in this invention are in effective amounts.

[0058] In another aspect, the present invention provides a method for increasing type I interferon expression in a subject, comprising applying to the subject newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese. Furthermore, the present invention provides a method for enhancing type I interferon activity in cells in vitro, comprising applying to the cells newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese, preferably, the method being non-therapeutic.

[0059] In another aspect, the present invention provides a method for inducing the cleavage of inflammatory factors to produce active forms in a subject, comprising applying to said subject newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese. Furthermore, the present invention also provides a method for inducing the cleavage of inflammatory factors to produce active forms in cells in vitro, comprising applying to said cells newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese, preferably, the method being non-therapeutic.

[0060] In another aspect, the present invention provides a method for stimulating antibody production in a subject, comprising applying to said subject newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese. Furthermore, the present invention also provides a method for stimulating antibody production in cells in vitro, comprising applying to said cells newly precipitated manganese, colloidal manganese, and / or a source capable of forming newly precipitated manganese and / or colloidal manganese, preferably, the method being non-therapeutic. Attached Figure Description

[0061] The foregoing and other aspects of the invention will be clearly illustrated below with reference to the detailed description and accompanying drawings. The embodiments shown in the drawings are presently preferred for illustrative purposes; however, it is to be understood that the invention is not limited to the specific embodiments disclosed.

[0062] Figure 1 The illustration shows that newly formed precipitated manganese and colloidal manganese (Mn2OHPO4) have good adjuvant effects. Figure 1 A shows the different states in which divalent manganese exists in solution; Figure 1 Image B shows a transmission electron microscope (TEM) image of colloidal manganese (Mn2OHPO4); Figure 1 C indicates the level of OVA antibody IgG1 after immunization; Figure 1 D shows a comparison of the residence time of MnCl2 solution and colloidal manganese (Mn2OHPO4) after intramuscular injection.

[0063] Figure 2 The diagram illustrates the different states and properties of MnCl2 solution mixed with other anions in physiological saline. The ability of these precipitates or colloids to activate type I interferon or inflammatory factors, and their adjuvant effects, were also measured. Figure 2 A shows that MnCl2 solution forms different states with different concentrations of carbonate ions, bicarbonate ions, phosphate ions, monohydrogen phosphate ions, and hydroxide ions. Figure 2 B shows the ability of manganese ions in different states to activate type I interferon, induce the production of the inflammatory factor IL-1β, and act as an adjuvant to mix with OVA protein to produce antibodies in immunization. Figure 2 C shows the products formed by manganese ions and different anions when irradiated with a laser pointer, to observe the Tyndall effect.

[0064] Figure 3 The figure illustrates the amount of anti-OVA antibodies produced in mice immunized by intramuscular injection of colloidal manganese (Mn2OHPO4) and aluminum adjuvant mixed with chicken ovalbumin (OVA) for 10 days (A), 17 days (B), 24 days (C), and 31 days (D).

[0065] Figure 4The figure illustrates the levels of anti-OVA antibodies in bronchoalveolar lavage fluid (A), oral lavage fluid (B), serum IgA (C), and serum IgG1 (D) after mice were immunized with OVA by intranasal administration of colloidal manganese (Mn2OHPO4) and cholera toxin B (CTB) adjuvant.

[0066] Figure 5 The diagram illustrates the following: Figure 1 and Figure 2 The method involved using colloidal manganese (Mn2OHPO4) as an adjuvant to mix OVA with intramuscular injection (A) or intranasal immunization (B) in mice, followed by determination of the amount of anti-OVA antibodies in the serum, oral lavage fluid, and bronchoalveolar lavage fluid of mice aged 1 to 6 months.

[0067] Figure 6 The diagram illustrates MnCl2 activation of the type I interferon pathway and inflammasome activation. Figure 6 AB shows the expression levels of type I interferon, downstream inducible factor ISG54, and viperin in mouse peritoneal macrophages after treatment with MnCl2, with aluminum adjuvant as a control. Figure 6 CD showed that MnCl2 could activate inflammasome activation, with aluminum adjuvant as a control. Figure 6 EF shows that MnCl2-activated inflammasome activation depends on NLRP3 and ASC.

[0068] Figure 7 The illustration shows how colloidal manganese (Mn2OHPO4) promotes CD4+. + T cell proliferation was assessed, with aluminum adjuvant serving as a control.

[0069] Figure 8 The illustration shows that the amount of antibodies produced in mice during intramuscular injection and mucosal immunization with colloidal manganese (Mn2OHPO4) depends on the cGAS-STING pathway and the inflammasome pathway.

[0070] Figure 9 The illustration shows that the ability of colloidal manganese (Mn2OHPO4) to promote dendritic cell maturation and T cell activation depends on the cGAS-STING pathway and the inflammasome pathway. Figure 9 A shows that colloidal manganese (MnOHPO4) induces the maturation of antigen-presenting cells (BMDCs). Figure 9 B shows colloidal manganese (MnOHPO4) plus antigen OVA stimulation of CD4 + The proliferative capacity of T cells. Figure 9 C indicates that colloidal manganese (MnOHPO4) was used as an adjuvant to stimulate CD8 in mice. + T cell activation.

[0071] Figure 10 The illustration shows that colloidal manganese (Mn2OHPO4) as an adjuvant can significantly enhance the immunoprotective effect against inactivated vesicular stomatitis virus (VSV) and herpes simplex virus (HSV-1). Figure 10 A shows the experimental procedure. Figure 10 B indicates that the inactivated VSV virus will be diluted to 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 Mice were immunized with a dose of [specific dosage], and the survival rate of the mice was observed after 14 days. Figure 10 C shows that it is diluted to 10. -3 After immunizing mice with a dose of VSV-inactivated virus, the mortality curve of the mice was observed over 14 days. Figure 10 D shows the viral titer in the mouse brain on day 4 after infection with VSV virus. Figure 10 EG showed that adding colloidal manganese to inactivated HSV-1 virus also had the same protective effect.

[0072] Figure 11 The illustration shows that colloidal manganese (Mn2OHPO4) as an adjuvant can significantly enhance the immunoprotective effect of inactivated influenza virus PR8. Figure 11 AB shows that inactivated influenza virus was diluted to 1, 10 -1 10 -2 10 -3 Mice were immunized once by intranasal drip of the appropriate dose. Seven days later, the mice were infected with influenza virus, and the survival rate and weight changes of the mice were observed. Figure 11 CD shows the survival rate and weight changes of mice infected with influenza virus after two intranasal immunizations.

[0073] Figure 12 The illustration shows that colloidal manganese (Mn2OHPO4) as an adjuvant can significantly enhance the immunoprotective effect of influenza subunit HA vaccine. Figure 12 A shows the levels of anti-HA protein IgG1 antibody in mouse serum at 14, 21, and 28 days after intramuscular injection. Figure 12 B shows the serum levels of anti-HA protein IgA antibodies in mice at 14, 21, and 28 days after intranasal immunization. Figure 12 C shows the weight changes in mice after infection with the influenza virus. Figure 12 D shows the lesions in the lung tissue of mice on day 5 of infection.

[0074] Figure 13 The illustration shows the protective effect of colloidal manganese Mn2OHPO4 as an adjuvant against inactivated influenza virus and subunit vaccines against homologous influenza virus (WSN) and heterologous influenza virus (H3N2). Figure 13 A shows the change in body weight in mice infected with WSN virus after immunization; Figure 13 B shows the changes in body weight of mice infected with H3N2 virus after immunization.

[0075] Figure 14 The illustration shows that colloidal manganese (Mn2OHPO4) can significantly inhibit the growth of both in situ tumors and metastatic tumors. Figure 14 A shows the size of the subcutaneous tumor captured by an imager at different times. Figure 14 B shows the change in tumor volume after tumor inoculation. Figure 14 C shows the survival curve of mice after tumor inoculation. Figure 14 Image D shows lung tissue with tumor metastasis 21 days after different immunization methods. Figure 14 E is displayed as Figure 14 Statistics on the number of tumor cells in D.

[0076] Figure 15 The illustration shows the efficacy of colloidal manganese (Mn2OHPO4) combined with the chemotherapeutic drug cyclophosphamide (CTX) in treating a subcutaneous tumor model, melanoma B16-F10. Figure 15 Image A shows tumor images 14 days after different treatments. Figure 15 B shows the growth curve of subcutaneous tumor B16F10 in mice. Figure 15 Figure C shows the tumor weight of the mice in the corresponding group on day 14 in Figure 15B.

[0077] Figure 16 The illustration shows the efficacy of colloidal manganese (Mn2OHPO4) combined with PD1 antibody in the treatment of a subcutaneous tumor model, melanoma B16-F10. Figure 16 Image A shows images of the tumor after different treatments. Figure 16 B shows the tumor growth curve. Figure 16 Figure C shows the tumor weight of the mice in the corresponding group in Figure B. Figure 16 DE map shows CD8 infiltrating tumor cells analyzed by flow cytometry. + T cell count.

[0078] Figure 17 The illustration shows the efficacy of colloidal manganese (Mn2OHPO4) combined with PD1 antibody drug in the treatment of a subcutaneous tumor model - colon cancer tumor MC38. Figure 17 Image A shows images of the tumor after different treatments. Figure 17 B shows the tumor growth curve. Figure 17 C shows the tumor weight of the corresponding group in Figure 17B. Figure 17 Figure D shows a tissue section of the tumor corresponding to the group in Figure A, with DAPI and CD8 assays performed. + Immunofluorescence staining of T cells. Detailed Implementation

[0079] definition

[0080] The term "innate immunity" as used in this article refers to the natural immune defense function that an organism develops during phylogenetic development and evolution; that is, the non-specific defense function possessed from birth, also known as 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-β), 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 chemokines; CX3C chemokines), lysozyme, etc.

[0081] The term "adaptive immunity" used in this article, also known as acquired immunity or specific immunity, refers to the specific immunity against an antigen that the body develops after stimulation by an antigen molecule. It involves cellular immunity and humoral immunity.

[0082] As used in this article, the term "adjuvant" refers to a reagent that does not constitute a specific antigen but enhances the strength and duration of the immune response to a co-administered antigen.

[0083] As used herein, the term "divalent manganese" or "divalent manganese compound" can refer to hydrochloride, carbonate, hydrobromide, sulfate, nitrate, phosphate, tartrate, fumarate, maleate, lactate, benzenesulfonate, pantothenate, ascorbate, hydroxide, or any combination thereof. Preferably, the divalent manganese compound is pharmaceutically acceptable.

[0084] As used herein, the term "colloidal manganese" refers to the state in which divalent manganese exists in a colloid (also known as a colloidal solution) with anions. The term "colloid" has the meaning understood by one of ordinary skill in the art. For example, the particle size of colloidal particles is typically 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 any two of these values, such as 1 to 20, 5 to 20 nm, or 10 nm. Typically, colloidal solutions exhibit the Tyndall effect when illuminated. Given that the particle size range of colloidal particles is in the nanometer range, "colloidal manganese" may also be referred to as "nano-manganese" based on its particle size range.

[0085] As used herein, the term "newly precipitated manganese" refers to the state of a divalent manganese compound after it has changed from a non-precipitated form (e.g., in solution) to a precipitated form over a short period of time. This short period generally does not exceed one 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 hour, for example, not exceeding 60, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute.

[0086] The terms used herein, such as “comprising,” “containing,” “containing,” and “including,” are not intended to be limiting. Furthermore, unless otherwise stated, “or” or “or” means “and / or.”

[0087] The phrase "derived from viruses, bacteria and / or parasites" as used in this article refers to inactivated viruses, bacteria and / or parasites, or proteins and / or nucleic acids extracted from the aforementioned pathogens (including the aforementioned substances after cutting, engineering, etc.), or purified recombinant proteins, or chemically synthesized peptides.

[0088] It should also be noted that, as used in this specification, the singular form includes the plural form of the object it refers to, unless it is clearly and explicitly limited to a single object. Furthermore, if a particular numerical value is mentioned, at least that value will be included, unless the text clearly indicates otherwise.

[0089] When a numerical value represents an approximation, it should be understood that a specific numerical value forms another implementation. As used, "approximately X" (where X is a numerical value) refers to ±10% (inclusive) of the listed value. All ranges are inclusive and can be combined, if applicable.

[0090] The term "pharmaceutically acceptable carrier" as used herein may be selected from: water, buffered aqueous solutions, isotonic saline solutions such as PBS (phosphate-buffered saline), glucose, mannitol, dextran glucose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The type of pharmaceutically acceptable carrier used depends particularly on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition according to the invention may contain lubricants, preservatives, stabilizers, humectants, emulsifiers, salts affecting osmotic pressure, buffers, coloring substances, flavoring substances, and / or aromatic substances as additives.

[0091] The pharmaceutical compositions according to the invention can be administered via any suitable route, such as orally, nasally, intradermally, subcutaneously, intramuscularly, or intravenously.

[0092] The term “application” as used in this article means the delivery of a substance to a subject in a pharmacologically feasible manner.

[0093] As used in this article, "effective dose" refers to a dose sufficient to demonstrate its benefit to the recipient. The actual amount administered, as well as the rate and duration of administration, will depend on the individual's condition and severity. Prescribing treatment (e.g., determining dosage) is ultimately the responsibility of and depends on the general practitioner and other physicians, who typically consider the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0094] As used in this article, the term "object" means animal, including warm-blooded mammals such as humans and primates; birds; domesticated or farm animals such as cats, dogs, sheep, goats, cattle, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; fish; reptiles; zoo animals and wild animals, etc.

[0095] Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0096] Unless otherwise stated, any component, element, property, or step disclosed in one embodiment of this method and product may be applied to any other method and product disclosed herein.

[0097] Every patent, patent application, publication cited, or description in this disclosure is incorporated herein by reference in its entirety.

[0098] The invention is further defined in the embodiments below. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. From the above discussion and these examples, those skilled in the art can determine the essential features of the invention, and can make various changes and modifications to adapt it to a wide range of uses and conditions without departing from its essence and scope.

[0099] Example

[0100] Materials and methods

[0101] Antibodies and reagents

[0102] The antibodies were obtained as follows: anti-GAPDH antibody (sc-25778) was purchased from Santa Cruz. Anti-Viperin antibody, anti-ISG54 antibody, anti-Casp1 / p20 antibody, anti-IL1β / p17 antibody, and anti-ASC antibody were all prepared and used by the published method (16). In short, the cDNA of the antigen fragment was inserted into the pET-21b vector (Novagen) and expressed in E. coli BL21(DE3). The recombinant protein was purified by Ni-NTA affinity column and then injected into mice or rabbits to obtain antiserum that could recognize the corresponding antigen.

[0103] All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Aluminum adjuvant (ImjectAlum, Thermo, 77161), mouse IL-1beta ELISA Kit (MULTI SCIENCES, EK201B2 / 2), mouse IL-18 ELISA Kit (MULTI SCIENCES, EK2181), lipopolysaccharide (Sigma, L4130), and ovalbumin (InvivoGen, #vac-pova) were all commercially available products.

[0104] cell

[0105] L929-ISRE (a cell line created in Jiang Zhengfan's laboratory, with pGL3 ISRE-Luciferase plasmid stably transfected 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 μg / ml penicillin, and 10 μg / ml streptomycin. Bone marrow-derived macrophages (BMDCs) were induced in RPMI-1640 (Gibco) medium containing 10 ng / mL GM-CSF, 10 ng / mL IL-4, and 10% FBS (Gibco), with half the medium replaced on day 3, and experiments performed on day 7. Peritoneal macrophages were harvested from mice 5 days after induced with mercaptoglycol (BD, Sparks, MD) injection and cultured in DMEM medium supplemented with 5% FBS.

[0106] mice

[0107] Tmem173 - / - The mouse model was established using the CRISPR-Cas9 method, by injecting Cas9 mRNA (100 ng / μl) and gRNA (50 ng / μl) into the cytoplasm of C57BL / 6J mouse zygotes. Cas9 mRNA and single guide RNA (gRNA) were transcribed in vitro using mMESSAGE mMACHINE T7 Ultra (Ambion, am1345). Nlrp3 - / - Nlrc4 - / - Pycard - / - and Aim2 - / - The gene knockout mice were generously provided by Vishva Dixit (Genentech Inc, USA). (Tmem173) - / - Pycard - / - Double knockout mice were created by using Tmem173 - / - Mice and Pycard - / - Obtained through identification after mating.

[0108] All mice were fed under sterile conditions at the Peking University Laboratory Animal Center in accordance with the NIH Guide for Care and Use of Laboratory Animals.

[0109] Type I IFN Bioassay

[0110] The concentration of type I IFN was determined according to a publicly available method (17). In short, an IFN-sensitive luciferase vector was constructed by cloning an IFN-stimulated response element (ISRE) into the pGL3-Basic vector (Promega), which was then stably transfected into L929 cells. L929-ISRE cells were seeded into 96-well plates and incubated with cell culture supernatant. Recombinant human and mouse IFN-β (R&D Systems) was used as a standard. After 4 hours, L929-ISRE cells were lysed, and the concentrations were measured using the Luciferase Reporter Assay System (Promega).

[0111] Viral infection

[0112] Herpes simplex virus 1 (HSV-1, from Hongbing Shu, Wuhan University) VR-1544) and vesicular stomatitis virus (VSV, Indiana strain, VR-1238 was kindly donated by the aforementioned colleagues. 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). HSV-1 and VSV viral titers were determined using BHK21 cells. Influenza virus titers were determined using MDCK cells.

[0113] Virus amplification and purification: HSV-1 and VSV viruses were amplified using Vero cells. PR8, WSN, and H3N2 influenza viruses were amplified using chicken embryos. The amplified viruses were purified using PEG8000, and the purified viruses were inactivated with 0.2% formaldehyde at 37°C for 24 hours. The complete inactivation of the virus was measured using the plaque assay. The inactivated viruses were then used to immunize mice.

[0114] Mouse survival experiment: using HSV-1 (1.4 x 10⁻⁶) 7 PFU / mouse) and VSV (8 x 10) 8 Intravenous infection of mice with PFU (1 x 10^9) for 8-12 weeks, or with PR8 (1 x 10^9) 5 PFU / mouse), WSN (1 x 10) 6 PFU / mouse), H3N2 (1 x 10) 6 PFU / mouse) was administered intranasally to mice for 8-12 weeks.

[0115] Plaque assay: BHK21 cells were incubated for 2 hours with homogenates from infected mouse organs (in a series of dilutions in serum-free DMEM). The medium was then replaced with serum-free DMEM containing 0.5% methylcellulose. After 60 hours, cells were fixed with 0.5% (vol / vol) glutaraldehyde and stained with 1% (wt / vol) crystal violet (dissolved in 70% ethanol). Plaques were counted to calculate the viral titer in plaque-forming units.

[0116] Protein expression and purification

[0117] The influenza virus HA1 protein purification plasmid was provided by the laboratory of Zhang Yonghui at Tsinghua University (18). The plasmid expresses amino acids 11-324 of the HA protein (Gene ID: 956529). When the bacterial culture was shaken to OD 600 When the concentration was 0.6, 0.5 mM IPTG was added for induction, and the mixture was incubated at 37°C for 5 hours. After harvesting the bacteria, inclusion bodies were collected by sonication, and the HA1 protein was obtained after denaturation and renaturation. The HA1 protein was then purified by gel filtration chromatography.

[0118] Ovalbumin (OVA) was used as an immunogen to immunize mice.

[0119] For intramuscular immunization with MnCl2 solution, add 10 μg MnCl2 and 10 μg OVA protein to 100 μL PBS, mix well, let stand for 5 minutes, and then inject intramuscularly into mice. For intranasal immunization with MnCl2 solution, add 5 μg MnCl2 and 10 μg OVA protein to 20 μL PBS, mix well, let stand for 5 minutes, and then inject intranasally into mice. For intramuscular immunization with colloidal manganese (using Mn2OHPO4 as an example), add 10 μg colloidal manganese and 10 μg OVA protein to 100 μL physiological saline, mix well, and then inject intramuscularly into mice. For intranasal immunization with colloidal manganese, add 5 μg colloidal manganese and 10 μg OVA protein to 20 μL physiological saline, mix well, and then inject intranasally into mice. For intramuscular immunization with aluminum adjuvant, add 10 μg / 20 μL aluminum adjuvant and 10 μg OVA protein to 80 μL physiological saline, emulsify using an emulsifier, and then inject intramuscularly into mice. The method for nasal immunization with cholera toxin B (CTB) is to add 5 μg CTB and 10 μg OVA protein to 20 μL of physiological saline, mix well, and then immunize mice with nasal drops.

[0120] ELISA method for determining OVA-specific IgG1, IgG2c, total IgG, and IgA antibodies: Diluted serum from immunized mice was incubated on an ELISA plate coated with 100 μg / ml OVA. After washing, the corresponding bound antibodies were detected using HRP-conjugated mouse IgG1 (eBioscience, #18-4015-82), IgG2c (GeneTex, GTX77297), IgG total (Invitrogen, G21040), and IgA (GeneTex, GTX77223). The plate was then incubated with the substrate TMB (eBioscience), the reaction was stopped with 1M H3PO4, and the absorbance was measured at 450 nm.

[0121] Statistical analysis

[0122] The data were analyzed using a t-test. The survival curves were compared using the Mantel-Cox test.

[0123] Example 1. The state of manganese compounds in solution and their effect as adjuvants

[0124] Experiment (a) MnCl2 reacts with PBS to produce a precipitate, as described below, to prepare colloidal manganese Mn2OHPO4.

[0125] like Figure 1 As shown in Figure A, the physiological saline (0.9% NaCl) used in the experiment was prepared by the experiment itself, and PBS (pH 7.4) was purchased from Gibco. There were three reaction systems: 100 μl MnCl2 (0.2 M) added to 900 μl physiological saline; 100 μl MnCl2 (0.2 M) added to 900 μl PBS; and 50 μl Na3PO4 added to 850 μl physiological saline, followed by the addition of 100 μl MnCl2 (0.2 M) to prepare colloidal manganese Mn2OHPO4. The molecular formula of this colloidal manganese was determined by X-ray photoelectron spectroscopy (XPS). No precipitate was formed in physiological saline, but a white precipitate formed after reacting overnight in PBS. The colloid formed in the third reaction did not precipitate. The Tyndall effect was observed in the product of the third reaction under light. The fine structure of the colloidal manganese was imaged using a transmission electron microscope, as shown below. Figure 1 As shown in B, the particle has a diameter of approximately 10 nm.

[0126] Experiment (b) compares the adjuvant effects of manganese compound precipitation and colloidal manganese in PBS.

[0127] Mice were randomly divided into 6 groups according to body weight: 1) PBS control group, 2) OVA antigen (10 μg), 3) MnCl2 reacted in physiological saline for 2 days (10 μg MnCl2 + OVA), 4) MnCl2 reacted in PBS for 5 minutes (10 μg MnCl2 + OVA), 5) MnCl2 reacted in PBS for 2 days (10 μg MnCl2 + OVA), and 6) MnCl2 reacted to generate colloidal manganese for 30 days (10 μg Mn2OHPO4 + OVA). Here, 10 μg refers to the mass of manganese. Each group was immunized intramuscularly according to the experimental design, with immunizations on days 0, 7, and 14. Blood was collected on day 21, serum was separated, and the level of anti-OVA IgG1 antibody was measured using ELISA. Figure 1 As shown in Figure C, MnCl2 particles reacting in PBS for a short time exhibit an adjuvant effect, but the effect weakens after a longer reaction time due to aggregation into larger particles. In contrast, colloidal manganese (abbreviated as MnJ in the figure) Mn2OHPO4 maintains a good effect even after one month. This indicates that colloidal manganese (Mn2OHPO4) is an effective and stable adjuvant.

[0128] Experiment (c) Comparison of residence time of MnCl2 solution and colloidal manganese Mn2OHPO4 at the injection site

[0129] Mice were randomly divided into two groups according to body weight: an intramuscular injection group of MnCl2 solution (20 μg MnCl2 dissolved in 100 μl physiological saline) and an intramuscular injection group of colloidal manganese (20 μg colloidal manganese suspended in 100 μl physiological saline). Here, 20 μg refers to the mass of manganese. At different time points after injection, 0.1 g of muscle sample was taken from the injection site, digested using a microwave digester, and then the manganese content was determined using ICP-MS (ThermoX SERIES II). Figure 1 As shown in D, colloidal manganese can remain at the injection site for a longer period of time, thus activating the body to produce antibodies more persistently, while solution manganese is quickly metabolized.

[0130] Example 2. Properties of precipitates formed by the reaction of manganese(II) ions with other anions and their cellular effects

[0131] Experiment (a) Reaction of fixed concentration of manganese ions (II) with different concentrations of anions

[0132] In a physiological saline reaction system, 20 mM MnCl2 is mixed with other anions from sodium salts (Na2CO3, NaHCO3, Na3PO4, Na2HPO4, NaOH) at different concentrations (2.5, 5, 10, 20, 30, 40 mM). Figure 2 As shown in A, divalent free manganese reacts with CO3. 2- The reaction produces a black precipitate of MnCO3; and reacts with HCO3 to form... - The reaction produces a white precipitate of Mn(HCO3)2; and reacts with PO4. 3- The reaction produces products in two states: a white precipitate at low concentrations and a slightly yellow colloidal substance at high concentrations. Analysis revealed that the product at low concentrations is Mn3(PO4)2, while the product at high concentrations is Mn2OHPO4; divalent free manganese reacts with HPO4. 2- The reaction produces white MnHPO4; and reacts with OH... - The reaction produces a slightly yellow precipitate of Mn(OH)2.

[0133] Experiment (b) compared the ability of different manganese ion (II) products to activate type I interferon and inflammatory cytokines to produce IL-1β and their adjuvant effects.

[0134] like Figure 2As shown in Figure B, following the order from left to right, in an Eppendorf tube, add 100 μL of 0.2 M MnCl2 to 900 μL of physiological saline and let it stand overnight (MnCl2), then add 100 μL of 0.5 M NaHCO3 + 800 μL of physiological saline and let it stand overnight (Mn(HCO3)2), then add 100 μL of 0.5 M Na2CO3 + 800 μL of physiological saline and let it stand overnight (MnCO3), then add 20 μL of 0.5 M Na2CO3 + 20 μL of 0.5 M NaOH + 860 μL of physiological saline and let it stand overnight (Mn2OHCO3), then add 100 μL of 0.5 M NaH2PO4 + 800 μL of physiological saline and let it stand overnight (Mn(H2PO4)2), then add 100 μL of 0.5 M... The reactions were: Na₂HPO₄ + 800 μL physiological saline overnight (MnHPO₄); 26.6 μL 0.5M Na₃PO₄ + 873.4 μL physiological saline overnight (Mn₃(PO₄)₂); 50 μL 0.5M Na₃PO₄ + 850 μL physiological saline overnight (Mn₂(OH)PO₄); and 100 μL 0.5M NaOH + 800 μL physiological saline overnight (Mn(OH)₂). The reactions produced colloidal or solid precipitates as shown in the figure. The precipitates were washed once with physiological saline before subsequent experiments (MnCl₂ was not washed because it did not precipitate in physiological saline; the solution was used for the experiments). Equimolar amounts (containing 500 μM Mn) were used. 2+ The various products of Mn(H2PO4)2 are readily soluble in water, with little precipitation (less than 500 μM). Other anions can cause Mn to precipitate. 2+ Complete precipitation) Stimulation 2×10 6 The level of type I interferon in the supernatant of THP1 cells was measured after 18 hours. Results are as follows: Figure 2 As shown in Figure B, Mn₂OHPO₄ and MnHPO₄ significantly activated the production of type I interferon. P-IRF3, shown in the figure, is the main transcription factor regulating type I interferon expression. Mn₂OHPO₄ and MnHPO₄ also significantly induced the production of the inflammatory factor IL-1β. In contrast, non-colloidal Mn(OH)₂ only activated the production of type I interferon and did not induce the production of the inflammatory factor IL-1β. Mice were immunized three times with 10 μg of each product (10 μg refers to the mass of manganese) mixed with 10 μg of OVA protein. The serum of the mice was then used to measure the content of anti-OVA IgG1 antibodies. Mn₂OHPO₄ and MnHPO₄ were significantly more effective than precipitated manganese compounds in stimulating antibody production. The leftmost MnCl₂ in the antibody test represents the adjuvant effect of precipitation after reacting MnCl₂ with PBS for 5 minutes. Other solid precipitates left overnight also showed some adjuvant effect. We selected the most effective colloidal manganese Mn₂OHPO₄ for subsequent experiments.

[0135] Experiment (c) Comparison of the Tyndall effect of different manganese ion (II) products

[0136] like Figure 2 As shown in Figure C, the reaction products were resuspended in physiological saline and then irradiated with a laser pointer. It can be seen that the Tyndall effect is most pronounced in Mn₂OHPO₄, while MnHPO₄ exhibits partial Tyndall effect, possibly because its diameter is slightly larger than 100 nm. The diameter of Mn₂OHPO₄ is approximately 10 nm, which conforms to the definition of a colloid.

[0137] Example 3. Colloidal manganese Mn2OHPO4 as an adjuvant for intramuscular and mucosal immunotherapy

[0138] Experiment (a) Colloidal manganese Mn2OHPO4 as an intramuscular adjuvant

[0139] Mice were randomly divided into six groups according to body weight: PBS control group, OVA antigen (10 μg), colloidal manganese Mn2OHPO4 group (10 μg Mn2OHPO4 + OVA), low-dose aluminum adjuvant group (440 μg aluminum adjuvant + OVA), medium-dose aluminum adjuvant group (800 μg aluminum adjuvant + OVA), and high-dose aluminum adjuvant group (1220 μg aluminum adjuvant + OVA). Each group was immunized intramuscularly according to the experimental design, twice a day, on day 0 and day 10. Blood samples were collected on days 10, 17, 24, and 31, serum was separated, and the level of anti-OVA IgG1 antibody was measured using ELISA. Figure 3 As shown, colloidal manganese (Mn₂OHPO₄) at a dose of 10 μg / mouse was more effective than a high dose (1220 μg / mouse) of aluminum adjuvant. This indicates that colloidal manganese can achieve better immune-enhancing effects at doses less than 100 times that of aluminum adjuvant, which could significantly reduce the side effects of currently widely used aluminum adjuvant.

[0140] Experiment (b) Colloidal manganese Mn2OHPO4 as a mucosal adjuvant

[0141] like Figure 4 As shown, equal volumes (5 μg / mouse) of colloidal manganese Mn2OHPO4 and the classic mucosal adjuvant cholera toxin B (CTB) were mixed with OVA antigen (10 μg) and administered via intranasal immunization to mice. Immunization was repeated on days 0, 7, and 14. On day 21, blood was collected to separate serum, and oral lavage fluid and bronchoalveolar lavage fluid were collected. The levels of IgA and IgG1 antibodies against OVA were measured using ELISA. Figure 4 As shown, when colloidal manganese Mn₂OHPO₄ was used as a mucosal adjuvant to immunize mice, the antibody levels in the bronchoalveolar lavage fluid were higher than those using cholera toxin B as an adjuvant. In oral lavage fluid and serum, Mn₂OHPO₄ was as effective as an equivalent amount of cholera toxin B. This indicates that colloidal manganese Mn₂OHPO₄ is also a good mucosal immune adjuvant.

[0142] Example 4. Colloidal manganese Mn2OHPO4, as an intramuscular and mucosal immunoadjuvant, can induce the body to produce specific antibodies in a sustained manner.

[0143] Experiment (a) Colloidal manganese Mn2OHPO4 as an intramuscular adjuvant has a long-lasting effect

[0144] Using the method described in Example 3, colloidal manganese Mn2OHPO4 was mixed with OVA and injected intramuscularly into mice. The levels of anti-OVA antibodies in mouse serum were measured at 1 week, 1 month, 3 months, and 6 months post-immunization. Figure 5 As shown in Figure A, serum anti-OVA antibodies remained at a high level until month 6, indicating that colloidal manganese Mn₂OHPO₄, as an adjuvant, can produce a durable protective effect. Mice typically live for two years, and it is expected that colloidal manganese Mn₂OHPO₄ can induce the production of protective antibodies throughout the mouse's lifespan.

[0145] Experiment (b) Colloidal manganese Mn2OHPO4 as a mucosal adjuvant has a long-lasting effect.

[0146] Following the method described in Example 3, mice were immunized with mucosal fluid, and blood, bronchoalveolar lavage fluid, and oral lavage fluid were collected at different time points to determine antibody levels. Figure 5 As shown in B, the concentration of specific antibodies remained at a high level until 6 months after immunization, indicating that colloidal manganese Mn2OHPO4 can also produce a long-lasting protective effect when used for mucosal immunization.

[0147] Example 5. Manganese ions (II) activate the production of inflammatory factors.

[0148] The inflammatory factor IL-1β can directly act on CD4. + T cells, promote T cell proliferation (8), IL-1α and IL-1β can promote neutrophil infiltration (19); the inflammatory factor IL-18 can effectively enhance the Th1 immune response, and can also promote the proliferation and cytotoxic effects of T cells and NK cells (9). It has been reported that the adjuvant effect of aluminum adjuvant is achieved by activating the NLRP3 inflammasome and releasing inflammatory factors (20-22), so we tested and compared the ability of MnCl2 / colloidal manganese Mn2OHPO4 and aluminum adjuvant to activate inflammatory factors.

[0149] MnCl2 and colloidal manganese Mn2OHPO4 have the same ability to activate inflammatory factors (see...) Figure 2B) and the mechanism, using MnCl2 as an example only. Primary cultures of mouse peritoneal macrophages were treated with manganese chloride or aluminum adjuvant at concentrations of 10, 20, 50, and 100 μg / mL, respectively. Western blotting was used to detect the interferon-inducible genes ISG54 and Viperin, and a Bioassay was used to detect the total type I interferon content (the sum of interferon α and interferon β concentrations) in the supernatant. Results are as follows... Figure 6 As shown in A and 6B, only MnCl2 could activate the expression of type I interferon, while aluminum adjuvant could not. Peritoneal macrophages pretreated with LPS were treated with the same concentrations of manganese chloride and aluminum adjuvant. Western blotting was used to detect the cleavage of caspase 1 and IL-1β in the supernatant (the upper band represents the precursor, and the lower band represents the active forms of caspase 1 and IL-1β). ELISA was used to detect the production of the inflammatory factors IL-1β and IL-18 in the supernatant. Results are as follows... Figure 6 As shown in C and 6D, MnCl2 significantly activated inflammatory factors more effectively than aluminum adjuvant. LPS-pretreated peritoneal macrophages were treated with MnCl2, ATP (the classic NLRP3 inflammasome activator), and VacV (the classic Aim2 inflammasome activator). Western blotting was used to detect the cleavage of caspase 1 and IL-1β in the supernatant (the upper band represents the precursor, and the lower band represents the active forms of caspase 1 and IL-1β). ELISA was used to detect the production of the inflammatory factors IL-1β and IL-18 in the supernatant. Results are as follows: Figure 6 As shown in E and 6F, in NLRP3 knockout cells, the ability of MnCl2 to activate inflammatory factors is significantly weakened; therefore, MnCl2 mainly activates the NLRP3 inflammasome.

[0150] Example 6. Colloidal manganese Mn2OHPO4 promotes T cell proliferation

[0151] 1×10 CFSE dye markings 6 OT-II cells were injected into mice via the tail vein. One day later, mice were immunized with OVA protein in the following groups: a PBS control group, control groups with different amounts of OVA (1 μg, 2 μg, and 5 μg), groups containing 10 μg colloidal manganese mixed with 1 μg, 2 μg, and 5 μg of OVA protein, and groups containing 1220 μg aluminum adjuvant mixed with 1 μg, 2 μg, and 5 μg of OVA protein. All dosages described here refer to a single immunization of one mouse. Three days after immunization, lymph nodes were harvested, and the proliferation of OT-II cells in the lymph nodes was analyzed by flow cytometry.

[0152] like Figure 7As shown in the figure, the rightmost peak represents the original OT-II cells injected into mice. After cell division, CFSE is evenly distributed between the two cells, causing a decrease in CFSE intensity. The cells with decreased CFSE on the left are the dividing cells. The results show that, using the same amount of antigen, colloidal manganese promotes T cell proliferation more effectively than aluminum adjuvant, requiring only 1 / 100th the amount of aluminum adjuvant to achieve a similar effect.

[0153] Example 7. Colloidal manganese Mn2OHPO4 activates antibody production, which depends on the STING and inflammasome pathways.

[0154] like Figure 8 As shown, Figure 8 In mice treated with 10 μg / mouse colloidal manganese and 10 μg / mouse OVA protein via intramuscular injection, WT, Pycard knockout (ASC protein deficiency), Sting knockout (STING protein deficiency), and double knockout mice (Pycard and Sting protein deficiency) were administered three injections. Serum samples were then collected and ELISA was used to measure the levels of anti-OVA antibodies IgG1, IgG2c, and total IgG. The results showed that knockout alone only partially eliminated the adjuvant effect of colloidal manganese, while double knockout significantly reduced the adjuvant effect. Figure 8 B involved immunizing mice three times with 5 μg / mouse colloidal manganese and 10 μg / mouse OVA protein via the mucosa. Serum, bronchoalveolar lavage fluid, and oral lavage fluid were then collected and the levels of secretory anti-OVA IgA antibodies were measured using ELISA. Results showed that the adjuvant effect was significantly reduced in double-knockout mice.

[0155] Example 8. Colloidal manganese Mn2OHPO4 promotes dendritic cell maturation

[0156] like Figure 9 As shown, Figure 9 A. Bone marrow-derived dendritic cells (BMDCs) were treated with 100, 200, and 400 μM colloidal manganese for 20 hours, and the CD86 protein, a maturation marker of dendritic cells, was detected by flow cytometry. The results showed that in wild-type mice, increasing concentration promoted dendritic cell maturation. However, double knockout cells did not promote cell maturation. Figure 9 In B, the efficiency of colloidal manganese in promoting T cell proliferation was tested in four mouse species using the method described in Example 6. The results showed that in double-knockout mice, the ability of colloidal manganese adjuvant to promote T cell proliferation was significantly reduced, which was consistent with dendritic cell maturation. Figure 9 In C, wild-type mice and double-knockout mice were injected intramuscularly with 10 μg / mouse of colloidal manganese and 10 μg / mouse of OVA protein. CD8 was analyzed by flow cytometry. + The content of OVA tetramer protein on the surface of T cells (characterizing adjuvant-activated OVA-specific CD8+)+ (T cells). The results showed that colloidal manganese could activate CD8. + T-cell immune responses depend on two pathways: STING and ASC.

[0157] Example 9. Colloidal manganese Mn2OHPO4 as an adjuvant to enhance the immunoprotective effect against inactivated vesicular stomatitis virus (VSV) and herpes simplex virus (HSV-1).

[0158] Experiment (a) VSV inactivated virus vaccine

[0159] The experimental procedure is as follows Figure 10 As shown in Figure A, mice were injected intramuscularly with the vaccine on day 0, infected with the virus on day 10, and the viral titer in mouse tissues was measured on day 14. Mice were observed and the mortality curve was recorded up to day 14. The VSV virus purified by PEG was inactivated and used as the vaccine, diluted to 10... -1 10 -2 10 -3 10 -4 10 -5 and 10 -6 Mice were immunized by intramuscular injection at various dilutions. When the viral concentration was sufficiently high, use alone was sufficient to produce adequate immunity and protect mice; mice were immunized after several 10-fold dilutions of the virus. At a concentration of 10... -3 At that concentration, the survival rate of mice was only 8.3%, and at a concentration of 10... -4 10 -5 10 -6 At 10, all the mice died; but at 10, all the mice died. -3 Adding Mn2OHPO4 colloid as an adjuvant to inactivated virus at a certain concentration increased the survival rate of mice to 75%. Figure 10 As shown in B and 10C, colloidal manganese, as an adjuvant, can enhance the immune effect of low-concentration immunogens, providing protection with small amounts of immunogen, and also reducing the titer of active viruses in mice. Figure 10 As shown in D.

[0160] Experiment (b) HSV-1 inactivated virus vaccine

[0161] HSV-1 virus purified by PEG was inactivated and used as a vaccine for intramuscular immunization of mice. When the viral dilution was 10... -1 10 -2 At that time, using it alone could produce sufficient immune effects to protect mice, but when the viral dilution was reduced to 10... -4 At 10, the survival rate of mice was only 16.7%, and in some cases, all mice died; while at 10 -4 Adding Mn2OHPO4 colloid to diluted virus increased the survival rate of mice to 100%. Figure 10As shown in E and 10F. Furthermore, the active viral titer in mice was reduced, as... Figure 10 As shown in G. 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.

[0162] Experiment (c) PR8 inactivated virus vaccine mucosal immunization once

[0163] The PEG-purified influenza PR8 virus was inactivated and diluted to 1:10. -1 10 -2 10 -3 Dilution, mice were immunized once intranasally. Seven days later, mice were infected with influenza virus, and survival rate and weight changes were recorded. All doses without adjuvant showed no protective effect; mice continued to lose weight until death. However, with the addition of Mn2OHPO4 colloid as adjuvant, the concentration was reduced to 10. -1 The viral load caused mice to regain their body weight after a decrease on days 4 and 5, and by day 10, their weight had returned to pre-infection levels, indicating improvement in their condition. Figure 11 As shown in A and 11B. This result indicates that colloidal manganese, as an adjuvant, can enable immunogens that are originally non-immunogenic or weakly immunogenic to produce a protective effect after a single immunization.

[0164] Experiment (d) Two mucosal immunizations with PR8 inactivated virus vaccine

[0165] like Figure 11 As shown in C and 11D, the PEG-purified influenza PR8 virus was inactivated and diluted to 1, 10... -1 10 -2 10 -3 Diluted mice were immunized twice intranasally (on days 0 and 7). Fourteen days later, mice were infected with influenza virus, and survival rate and weight changes were recorded. All doses without adjuvant showed no protective effect; mice continued to lose weight until death. However, with the addition of Mn2OHPO4 colloid as adjuvant, the dilution to 10... -1 When mice were immunized twice with the diluted viral dose, their body weight only decreased by 10% after infection and quickly recovered; however, immunization with the undiluted viral dose did not result in a decrease in body weight or the development of infection symptoms. These results indicate that when colloidal manganese is added as an adjuvant to a vaccine that initially had no immunizing effect, two immunizations can achieve a significant preventative effect.

[0166] Example 10. Colloidal manganese Mn2OHPO4 as an adjuvant to enhance the protective effect of influenza subunit vaccines.

[0167] Mice were randomly divided into four groups according to body weight: a blank control group (ctrl), a HA antigen group (HA), a HA antigen and colloidal manganese group (HA+Mn), and a HA antigen and aluminum adjuvant group (HA+Al). After intramuscular injection, the serum levels of anti-HA protein IgG1 antibodies were measured on days 14, 21, and 28. Figure 12 As shown in Figure A, no detectable specific antibodies were produced when HA protein (5 μg / mouse) was used alone. However, when Mn2OHPO4 colloid (50 μg / mouse) was added as an adjuvant, the amount of anti-HA IgG1 antibodies produced by intramuscular immunization was significantly higher than that produced by aluminum adjuvant (800 μg / mouse). Comparing the effects of colloidal manganese and CTB as adjuvants for intranasal immunization, when Mn2OHPO4 colloid (20 μg / mouse) was added as an adjuvant, the anti-HA IgA antibody produced by intranasal immunization was significantly higher than that produced by CTB (5 μg / mouse) as an adjuvant after 4 weeks of immunization. Furthermore, immunization of mice with colloidal manganese as an adjuvant did not result in any change in mouse weight or lung inflammation; while immunization of mice with aluminum adjuvant and CTB as adjuvants resulted in a slight decrease in mouse weight followed by recovery, and a small amount of lung inflammation. Figure 12 As shown in C and 12D. These results indicate that colloidal manganese can serve as a subunit vaccine adjuvant with higher safety and better efficacy than traditional adjuvants.

[0168] Example 11. Colloidal manganese Mn2OHPO4 as an adjuvant enhances the protective effect of inactivated influenza virus and subunit vaccines against both homologous influenza virus (WSN) and heterologous influenza virus (H3N2).

[0169] Experiment (a) showed that a vaccine made from colloidal manganese Mn2OHPO4 mixed with PR8 inactivated virus or PR8-HA1 protein could prevent WSN virus infection.

[0170] Mice were randomly divided into 5 groups according to body weight: 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). Mice were immunized once on day 0, day 7, and day 14. Mice were infected with WSN virus on day 21, and mouse body weight was recorded for 14 consecutive days. Figure 13 As shown in Figure A, the mice in the experimental group with added colloidal manganese did not lose weight, indicating excellent protection against the virus. In contrast, the mice in the control group without adjuvant and the blank control group lost weight rapidly.

[0171] Experiment (b) showed that a vaccine made from colloidal manganese Mn2OHPO4 mixed with PR8 inactivated virus or PR8-HA1 protein could prevent H3N2 virus infection.

[0172] Mice were randomly divided into 5 groups according to body weight: 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). Mice were immunized once on day 0, day 7, and day 14. Mice were infected with H3N2 virus on day 21, and mouse body weight was recorded for 14 consecutive days. Figure 13 As shown in B, the toxicity of the H3N2 virus did not cause death in mice. In the blank control group and the inactivated virus group alone, the weight of mice dropped to 80% before recovering. In the HA1 group alone, the weight dropped to 90% before recovering. However, the weight of the HA1 group with added colloidal manganese did not drop much, and the weight of the inactivated virus group with added colloidal manganese only dropped by 5% before recovering.

[0173] Example 12. Colloidal manganese Mn2OHPO4 as an immune enhancer to enhance the body's anti-tumor effect.

[0174] Experiment (a) Colloidal manganese Mn2OHPO4 vaccine inhibits in situ tumor growth

[0175] Mice were randomly divided into four groups according to body weight: a blank control group (con), an OVA control group, an OVA and colloidal manganese group (OVA+Mn), and an OVA and aluminum adjuvant group (OVA+Al). After immunizing mice three times (every 7 days), a melanoma B16-OVA was subcutaneously injected to establish an orthotopic tumor model. Figure 14 As shown in A to 14C, pretreatment with aluminum adjuvant did not inhibit tumor growth or improve mouse survival; however, pretreatment with colloidal manganese significantly inhibited tumor growth and improved mouse survival. Twenty-one days after tumor inoculation, the average tumor size in the control group mice was 1906 mm. 3 The average tumor size in the OVA group mice was 925.5 mm. 3 The average tumor size in mice immunized with colloidal manganese was 222.9 mm. 3 The average tumor size in the aluminum adjuvant group mice was 1946 mm. 3 ,like Figure 14 As shown in B; the survival time of control mice was 26 days, while the survival time of mice immunized with colloidal manganese was 49 days, significantly longer than the 27 days of mice immunized with adjuvant. Figure 14 As shown in Figure C. This result indicates that colloidal manganese can act as an immune enhancer to strengthen the body's antigenic tumor response.

[0176] Experiment (b) Colloidal manganese Mn2OHPO4 vaccine inhibits tumor metastasis

[0177] Mn2OHPO4 colloid and aluminum adjuvant were mixed with OVA antigen and immunized in mice, with immunizations repeated every 7 days for 3 times to activate the in vivo immune response. B16-OVA tumors were injected via tail vein to establish a tumor metastasis model. Mice were sacrificed 21 days after tumor injection, and lung tissue was removed. The lungs of mice in the blank control group were found to be covered with melanomas, while mice immunized only with OVA showed a certain number of melanomas in their lungs. Mice immunized with OVA + colloidal manganese showed almost no tumors in their lungs. Figure 14 As shown in D. After grinding the tumor tissue, the number of tumor cells was counted using flow cytometry. The results showed that the number of tumor cells in the lungs of mice immunized with OVA + colloidal manganese was significantly lower than in the other two groups. Figure 14 As shown in Figure E. This result indicates that colloidal manganese can act as an immune enhancer to strengthen the body's anti-metastatic tumor effect.

[0178] Experiment (c) Antitumor effect of colloidal manganese Mn2OHPO4 combined with cyclophosphamide

[0179] Mice were subcutaneously inoculated with melanoma B16F10 and randomly divided into four groups: control group (con), Mn2OHPO4 colloid group (5 mg / kg), Cyclophosphamide Monohydrate (CTX) group (140 mg / kg), and combination therapy group (CTX + Mn). Cyclophosphamide (CTX) is a widely used antitumor chemotherapeutic drug in clinical practice. Mn2OHPO4 colloid was administered intramuscularly (every two days after tumor inoculation), CTX was administered intraperitoneally (on days 6, 9, and 12 after tumor inoculation), and the control group received an equal volume of physiological saline. Figure 15 As shown, the combination of colloidal manganese and CTX significantly inhibited tumor growth. Fourteen days after tumor inoculation, mice were sacrificed, and the tumors were dissected and their volume measured. The average tumor size in the control group was 960.1 mm. 3 In mice, the tumor size decreased to 659.5 mm when treated with colloidal manganese alone. 3 In mice treated with CTX alone, the tumor size was 393.9 mm. 3 The combined treatment resulted in a tumor size of 238 mm in mice. 3 ,like Figure 15 As shown in Figure B; simultaneously, the weight of each tumor was measured, and the results showed that the combined use of colloidal manganese and CTX inhibited tumor growth, such as... Figure 15 As shown in Figure C, this indicates that colloidal manganese, as an immune enhancer, enhances traditional treatments such as chemotherapy, and when used in combination, has a significant inhibitory effect on tumor growth.

[0180] Experiment (d) Combination of colloidal manganese Mn2OHPO4 with PD-1 antibody for anti-melanoma

[0181] Mice were subcutaneously inoculated with melanoma B16F10 and randomly divided into four groups: control group (con), Mn2OHPO4 colloid group (5 mg / kg), anti-PD1 antibody group (200 μg / mouse), and combination therapy group (anti-PD1 + Mn). PD1 antibody is a relatively new drug used in immunotherapy in recent years. PD1 antibody (Clone 29 F.1A12, BioXCell) was administered once each on days 3, 7, and 11 after tumor inoculation, with 200 μg of antibody dissolved in 200 μL of PBS and injected intraperitoneally each time. Figure 16 As shown in AC, 15 days after tumor inoculation, mice were sacrificed, and the tumors were dissected and measured. The average tumor size in the control group mice was 1847 mm. 3 The average tumor size in mice treated with colloidal manganese alone was 797.3 mm. 3 The average tumor size in mice in the PD1 antibody-only group was 687.3 mm. 3 The average tumor size in the combination treatment group of mice was 342 mm. 3 The levels were significantly lower than those obtained using PD1 antibody alone. Flow cytometry analysis of infiltrating CD8+ cells in tumors... + The proportion of T cells was increased by colloidal manganese, which improved the efficacy of PD-1 antibody drugs from 27.7% to 45.4%. This indicates that colloidal manganese can synergistically treat melanoma with PD-1 antibody drugs.

[0182] Experiment (e) Combination of colloidal manganese Mn2OHPO4 with PD-1 antibody for anti-colon cancer tumors

[0183] Mice were subcutaneously inoculated with MC38 colon cancer tumors and randomly divided into four groups: a control antibody group (Isotype, 200 μg / mouse), a Mn2OHPO4 colloid and control antibody group (Isotype + Mn), an anti-PD1 antibody group (200 μg / mouse), and a combination therapy group (anti-PD1 + Mn). PD-1 administration was the same as in experiment (d). Twenty days after tumor inoculation, mice were sacrificed, and the tumors were dissected and measured. The average tumor size in the control group was 319.8 mm. 3 The size of mice treated with manganese alone was 126.9 mm. 3 In mice treated with PD1 antibody alone, the tumor size was reduced to 143.1 mm. 3 The combined treatment reduced the tumor size in mice to 66.74 mm. 3 ,like Figure 17 As shown in B. Tumor tissue sections were prepared, and DAPI and CD8 were analyzed. + Immunofluorescence staining of T cells revealed CD8 cells infiltrating tumors in mice treated with a combination of PD1 antibody and colloidal manganese. + The proportion of T cells increased significantly, such as Figure 17As shown in Figure D, this indicates that colloidal manganese can synergistically treat colon cancer with PD1 antibody drugs.

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Claims

1. A vaccine composition comprising A) Vaccine immunogen, and B) Colloidal manganese, wherein the colloidal manganese is Mn2OHPO4 colloid or MnHPO4 colloid; Optionally, components A and B are in the same and / or separate containers.

2. The vaccine composition according to claim 1, wherein the vaccine immunogen is derived from a virus, bacteria, and / or parasite.

3. The vaccine composition according to claim 2, wherein the virus is selected from DNA viruses and RNA viruses.

4. The vaccine composition according to claim 3, wherein the virus is selected from the Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Picornaviridae, Hepatoviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae families.

5. The vaccine composition according to claim 3, wherein the virus is selected from herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV.

6. The vaccine composition according to claim 2, wherein the bacteria are selected from Gram-negative and Gram-positive bacteria.

7. The vaccine composition according to claim 6, wherein the bacteria are selected from Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Meningococcus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumanni.

8. The vaccine composition according to claim 2, wherein the vaccine immunogen is derived from influenza virus, hepatitis virus, poliovirus, rabies virus, HPV virus, encephalitis virus, mumps virus, rubella virus, tetanus bacillus, pertussis bacillus, diphtheria bacillus, leprosy bacillus, tuberculosis bacillus, meningococcus, pneumococcus, and any combination thereof.

9. A method of manufacturing a composition for immune enhancement, comprising: 1) Provide a source capable of forming colloidal manganese; and 2) Convert the source capable of forming colloidal manganese into colloidal manganese, wherein the colloidal manganese is Mn2OHPO4 colloid or MnHPO4 colloid.

10. A method for manufacturing a vaccine composition, comprising: 1) Provide vaccine immunogen; and 2) Provide colloidal manganese and / or a source capable of forming colloidal manganese, wherein the source capable of forming colloidal manganese is converted into colloidal manganese, wherein the colloidal manganese is Mn2OHPO4 colloid or MnHPO4 colloid.

11. The method of claim 10, wherein the vaccine immunogenicity is derived from a virus, bacteria, and / or parasite.

12. The method of claim 11, wherein the virus is selected from DNA viruses and RNA viruses.

13. The method of claim 12, wherein the virus is selected from the Herpesviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Picornaviridae, Hepatoviridae, Flaviviridae, Papillomaviridae, Poxviridae, and Retroviridae families.

14. The method of claim 13, wherein the virus is selected from herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV, and HBV.

15. The method of claim 11, wherein the bacteria are selected from Gram-negative and Gram-positive bacteria.

16. The method of claim 15, wherein the bacteria are selected from Streptococcus pneumoniae, Haemophilus influenzae, Salmonella, Meningococcus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Acinetobacter baumanni.

17. The method of claim 11, wherein the vaccine immunogenicity is derived from influenza virus, hepatitis virus, poliovirus, rabies virus, HPV virus, encephalitis virus, mumps virus, rubella virus, tetanus bacillus, pertussis bacillus, diphtheria bacillus, leprosy bacillus, tuberculosis bacillus, meningococcus, pneumococcus, and any combination thereof.

18. Use of colloidal manganese in the preparation of an immune-enhancing composition or a vaccine composition, wherein the immune-enhancing composition is an immune adjuvant, and the colloidal manganese is Mn2OHPO4 colloid or MnHPO4 colloid.

19. The use according to claim 18, wherein the immune enhancement is an improvement of innate immunity and / or adaptive immunity.

20. The use according to claim 18, wherein the immune enhancement is an increase in type I interferon expression.

21. The use according to claim 18, wherein the immune enhancement is the induction of inflammatory cytokine cleavage to produce an active form.

22. The use according to claim 18, wherein the immune enhancement is to promote antibody production.

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