CN107412260A

CN107412260A - CGAS-STING Pathway Activation agent and application thereof

Info

Publication number: CN107412260A
Application number: CN201610347815.6A
Authority: CN
Inventors: 蒋争凡; 管玉坤; 王晨光
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2016-05-23
Filing date: 2016-05-23
Publication date: 2017-12-01
Anticipated expiration: 2036-05-23
Also published as: CN107412260B

Abstract

The invention provides the purposes that the activator of a kind of new cGAS STING paths and the activator are used to activate cGAS STING paths.It is used for the purposes for improving inherent immunity and/or adaptive immunity present invention also offers the activator.

Description

CGAS-STING Pathway Activation agent and application thereof

Technical field

The invention provides the activator of a kind of new cGAS-STING paths and the activator For activating the purposes of cGAS-STING paths.It is used to change present invention also offers the activator The purposes of kind inherent immunity and/or adaptive immunity.

Background technology

Pathogenic process of the natural immune system in resistance infection, suppression tumour growth and autoimmunity disease In play vital effect, mainly pass through pattern recognition receptors and identify pathogenic microorganism and cancer cell Component, start downstream signaling pathway, expressed eventually through inducing cytokine, on the one hand kill disease Pathogenic microorganism and cell infected or that canceration occurs, on the other hand activate acquired immune system and promote Enter the generation of antibody and T lymphocyte specific, so as to protect body from pathogenic microorganism and swell The infringement of knurl.

Specifically, inherent immunity system is referred to as pattern recognition receptors (pattern with a series of Recognition receptors, PRR) cause of disease identification receptor monitor extracellular danger signal, endosome (endosome) and the non-composition of kytoplasm and self composition, as LPS, bacterium flagellin, It is cyclized dinucleotides, CpG DNA, DNA, dsDNA, ssDNA rich in AT etc..PRR Pathogen can be directed to or the tissue damage of itself produces the immune response of appropriateness, and enter cell cytoplasm DNA is considered a kind of danger signal of pathogenic infection by body, if cytoplasmic DNA can not be reasonable Removing can then be accumulated in kytoplasm, will trigger pathological inflammatory and autoimmunity disease, such as systemic red yabbi Sore (systemic lupus erythematosus, SLE).

Dissociative DNA present in cell cytoplasm can be by the inherent immunity system of host as potential danger Dangerous signal, but the mechanism that immune system identifies and removes these danger signals is still not clear at present.DNA Receptor (DNA sensor) is that host experiences DNA and the bridge of immune defense, has been had at present More than 10 kinds DNA receptors are found, and interferon-stimulated gene (stimulator of Interferon genes, STING) as a kind of DNA experience the crucial linkers of passage downstream, Important signal transmission effect is played in terms of cytoplasmic DNA and immune defense is experienced.Especially, most A kind of Novel DNA receptor, i.e. cGAS (cGMP-AMP are closely found that in mammalian cell Synthase), it is a kind of nucleic acid transferase, it can produce endogenic CDN：2 ' -3 ' ring AMP-GMP (cGAMP), and then activate STING.The path is referred to as cCAS-STING Path, it is played an important role in the startup of organism immune response.

The content of the invention

Present inventors have surprisingly discovered that a kind of new cGAS-STING activator, it can CCAS-STING paths are effectively activated, so as to improve inherent immunity and/or adaptive immunity.It is special Not, the cGAS-STING activator be selected from bivalent manganese, divalence manganese element, bivalent manganese source, (it is in object with bivalent manganese for manganese (its presence in the form of bivalent manganese in object) and manganese element Form presence).

In one aspect, it is used to improve inherent immunity and/or fits preparing the invention provides bivalent manganese Purposes in the immune medicine of answering property.In some embodiments, the bivalent manganese passes through activation CGAS-STING paths improve inherent immunity and/or adaptive immunity.In other embodiments, The bivalent manganese improves inherent immunity and/or adaptive immunity by strengthening endochylema DNA impressions.Again In some embodiments, the bivalent manganese is improved inherently by improving cGAS to DNA sensitiveness Immune and/or adaptive immunity.

In another aspect, the invention provides bivalent manganese source to prepare for improving inherently in object Purposes in the immune and/or medicine of adaptive immunity.Preferably, the divalence in the medicine Manganese source exists in the object in the form of bivalent manganese.In some embodiments, the bivalent manganese Improve inherent immunity and/or adaptive immunity by activating cGAS-STING paths.In other realities Apply in scheme, the bivalent manganese improves inherent immunity and/or adaptability by strengthening endochylema DNA impressions It is immune.In other embodiment, the bivalent manganese is by improving sensitivities of the cGAS to DNA Property improve inherent immunity and/or adaptive immunity.

In another aspect, it is used to improve in object inherently to exempt from preparation the invention provides manganese element Purposes in the medicine of epidemic disease and/or adaptive immunity, wherein the manganese element in the medicine is in institute State in object and exist in the form of bivalent manganese.In some embodiments, the manganese element passes through activation CGAS-STING paths improve inherent immunity and/or adaptive immunity.In other embodiments, The manganese element improves inherent immunity and/or adaptive immunity by strengthening endochylema DNA impressions.Again In some embodiments, the manganese element is improved inherently by improving cGAS to DNA sensitiveness Immune and/or adaptive immunity.

In one aspect, the invention provides improve inherent immunity and/or adaptive immunity in object Method, it includes applying bivalent manganese or its prodrug to the object.

In another aspect, the invention provides improve inherent immunity and/or adaptability in object to exempt from The method of epidemic disease, it includes applying bivalent manganese source to the object.Preferably, the bivalent manganese source is in institute State in object and exist or be changed into the form of bivalent manganese in the form of bivalent manganese.

In another aspect, the invention provides improve inherent immunity and/or adaptability in object to exempt from The method of epidemic disease, it includes applying manganese element to the object, wherein the manganese element is in the object Exist or be changed into the form of bivalent manganese in the form of bivalent manganese.

In one aspect, the invention provides bivalent manganese or its prodrug, it improves in object and inherently exempted from Epidemic disease and/or adaptive immunity.

In another aspect, the invention provides bivalent manganese source, its improve in object inherent immunity and / or adaptive immunity.Preferably, the bivalent manganese source is deposited in the object in the form of bivalent manganese Or be changed into the form of bivalent manganese.

In another aspect, the invention provides manganese element, its improve in object inherent immunity and/ Or adaptive immunity, wherein the manganese element exists or turned in the form of bivalent manganese in the object It is changed into the form of bivalent manganese.

In some embodiments, the bivalent manganese is the form of manganous salt, preferably described divalence Manganese salt is selected from：Manganese chloride, manganous bromide, manganese iodide, manganese sulfate, manganese nitrate, Manganese perchlorate, acetic acid Manganese, manganese carbonate, manganese borate, manganese phosphate, hydrobromic acid manganese, manganese tartrate, fumaric acid manganese, maleic acid Manganese, manganese lactate, benzene sulfonic acid manganese, pantothenic acid manganese, Manganese ascorbate and its any combination.

In other embodiments, the bivalent manganese is the form of free divalent manganesetion.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source or manganese element to prepare use Purposes in the medicine of activation cGAS-STING paths.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source or manganese element to prepare use Purposes in the medicine of enhancing endochylema DNA impressions.Preferably, with bivalent manganese in the absence of when compared with, Bivalent manganese cause endochylema DNA impression enhancing at least 10,50,100,500,1000,5000 or 10⁴Times.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source or manganese element to prepare use In raising cGAS to the purposes in the medicine of DNA sensitiveness.Especially, cGAS is to DNA's Sensitiveness improve mean, with bivalent manganese in the absence of when compared with, in lower level DNA (such as endochylemas DNA in the presence of), cGAS is activated.Preferably, with bivalent manganese in the absence of when compared with, cGAS At least 10,50,100,500,1000,5000 or 10 are improved to DNA sensitiveness⁴Times, Or cGAS is as little as 10^-7、10^-6、10^-5Or 10^-4Swashed under mg/ml dsDNA concentration It is living.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source or manganese element to prepare use In disease of the treatment selected from bacterium infection, viral infection, parasite, autoimmune disease and cancer Medicine in purposes.

In some embodiments, the virus is selected from：DNA virus and RNA virus, preferably The virus is selected from：Herpetoviridae, Rhabdoviridae, filamentous virus section, orthomyxoviridae family, pair Myxovirus section, coronaviridae, Picornaviridae, Hepadnaviridae, flaviviridae, Papillomaviridae, Poxviridae and Retroviridae, more preferably described virus are selected from：It is single Pure herpesviral, vesicular stomatitis virus, vaccinia virus, HIV and HBV.

In some embodiments, the bacterium is selected from Gram-negative bacteria and gram-positive bacteria, excellent Bacterium described in selection of land is selected from streptococcus pneumonia (Streptococcus pneumoniae), the bloodthirsty bar of influenza Bacterium (Haemophilus influenzae), salmonella (Salmonella), diplococcus meningitidis (Meningococcus), MRSE (Staphylococcus epidermidis), golden yellow Color staphylococcus (Staphylococcus aureus), Escherichia coli (Escherichia coli), lung Scorching Klebsiella (Klebsiella pneumoniae), acid-producing Klebsiella bacterium (Klebsiella Oxytoca), enterobacter cloacae (Enterobacter cloacae), citrobacter freundii (Citrobacter Freundii), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Baume acinetobacter calcoaceticus (Acinetobacter baumanni)。

In some embodiments, the autoimmune disease be selected from type i diabetes, psoriasis, Rheumatoid arthritis, systemic loupus erythematosus and multiple sclerosis.

In some embodiments, the cancer is selected from oophoroma, lung cancer, stomach cancer, breast cancer, liver Cancer, cancer of pancreas, cutaneum carcinoma, malignant mela noma, head and neck cancer, sarcoma, cholangiocarcinoma, carcinoma of urinary bladder, Kidney, colon cancer, Chorionic villi of placenta cancer, cervix cancer, carcinoma of testis, uterine cancer and leukaemia.

In some embodiments, the parasite is cytozoon, be preferably chosen from plasmodium, Infection of Toxoplasma Gondii, trypanosome, blood fluke, filaria and Leishmania.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source or manganese element to exempt from preparation Purposes in epidemic disease adjuvant.In some embodiments, immunologic adjuvant activation T cell activation and/ Or antibody producing.Preferably, the immunologic adjuvant is selected from bacterium infection for treatment, virus infects, In the vaccine combination of the disease of parasite, autoimmune disease and cancer.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source or manganese element to prepare epidemic disease Purposes in seedling composition, wherein the vaccine combination also includes one or more of antigens.One In a little embodiments, bivalent manganese, bivalent manganese source or manganese element activation T in the vaccine combination Cell activation and/or antibody producing.Preferably, the vaccine combination, which is used to treat, is selected from bacterium sense Dye, viral infection, parasite, the disease of autoimmune disease and cancer.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source or manganese element to prepare use Purposes in the medicine of induced chemokine CCL3 productions.In some embodiments, it is described become Change factor CCL3 production and then suppress HIV.

In yet another aspect, the invention provides a kind of vaccine combination, it is included：It is one or more of Kind antigen, and bivalent manganese.Preferably, vaccine combination of the invention is also comprising pharmaceutically acceptable Carrier.

In yet another aspect, the invention provides a kind of kit for immunity inoculation, it is included： First container, wherein accommodating one or more of antigens；And second container, wherein accommodating bivalent manganese. Preferably, first container and/or second container also include pharmaceutically acceptable carrier.Especially Ground, the kit are used for one or more of purposes of the present invention.

In some embodiments, the antigen used in the present invention is selected from：Virus or bacterium or parasitism Thing antigen, for example, A type, B-mode, the third type, fourth type and penta 3 Hepatitis virus, HIV, blebs Viral 1,2,6 and 7 types, cytomegalovirus, varicellazoster virus, papillomavirus, Epstein-Barr virus, influenza virus, parainfluenza virus, adenovirus, bunyavirus (Hantaan virus), Ke Sa Qi viruses, picornavirus, rotavirus, Respiratory Syncytial Virus(RSV), poxvirus, rhinovirus, Rubella virus, papovavirus, mumps virus and measles virus, point for causing tuberculosis and leprosy Branch bacillus, pneumococcus, aerobic gram-Negative bacillus, mycoplasma, staphy lococcus infection, hammer Bacterium infection, salmonella and Chlamydia, helicobacter pylori, malaria, leishmaniasis, trypanosomiasis, bow Shape parasitosis, snail fever and filariasis.

In some embodiments, the bivalent manganese in the present invention, bivalent manganese source, manganese element and/or anti- Original is effective dose.

Preferably, the bivalent manganese source and/or the manganese element in the object with the shape of bivalent manganese There is or be changed into the form of bivalent manganese in formula.

In yet another aspect, the invention provides for activating cGAS-STING paths in object Method, it includes applying bivalent manganese, bivalent manganese source and/or manganese element to the object.In addition, Present invention also offers the method for cGAS-STING paths in Activation In Vitro cell, it includes Apply bivalent manganese to the cell, it is preferable that methods described is non-treatment purpose.

In yet another aspect, the invention provides the side for strengthening endochylema DNA impressions in object Method, it includes applying bivalent manganese, bivalent manganese source and/or manganese element to the object.In addition, this hair Bright to additionally provide for strengthening the method that endochylema DNA experiences in cell in vitro, it is included to described thin Born of the same parents apply bivalent manganese, it is preferable that methods described is non-treatment purpose.

In yet another aspect, the invention provides sensitive to DNA for improving cGAS in object The method of property, it includes applying bivalent manganese, bivalent manganese source and/or manganese element to the object.In addition, Present invention also offers for improving cGAS in vitro to the method for DNA sensitiveness, it is included to institute State cell and apply bivalent manganese, it is preferable that methods described is non-treatment purpose.

In yet another aspect, the invention provides for being treated in object selected from bacterium infection, virus Infection, parasite, autoimmune disease and cancer disease method, it is included to the object Using bivalent manganese, bivalent manganese source or manganese element.

In yet another aspect, the invention provides for activating T cell activation in object and/or resisting The method of body production, it includes applying bivalent manganese, bivalent manganese source or manganese element to the object.

In yet another aspect, the invention provides in object induced chemokine CCL3 give birth to The method of production, it includes applying bivalent manganese, bivalent manganese source or manganese element to the object.

In yet another aspect, the invention provides bivalent manganese, bivalent manganese source and/or manganese element, it is used In activation cGAS-STING paths, enhancing endochylema DNA impressions, raising cGAS in object To DNA sensitiveness, treatment selected from bacterium infection, viral infection, parasite, LADA disease The disease of disease and cancer, activation T cell activation and/or antibody producing, and/or induced chemokine CCL3 is produced.

Brief description of the drawings

Chatted below by detailed description of the present invention and accompanying drawing to clearly demonstrate before the present invention The aspect stated and other aspects.In order to demonstrate the invention, embodiment in the accompanying drawings is mesh It is preceding preferable, it being understood, however, that the present invention is not limited to disclosed particular.

Fig. 1 shows Mn²⁺Pretreatment imparts resistance of the host cell to virus infection.Wherein with 100 μM MnCl₂To THP1 cell pretreatments 12 hours, then with HSV-1-GFP, VSV-GFP Or NDVGFP infection cells.After 24 hours, cell is taken pictures (left side) and use flow cytometry Measure on (right side).

Fig. 2 shows Mn²⁺Pre-process the dose dependent of antiviral activity.Wherein with shown concentration MnCl₂Or DMXAA (DMX) handles THP1 cells 24 hours, then with shown GFP- Virus infected cell 24 hours, GFP expression is measured by western blot afterwards.

Fig. 3 shows Mn²⁺Pre-process the dose dependent of antiviral activity.Wherein with shown concentration MnCl₂Or DMXAA (DMX) handles THP1 cells 24 hours, then with shown GFP- Virus infected cell 24 hours, the median fluorescent by flow cytometry measure virus-GFP is strong afterwards (Median Fluorescence Intensity, MFI) distribution is spent, wherein by unused MnCl₂ The MFI of the THP1 cells of processing is normalized to 1.

Fig. 4 display intravenous injections Mn²⁺Mouse is assigned with virus resistance.Wherein with 2mg/kg's MnCl₂Mouse is injected intravenously, after 24 hours, mouse is attacked with VSV, VACV or HSV-1, Monitor the survival of mouse.

Fig. 5 display intravenous injections Mn²⁺Mouse is assigned with virus resistance.Wherein with 2mg/kg's MnCl₂Mouse is injected intravenously, after 24 hours, mouse is attacked with VSV, VACV or HSV-1, Infection takes lung and spleen after four days, is homogenized, and is then measured by plaque assays (plaque assay) Virus titer, wherein data are expressed as average value ± SEM, are obtained at least three independent experiment Similar result.

Fig. 6 shows Mn²⁺Handle the influence to gene expression.Wherein, A figures, which are shown, uses MnCl₂、 VACV, IFN β or culture medium (control) handle THP1 cells 12 hours, extract RNA And it is sequenced.By calculating log₂((through handling RPKM)/(control RPKM)) obtains gene table The thermal map reached；B figures show Mn²⁺Processing induction I-IFN productions, wherein with 200 μM of MnCl₂ THP1 cells are handled, were discarded after 2,12,24 hours containing MnCl₂Culture medium and more Change fresh culture into, NW represents not discarding containing MnCl₂Culture medium；C figures are shown using different Manganese salt processing induction I-IFN productions, wherein the MnCl with shown concentration₂, Mn (OAc)₂Or Mn(OAc)₃Handle THP1 cells 24 hours, then analyzed using I-IFN biologicall tests Supernatant；D figures are shown in injection MnCl in mouse medium sized vein₂I-IFN productions are induced, wherein using 0,2 or 5mg/kg MnCl in PBS₂Solution is injected intravenously mouse, separates serum and uses I-IFN biologicall tests are analyzed；E figures are shown in injection MnCl in mouse medium sized vein₂ISG afterwards Organ is distributed, wherein using in PBS 0,2 or 5mg/kg MnCl₂Solution intravenous injection is small Mouse, organ is taken, ISG is determined by western blot；F figures are shown with various concentrations MnCl₂ PBMC I-IFN productions and ISG are expressed in healthy adult donor after handling 36 hours, are used I-IFN biometric analysis I-IFN is produced, and ISG expression is determined by western blot；G schemes 200 μM of MnCl of display₂PBMC I-IFN gives birth in healthy adult donor after processing different time Production and ISG expression, are produced using I-IFN biometric analysis I-IFN, are printed by Western Mark measure ISG expression.Data are expressed as average value ± SD.

Fig. 7 shows Mn²⁺Processing inducing cytokine, which produces, depends on cGAS-STING paths.A Figure is shown, with various concentrations MnCl₂Solution handled HeLa cells and THP1 cells after 24 hours IRF3 phosphorylations and vipoxin (Viperin) production；B figures are shown with 500 μM of MnCl₂ IRF3 phosphorylation and vipoxin of the HeLa cells of gene knockout after 24 hours shown in solution processing Production；C figures are shown with 500 μM of MnCl₂Solution, VACV or gene deletion shown in SeV processing With the IRF3 phosphorylations (being determined by western blot) after the HeLa cells 24 hours of redemption； D figures are shown with 500 μM of MnCl₂Solution, VACV or SeV processing WT or shown clpp genes Except after mouse 24 hours, vipoxin and ISG productions are (logical in the peritoneal macrophages therefrom obtained Cross western blot measure)；E figures show that the IFN productions after being handled in D (use I-IFN Biologicall test).Data are expressed as average value ± SD, are obtained at least three independent experiment similar Result.

Fig. 8 shows Mn²⁺Directly activate cGAS.A figures show total length (1-522) the people cGAS of purifying With MnCl₂, nucleic acid shown in figure be incubated together, the reaction uses the THP1 of digitonin permeabilization, Cell lysis, lysate to be used for the IRF3 phosphorylations for determining cGAMP inductions；B figures are shown entirely Long or mutation people cGAS and MnCl₂/MgCl₂, a variety of different amounts of dsDNA are incubated together； C figures show people cGAS albumen and 0.5mM MnCl₂Or 5mM MgCl₂, and 10-2 Mg/ml dsDNA are incubated together, are produced using Mono Q ion exchange columns analysis cGAMP, With cGAMP, ATP and GTP as control.Obtained at least three independent experiment similar As a result.

Fig. 9 shows Mn²⁺Strong adjuvanticity.A figures are shown in the 0th day and the 10th day and use OVA (10 μ g), OVA+10 μ g MnCl2 or OVA+30 μ g DMXAA intramuscular immunisations are small Mouse, serum and splenocyte were taken at the 17th day, determining OVA specific IgGs 1 using serum (passes through ELISA), AU：OD450 absorbance unit, data are expressed as average value ± SD, from three Independent experiment, the spleen leucocyte in A is stimulated with OVA peptides H-2Kb or I-Ab, takes supernatant IFN γ (B figures) and IL-2 (C figures) secretions are determined by ELISA, data are expressed as average value ±SD。

Figure 10 shows Mn²⁺Produced by CCL3 and suppress HIV.(A and B figures) is as in figure Shown processing THP1 cells, measure I-IFN (A figures) and CCL3 (B figures) secretions.(C Figure) using come the Mn that hangs oneself²⁺The supernatant of the THP1 cells of processing triggers MAGIC5 cells 4 hours, Then CCR5- preferendums (CCR5-tropic), CXCR4- preferendums or VSV-G- vacation types are used (pseudotyped) HIV-Luc virus infected cells.After 3 days, luciferase, display are determined Virus infection.The luciferase activity value for compareing the MAGIC5 cells that supernatant triggers is normalized to 1.(D and E figures) is tested as shown in (A and B figures), is different only in that use comes from The PBMC of healthy adult, uses Mn²⁺PBMC cells 18 or 36 hours are handled, are then determined I-IFN and CCL3.F figures are similar with C figures, are different only in that use come the Mn that hangs oneself²⁺Handle PBMC The supernatant of cell triggers MAGIC5 cells.(G figures) processing comes from WT or base as shown in FIG. Because of the peritoneal macrophages of knock-out mice, CCL3 secretions are determined by ELISA.Data are expressed as Average value ± SD.

Figure 11 shows Mn²⁺Mouse peritoneal macrophages are activated with DMXAA.Shown in (A figures) is used The Mn of amount²⁺Mouse peritoneal macrophages are handled with DMXAA 36,48 hours, then determine viper Ophiotoxin.

Figure 12 shows Mn²⁺Directly activate cGAS.A figures show the MnCl with amount shown₂/MgCl₂ Handle THP1 cells 24 hours, collect supernatant, determine I-IFN.B figures show Coomassie brilliant blue Purifying total length (1-522), mutant (E225A/D227A) and the truncated mutant (161-522) of dyeing People cGAS.C figures are shown, use truncation (161-522) people cGAS and MnCl₂/MgCl₂, it is more The different amounts of dsDNA of kind is incubated together, similar with Fig. 8 B.D figures are shown in 1mM MnCl₂ Or 1mM MgCl₂In the presence of, total length people cGAS is determined by identical titration calorimetry (ITC) And dsDNA combination.

Figure 13 shows Mn²⁺The cGAS activation of induction is not related to injury of mitochondria.(A figures) is with 20 μM ABT-263,20 μM of ABT-737 or MnCl₂(50,100,200 μM) processing THP1 Cell 24 hours, pass through Annexin V-FITC/PI double dyeing FCM, DNA gel and Western Trace determines apoptosis.(B scheme and C figure) handle as shown in the figure wild type (WT) and Bax-/- Bak-/- MEF, then determine Casp3 cuttings and vipoxin induction.(D figures) is with 500 μM of MnCl2 The BMDM from C57BL/6J mouse is handled, mitochondria is shown by TEM, uses supernatant To determine I-IFN productions.(E and F figures) is triggered with 1 μ g/ml LPS comes from WT or shown bases Because of the peritoneal macrophages 5 hours of knock-out mice, then processing 6 hours as shown.Use supernatant (Sup) activated with full cell lysate (WCL) measure inflammation corpusculum (inflammasome). Data are expressed as average value ± SD.

Embodiment

Definition

Term " inherent immunity " used herein refers to body in germline development and evolutionary process The innate immune defence function of formation, that is, the nonspecific defense function of just having possessed after being born, also referred to as For nospecific immunity (non-specific immunity).Inherent immunity is related to various kinds of cell and molecule, Such as macrophage, NK, complement, cell factor (IL, CSF, IFN, TNF, TGF-β), chemotactic factor (CF) (including CC chemotactic factor (CF)s, for example, CCL1, CCL2, CCL3, CCL4、CCL5、CCL6、CCL7、CCL8、CCL9、CCL10、CCL11、CCL12 Deng, Gro-beta-T, for example, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9 etc., C chemotactic factor (CF)s, CX3C chemotactic factor (CF)s), Lysozyme etc..

Term " adaptive immunity " used herein is also known as acquired immunity or specific immunity, is Referring to body stimulates the specific immunity for the antigen formed afterwards in antigen molecule, and it is related to cell Immune and humoral immunity.

Term " adjuvant " used herein refers to not form specific antigen, but strengthens to altogether With the intensity of immune response and the reagent of duration of the antigen of administration.

Term " manganous salt " used herein, can be hydrochloride, hydrobromate, sulfate, Nitrate, phosphate, tartrate, fumarate, maleate, lactate, benzene sulfonate, Pantothenate, ascorbate etc., or its any combination.Preferably, the salt is can pharmaceutically to connect The salt received.

Terms used herein such as "comprising", " containing ", " containing " and " comprising " are not intended to limit.This Outside, unless otherwise indicated, "or", "or" mean "and/or".

It is further noted that as used in this description, singulative includes answering for its referent Number form formula, unless understanding and being clearly limited to a referent.If mention one specifically Numerical value, it can at least include the numerical value, unless article has clearly showed that it refers else.

When numerical value represents approximation, it is thus understood that specific numerical value forms another embodiment.Just As used, " about X " (wherein X is a numerical value) refers to value listed by ± 10% (including). If it does, all scopes all include and can combined.

Terms used herein " pharmaceutically acceptable carrier " may be selected from：Water, aqueous buffer solution, Isotonic salting liquid such as PBS (phosphate buffer), glucose, mannitol, D-glucose, breast Sugar, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerine, hyaluronic acid, ethanol or PAG such as polypropylene glycol, triglycerides etc..The type of pharmaceutical acceptable carrier used is among others dependent on In whether being formulated as being used for oral, nose, intracutaneous, subcutaneous, intramuscular or quiet according to the composition of the present invention Arteries and veins is applied.According to the present invention composition can include lubricant, preservative, stabilizer, wetting agent, Emulsifying agent, salt, buffer, coloring material, flavor ameliorating substances and/or the aromatic substance for influenceing osmotic pressure Deng being used as additive.

Can be applied according to the pharmaceutical composition of the present invention by any suitable approach, for example, can orally, Nose, intracutaneous, subcutaneous, intramuscular or intravenous administration.

Terms used herein " administration " means to provide thing to object in a manner of pharmacologically available Matter.

" medicine effective quantity " used herein, " effective dose " refer to be enough to show it for institute's subject The dosage of benefit.The actual amount of administration, and the speed and time course applied can depend on being treated The own situation and the order of severity of person.The prescription (such as decision to dosage etc.) for the treatment of is finally complete The responsibility of section doctor and other doctors simultaneously rely on it and made a decision, and generally consider treated disease, patient Situation, site of delivery, application process and the known other factorses for doctor of individual.

Term as used herein " object " means animal, including warm-blooded mammals, such as people and Ling Long class animal；Birds；The domestic or farm-animals raised and train, for example, cat, dog, sheep, goat, ox, Horse and pig；Laboratory animal, such as mouse, rat and cavy；Fish；Reptile；Move in zoo Thing and wild animal etc..

Unless otherwise defined, all scientific and technical terminologies used herein have those of ordinary skill in the art institute The identical meanings of understanding.

Unless otherwise indicated, it is any on component, member disclosed in this method and a kind of embodiment of product Element, attribute or step can apply to any other method disclosed herein and product.

Description in each patent of the disclosure, patent application, the publication or this document quoted with The mode of reference is integrally incorporated in text.

The present invention further definition in the following embodiments.These embodiments are should be appreciated that only to lift The mode of example illustrates, it is no intended to limits the scope of the present invention.From the discussion above and these examples In son, those skilled in the art can determine the substantive characteristics of the present invention, and without departing from its spirit In the case of scope, change and the modification that each side can be made to the present invention are various to be allowed to adapt to The usage and condition of various kinds.

Material and method

Antibody and reagent

Antibody sources are as follows：Anti- GFP (Sungene Biotech, KM8009), anti-GAPDH (Santa Cruz, sc-25778), anti-cGAS (Santa Cruz, sc-245858), anti-p-IRF3 (Epitomics, 2562-1), anti-pIKK α/βs (Cell signaling, #2078S), anti-I κ B α (Cell signaling, #4814), anti-TBK1 (Santa Cruz, sc-52957, Cell signaling, #3504), anti-STING (Proteintech, 19851-1-AP；Abgent, AP9747b), resist RIG-I (Cell signaling, #3743S), anti-MAVS (Santa Cruz, sc-166583), resist Caspase-3 (Santa Cruz, sc-271759).Other antibody are by described in prior art Method is made and uses (7,34,35), vipoxin (Viperin) (people 75-361aa, mouse 243-360aa), ISG54 (people 6-285aa, mouse 3-289aa), IRF3 (people's total length), Lamin A (people's total length), Casp1/p20 (mouse 121-296aa), IL1 β/p17 (mouse 118-269aa) And ASC (mouse total length) cDNA is inserted into pET-21b carriers (Novagen) and expressed In E. coli BL21 (DE3).By Ni-NTA affinity column purification of recombinant proteins, so Mouse or rabbit are injected into afterwards to produce antiserum.

All chemicals are purchased from Sigma-Aldrich (St.Louis, MO), unless otherwise stated.It is small Mouse CCL3 ELISA Kit (eBioscience, #88-56013-22), people CCL3 ELISA Kit (Liankebio, EK1612), DMXAA (Selleck, S1537), ABT-737 (Selleck, S1002), ABT-263 (Selleck, S1001), LPS (Sigma, L4130), Digitonin (Sigma, D141), Ovalbumin (InvivoGen, #vac-pova), poly (I：C) (Amersham, #27-4723-01), dsDNA/ssDNA (Sangon Biotech, there is adopted sequence： 5-tacagatctactagtgatctatgactgatctgtacatgatctaca-3), dsRNA/ssRNA (Dharmacon, there is adopted sequence： 5-uacagaucuacuagugaucuaugacugaucuguacaugaucuaca-3).According to there is adopted sequence The equimolar amounts between antisense sequences, dsDNA and dsRNA is annealed.

Plasmid

People cGAS expression vectors with Flag labels are by Dr.Zhijian Chen (UT Southwestern Medical Center) give.PET-21b-hcGAS is by Xiaodong Su (Peking University) give.PU6-sgRNA and pcDNA3.1-CAS9 are by Jianzhong Jeff Xi (Peking University) give.PLVX si3LTR Luc, pHIV JRFL env and PRE11 NL43env are by Takaomi Ishida (Institute of Microbiology, CAS) favour Give.PsPAX2 and pMD2.G are derived from Addgene.PGL3-Basic carriers and PRL-SV40-Renilla is derived from Promega.Encoding human IRF3, STING, TBK1 and The cDNA of RIG-I N-terminal is amplified from THP1 cells, clones into pcDNA3.1 (Invitrogen). All constructs are confirmed by sequencing.

Cell

HEK293T, L929-ISRE, 2fTGH and its derivative mutant strain (U3A, U4A, U5A and 2A), 2fTGH-ISRE, HeLa and its derivative knockout strain, iBMDM, BHK21, MAGIC5 Cell culture is in added with 10%FBS (Gibco), 5 μ g/ml penicillin and 10 μ g/ml streptomysins DMEM (Gibco) in.THP1 is incubated at added with 10%FBS's (Gibco) In RPMI-1640 (Gibco) culture medium.Human peripheral blood single nucleus cell (PBMC) is isolated from Healthy People adult human's peripheral blood, pass through company using Histopaque-1077 (Sigma, 10771) Continuous centrifugation is separated, and is then incubated at the RPMI-1640 culture mediums added with 5%FBS.It is wild Raw type and the MEF knocked out were prepared by the 15th day embryo, were incubated at added with 10%FBS's In DMEM.Peritoneal macrophages harvest is injected from thioglycolate (BD, Sparks, MD) The mouse of 5 days after induction, it is incubated in the DMEM added with 5%FBS.Derived from bone marrow macrophage Cell (BMDM) is incubated at added with 20%FBS and 30%L929 collected from femur and shin bone DMEM adjustment culture medium in 5-7 days.

C57BL/6 mouse source wild type iBMDM cells (Katherine Fitzgerald, University of Massachusetts Medical School), Tlr4-/- iBMDM cells (Aihao Ding, Weill Cornell Medical College), Tbk1-/- MEFs (Wen-Chen Yeh, Amgen), MAGIC5 (Takaomi Ishida, Institute of Microbiology, CAS), 2fTGH and its derivative mutant strain (George Stark, Cleveland Clinic) and Bax-/- Bak-/- Source is such as cell (Kevin M Ryan, Beatson Institute for Cancer Research, UK) It is shown.

Mouse

CGas-/-, be derived from cGAS and STING with cGas-/- Sting-/- mouse establishes Sting-/- The hybridization of mouse (founder mice), it is described to establish mouse by by Cas9 mRNA (100ng/ μ l) C57BL/6J embryonated eggs are expelled to gRNA (50ng/ μ l) kytoplasm and are obtained.Pass through MMESSAGE mMACHINE T7 Ultra (Ambion, am1345) in-vitro transcriptions Cas9 MRNA and single guiding (singleguide) RNA (gRNA).Sod1-/- (#002972) and Sod2-/- (#002973) mouse is purchased from Jackson Laboratory.Nlrp3-/-, Asc-/- and Aim2-/- given by Vishva Dixit (Genentech Inc, USA).Mavs-/- mouse derives from Zhijian Chen (UT SouthWestern Medical Center), Irf3-/- and Irf3-/- Irf7-/- Mouse derives from Tadatsugu Taniguchi (The University of Tokyo).

All mouse are according to NIH management of laboratory animal guide for use (National Institute of Health Guide for Care and Use of Laboratory Animals) aseptically feed Support in Peking University's Experimental Animal Center.

I types IFN (Type I-IFN) biologicall test

I types IFN concentration (36) is determined as described in forefathers.In short, should by the way that IFN- is stimulated Element (IFN-stimulated response element, ISRE) is answered to clone into pGL3-Basic loads Body (Promega) builds IFN sensitive luciferase carriers, and its stable transfection then is entered into 2fTGH Or L929 cells.2fTGH-ISRE or L929-ISRE cells are inoculated into 96 orifice plates, respectively It is incubated with together with people or mouse cell culture supernatant.Use recombined human and mouse IFN β (R＆D Systems standard items) are used as.After 4 hours, cell lysis, pass through Luciferase Reporter Assay System (Promega) is measured.

Luciferase reporter thing determines

Luciferase reporter thing measure implements (7) as described in forefathers.In short, people CCL3 is opened - the 1076 of sub-area to -1, -800 to -1, -700 to -1 and -601 to -1, and mouse IFN β Clone into pGL3-Basic carriers (Promega) to-1-the 155 of promoter region.By HEK293T Cell is with 1x10⁵The density in/hole is inoculated into 24 orifice plates, is transfected with the expression vector, uses PGL3-Basic derives luciferase reporter vector and pRL-SV40-Renilla (Promega) makees For internal contrast.After 24 hours, double Luciferase assay system (Dual-Luciferase are used Reporter Assay System) measure reporter gene activity.

Virus infection

Herpes simplex virus 1-GFP (HSV-1-GFP, Kos strains, from Chinese Academy of Medical Sciences, Beijing), vesicular stomatitis virus-GFP (VSV-GFP, comes from Rongbin Zhou, University of Science and Technology of China), Newcastle Disease Poison-GFP (NDV-GFP, from Chen Wang, Institute of Biochemistry and Cell Biology, SIBS, CAS), sendai virus (SeV, from Congyi Zheng, Wuhan University, China), vaccinia virus (VACV, Western Reserve strains, from Min Fang, Institute of Microbiology, CAS；Or Western Reserve-Vvt7 strains, come From Meilin Jin, Huazhong Agricultural University), herpes simplex virus 1 (HSV-1, wild type F strains, from Hongbing Shu, Wuhan University；F strains, come From Bernard Roizman, The University of Chicago) and vesicular stomatitis virus (VSV, Indiana strain) given by above-mentioned colleague.Virus titer is determined by Plaque Technique Detected using BHK21.

Virus resistance determines：Cell is handled using MnCl2 or is triggered using supernatant, is used HSV-1-GFP, VSV-GFP and NDV-GFP are with 0.5 MOI infection cells.After 24 hours, Flow cytometry is carried out by FACSCalibur equipment (BD Biosciences) so as to determine difference Virus-the GFP of virus infected cell.Use FlowJo software analysis FACS data.

Cytositimulation：With SeV (MOI 0.1), VACV (Western Reserve-Vvt7 strains, MOI is 0.1) HSV-1 (F strains, MOI 0.1) and NDV-GFP (MOI of 1) infection Cell 1 hour, elutes and is incubated in fresh culture.

Mouse survival is tested：With HSV-1 (wild type F strains, 1.4x10⁷Pfu/ mouse) and VSV (8x10⁸Pfu/ mouse) intravenously 8-12 weeks mouse of infection, or with VACV (Western Reserve strains, 1x10⁷Pfu/ mouse) 8-12 weeks mouse of intranasal infection.

Plaque assay：By BHK21 cells and the homogenate from infected mouse organs (in serum-free A series of dilutions in DMEM) it is incubated 2 hours together.Then, with serum-free DMEM In 0.5% methylcellulose replace culture medium.It is solid with 0.5% (vol/vol) glutaraldehyde after 60 hours It is fixed, dyed with 1% (wt/vol) crystal violet (being dissolved in 70% ethanol).Plaque is counted, So as to calculate the virus titer in terms of plaque forming unit.

Protein expression and purification

Total length (1-522) and (161-522) the people cGAS (hcGAS) truncated are subcloned into PET-21b carriers.Expression His6-hcGAS, uses 0.3mM in E.coli BL21 (DE3) Isopropyl ss-D-1- thiogalactosides (IPTG) are in 18 degrees Celsius of overnight inductions.Buffered in cracking Cell lysis in liquid (25mM Tris-HCl, 500mM NaCl, pH 7.5), passes through centrifugation (20,000rpm, 1h) removes cell fragment.With 0.2 μm of low protein binding film (Pall Corporation supernatant) is filtered.The supernatant of clarification is loaded onto HisTrap HP posts (GE Healthcare), eluted with lysis buffer 0 to 300mM imidazole gradients.

Stayed overnight using 1U/ μ l Benzonase (Sigma) processing protein to remove DNA and RNA. 2mM EDTA are added to remove bivalent cation.Afterwards, after being balanced using lysis buffer The posts of Superdex 200 (GE Healthcare) carry out gel filtration to protein.Finally, use Amicon Ultra-410K centrifugal filters (Merck Milipore) by the protein concentration of purifying extremely 10mg/ml。

CGAS determinations of activity

By the recombinant full-lenght of purifying, truncation or the mutation hcGAS and 40 μ l containing following ingredients Reaction solution is incubated 90 minutes together under 37 degrees Celsius：1 μM of hcGAS, 1mM ATP, 1mM GTP, 100mM NaCl, 40mM Tris-HCl pH7.5,10-2-10-6mg/ml dsDNA and 0.05-1mM MnCl2 or 0.5-10mM MgCl₂.In 99 degrees Celsius of lower heating response liquid, So that protein denaturation, is centrifuged 10 minutes under 16,000g.By supernatant and 10 μ g/ml digitalis The permeabilized 6x10 of saponin(e (digitonin)⁵THP1 cells are incubated 30 minutes under 37 degrees Celsius, Then cell is cracked, carries out western blot, so as to determine the IRF3 phosphorus of cGAMP inductions Acidifying.In addition, use the Mono balanced through working buffer solution (50mM Tris-HCl, pH 8.5) Q ion exchange columns (GE Healthcare) analyze the supernatant, with 0 to 500mM NaCl Gradient elution.

Identical titration calorimetry (ITC)

By under 25 degrees Celsius using iTC200 equipment (GE Healthcare) carry out ITC come Measure dissociation composition.By gel filtration chromatography come protein purification and ISD, wherein containing or not MnCl containing 1mM₂Or MgCl₂Lysis buffer (25mM Tris-HCl, 500mM NaCl, PH 7.5) in elute.ISD (50 μM in syringe, 18x2 μ l injections) and hcGAS (reactions 5 μM in hole) run using following parameter：With reference to biasing (reference offset) 10 μ cal/s, note Emitter rotating speed 500rpm, delay 180s before injecting, and 5s intra-record slack bytes.Use Origin 7 (Microcal) analyze data.

RT-PCR and real-time PCR analysis

Total serum IgE is separated according to explanation using TRIzol reagents (Invitrogen).1 microgram is total RNA is converted into cDNA, uses random primer and Superscript III reverse transcriptases (Invitrogen).Enter performing PCR using gene-specific primer.It is right on 1.5% Ago-Gel RT-PCR products carry out gel electrophoresis, are shown by EB dyeing.

Sybr green are used to carry out in the systems of LightCycler 96 (Roche) quantitatively real-time PCR, data are expressed as mRNA accumulation indexes (2^ΔΔCt)。

Apoptosis is analyzed

Pass through annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis Determine kit (Solarbio, CA1020) and apoptosis analysis is carried out according to explanation.In short, will be thin Born of the same parents are resuspended in 500 μ l combination buffers, and (it contains extra 5 μ l Annexin V-FITC and 5 μ L Propidium iodide), it is being incubated at room temperature 15 minutes, without direct illumination.Supernatant discarding, washed carefully with PBS Born of the same parents.Then, cell is resuspended in 400 μ l PBS.By flow cytometry sample, make With FlowJo analyze datas.Annexin V positive and PI negative colony represents that early apoptosis is thin Born of the same parents, and annexin V and PI positive populations represent apoptotic cell.

DNA fragmentationization determines：By 10⁵THP1 cells are resuspended in 500 μ l buffer solution D (100mM Tris-HCl pH 8.0,5mM EDTA, 0.2M NaCl, 0.4%SDS, 0.2mg/ml protease K) and under 37 degrees Celsius it is incubated overnight.Removed using phenol and phenol chloroform (1: 1) in DNA Albumen, precipitated with 2 times of volume ethanols.DNA precipitations are resuspended in containing 1 μ g/ μ l RNase's 15μl TE(pH 8.0).After 37 degrees Celsius are incubated 2 hours, DNA is loaded onto 1% agar Sugared gel, electrophoresis is carried out, shown with EB dyeing and short wavelength UV light irradiation.

It is immunized using ovalbumin

Mouse is immunized as previously described, is measured (3,37,38).In short, at the 0th day and 10 days using 10 μ g OVA individually or with 10 μ g MnCl in PBS₂Or 30 μ g DMXAA Intramuscular immunisation mouse together.Serum and splenocyte were taken at the 17th day, to determine OVA specificity respectively IgG1 and t cell response.

OVA specific IgGs 1 detect：Dilute serum from immune mouse is incubated in and is coated with On 100 μ g/ml OVA elisa plate.After cleaning, the conjugated antibody mouse IgG1 of HRP- are used The IgG1 that (eBioscience, #18-4015-82) detection combines.Then, by plate and substrate TMB (eBioscience) it is incubated together, with 1M H₃PO₄Stop reaction, then determine absorbance.Make Standard items are used as with OVA specific IgGs 1 (abcam, ab17293).

OVA specific T-cells responses：Splenocyte is inoculated in 24 orifice plates, 1x10⁶/ hole, with 10 The OVA peptides of μ g/ml synthesis are stimulated, I-Ab (ISQAVHAAHAEINEAGR), H-2Kb Or control peptide (FAPGNYPAL) (SIINFEKL).After 24 hours, cell culture is collected Supernatant, by ELISA determine IFN-γ (Liankebio, EK2801) or IL-2 (eBioscience, #88-7024-22) concentration.

False type HIV-1 (HIV-Luc) production, infection and detection

Tested as previously described (33,39-41).In short, with Lipofectamine 2000 (Invitrogen) 10 μ g, are taken to transmit carrier (pLVX si3LTR Luc), 7.5 μ g packagings (VSV-G- vacation types HIV is pMD2.G, CCR5- for carrier (psPAX2) and 5 μ g envelope vectors Preferendum HIV is pHIV JRFL env, CXCR4- preferendums HIV is pRE11NL43env) turn Contaminate HEK293T cells in the 10cm culture dishes of degree of converging~70%.After 6 hours, by culture medium It is replaced by DMEMs of the 10mL containing 20%FBS.After 48-72 hours, harvest contains pseudotype virus Through transfectional cell supernatant, filtered with 0.45 syringe filter.Pseudotype virus sense is used at once MAGIC5 cells are contaminated, or pseudotype virus is frozen in -80 degrees Celsius.By infected The luciferase assays of MAGIC5 cells measures virus titer.In order to infect MAGIC5, Cell is inoculated in 12 orifice plates by infection the previous day with~30% degree of converging.Using from THP1 or PBMC supernatant triggers cell, then with CCR5- preferendums, CXCR4- preferendums or VSV-G- False type HIV is incubated 6h together.Then, culture medium is replaced by the DMEM containing 10%FBS, Culture 3 days.Culture medium is removed, with 50 μ l 1x Passive Lysis Buffer (Promega)/hole Cell is cracked 30 minutes, carried out with Firefly Luciferase Assay System (Promega) Measurement.

Transmission electron microscope (TEM)

Cell is washed using 0.1M phosphate buffers (pH7.4) twice, then uses same buffer In the glutaraldehyde of 2% paraformaldehyde/2.5% fix 2 hours, with 1%OsO4 after room temperature it is fixed (postfix) 1 hour.With phosphate buffer and distillation water wash several times after, by cell 0.1% It is incubated 30 minutes in tannic acid (in phosphate buffer).With water wash cell is distilled, 2% Dyed 1 hour in uranyl acetate.Wash in distilled water, be dehydrated in ethanol again, be embedded in The resins of SPI-Pon 812 (SPI Supplies, PA, USA).With uranyl acetate and lead citrate to super Thin (75nm is thick) section is dyed, observed under Tecnai G2 20TWIN transmission electron microscopes, 120kV accelerating potentials.With Eagle (4k x 4k) digital camera (FEI, Oregon, USA) place Manage image.

Statistical analysis

Use t check analysis data.Examined using Mantel-Cox and compare survival curve.

Embodiment

Embodiment 1.Mn²⁺Processing imparts resistance of the host cell to virus infection

Test (a)

With 100 μM of MnCl₂10%FBS RPMI-1640 culture mediums are with the addition of to being incubated at (Gibco) the THP1 cell pretreatments in 12 hours, it is then 0.5 with MOI HSV-1-GFP (is derived from the Chinese Academy of Medical Sciences), and VSV-GFP (is derived from Chinese University of Science and Technology Rongbin zhou) or NDV-GFP (be derived from Chinese Academy of Sciences's biochemistry and cell biological Learn research institute Chen Wang) infection cell.After 24 hours, cell is taken pictures and uses streaming thin Born of the same parents' art (FACSCalibur) is measured and analyzed (as shown in Figure 1) using FlowJo. Clearly illustrated in Fig. 1 using Mn²⁺Pretreatment imparts resistance of the host cell to virus infection.

As described above, shown in Fig. 2 or Fig. 3 concentration MnCl₂Or DMXAA (DMX) place Manage THP1 cells 24 hours, then GFP- virus infected cells 24 hours, Zhi Houtong shown in Cross western blot measurement GFP expression and by flow cytometry measure virus-GFP Position fluorescence intensity (Median Fluorescence Intensity, MFI) distribution, wherein will be not used MnCl₂The MFI of the THP1 cells of processing is normalized to 1, it is shown that Mn²⁺Pre-process antiviral The dose dependent of activity.

Test (b)

That takes the logarithm growth period is incubated at the DMEM culture mediums (Gibco) that with the addition of 10%FBS In human cervical cancer cell line HeLa, by A liquid and B liquid (A liquid compositions：PBS (NaCl 137mmol/L, KCl2.7mmol/L, Na₂HPO₄10mmol/L, KH₂PO₄2mmol/L) Or physiological saline, pH value 7.4.B liquid compositions：MnCl₂100mmol/L) according in table Ratio is mixed and added in cultivating system.After 24 hours, Green Fluorescent Protein (GFP) is added Virus infected, be respectively：I herpes simplex virus types (HSV-1), New Jersey's type blister Stomatovirus (VSV) and NDV (NDV).After virus infection 24 hours, stream is used Formula cytometry viral infection rate (GFP positive rates), it is as shown in table 1 below.

Table 1.Mn²⁺Processing significantly reduces viral infection rate

A liquid	B liquid	Culture medium	HSV-1 infection rates	VSV infection rates	NDV infection rates
						10μL	0μL	1mL	99.5% ± 0.1%	98.2% ± 1.0%	97.7% ± 2.0%
9μL	1μL	1mL	65.9% ± 0.8%	71.3% ± 0.8%	64.3% ± 1.0%
						8μL	2μL	1mL	40.8% ± 0.4%	51.1% ± 0.6%	47.7% ± 0.5%
5μL	5μL	1mL	32.4% ± 0.6%	21.0% ± 0.5%	18.8% ± 0.5%
						0μL	10μL	1mL	10.1% ± 0.1%	3.3% ± 0.4%	5.1% ± 0.7%

Test (c)

The Mn measured shown in Figure 11²⁺Mouse peritoneal macrophages 36,48 are handled with DMXAA Hour, then determine vipoxin, the results showed that display Mn²⁺Mouse peritoneum is activated with DMXAA Macrophage (as shown in Figure 11 A).

Embodiment 2. is injected intravenously Mn²⁺Mouse is assigned with virus resistance

Test (a)

Eight week old SPF level experiments C57BL/6 (buying from Beijing company of dimension tonneau China) are taken, by A Liquid and B liquid (as described above) mix according to the ratio in table, and pass through tail vein injection.16-24 After hour, I herpes simplex virus types (HSV-1), New Jersey's type vesicular stomatitis disease are injected respectively Malicious (VSV) and vaccinia virus (VacV).Virus drop in Plaque Technique Detected detection mouse is used after 48 hours Degree, and monitor viral fatal rate.

Table 2. is injected intravenously Mn²⁺Virus titer in mouse afterwards

A liquid	B liquid	Mncl₂Usage amount	HSV-1 titres	VSV titres	VacV titres
						200μL	0μL	0mg/kg
198μL	2μL	1mg/kg
						196μL	4μL	2mg/kg
190μL	10μL	5mg/kg			Decline hundreds times
						180μL	20μL	10mg/kg	Decline hundreds times

Table 3. is injected intravenously Mn²⁺Mouse lethal rate afterwards

A liquid	B liquid	Mncl₂Usage amount	HSV-1 fatal rates	VSV fatal rates	VacV fatal rates
						200μL	0μL	0mg/kg	100%	100%	100%
198μL	2μL	1mg/kg	80%	50%	55%
						196μL	4μL	2mg/kg	75%	25%	35%
190μL	10μL	5mg/kg	50%	5%	15%
						180μL	20μL	10mg/kg	25%	0%	0%

Test (b)

Eight week old SPF level experiments C57BL/6 (buying from Beijing company of dimension tonneau China) are taken, are used 2mg/kg MnCl₂Mouse is injected intravenously, after 24 hours, with VSV (8x10⁸Pfu/ is only Mouse), VACV (Western Reserve strains, 1x10⁷Pfu/ mouse) or HSV-1 (open countries Raw type F strains, 1.4x10⁷Pfu/ mouse) mouse is attacked, monitor survival (such as Fig. 4 of mouse It is shown).

Test (c)

2mg/kg MnCl is used as described above₂Be injected intravenously mouse, after 24 hours, with VSV, VACV or HSV-1 attack mouse, infection take lung and spleen after four days, are homogenized, then pass through sky Patch test (plaque assay) measures virus titer, as a result as shown in Figure 5.

Embodiment 3.Mn²⁺Handle the influence to downstream gene expression

Test (a)

With 100 μM of MnCl₂, VACV, IFN β or culture medium (control) processing THP1 Cell 12 hours, extract RNA and be sequenced.By calculating log₂((through handling RPKM)/it is (right According to RPKM)) obtain the thermal map (as shown in Figure 6A) of gene expression.

Test (b)

With 200 μM of MnCl₂Handle THP1 cells, discard after 2,12,24 hours containing MnCl₂Culture medium and be replaced with fresh culture, NW represent do not discard containing MnCl₂Culture medium, Fig. 6 B show I-IFN production.

Test (c)

With the MnCl of shown concentration₂, Mn (OAc)₂Or Mn (OAc)₃Handle THP1 Cell 24 hours, supernatant then is analyzed using I-IFN biologicall tests, Fig. 6 C displays are not using With manganese salt processing induction I-IFN productions, show that manganous salt can induce I-IFN to produce, and three Valency manganese salt effect much weaker.

Test (d)

Use in PBS 0,2 or 5mg/kg MnCl₂Solution is injected intravenously mouse, separates blood Analyzed clearly and using I-IFN biologicall tests, be additionally carried out identical experiment, but put to death mouse, taken Organ, ISG is determined by western blot.Fig. 6 D are shown in injection MnCl in mouse medium sized vein₂ I-IFN productions are induced, Fig. 6 E figures are shown in injection MnCl in mouse medium sized vein₂ISG organ afterwards Distribution, wherein all induction of vipoxin and ISG54 in multiple organs.

Test (e)

Fig. 6 F are shown with various concentrations MnCl₂PBMC from healthy adult donor (is used Histopaque-1077 from Sigma is obtained by continuous centrifugal, is incubated at containing 5%FBS's In RPMI-1640 culture mediums) processing 36 hours after I-IFN production and ISG expression, use I-IFN Biometric analysis I-IFN is produced, and ISG expression is determined by western blot.Fig. 6 G are shown With 200 μM of MnCl₂To the I-IFN after handling different time from the PBMC of healthy adult donor Production and ISG expression.These data show Mn²⁺I-IFN productions and ISG tables caused by processing The dose dependent and time dependence reached, so as to prove Mn²⁺No matter processing is in vitro or in body Downstream gene expression is inside triggered.

Test (f)

Take the logarithm growth period THP1 cells or people's primary peripheral blood mononuclear cell (from health Adult donor).A liquid and B liquid (as described above) are mixed and added into according to the ratio in table Into cultivating system.After 16-24 hours, cells and supernatant is taken, is examined using enzyme linked immunosorbent assay Wherein I types interference cellulose content is surveyed, as a result as shown in Table 4 below.

Table 4.Mn²⁺Extracorporeal treatment inducing interferon produces

Test (g)

Eight week old SPF level experiments C57BL/6 (buying from Beijing company of dimension tonneau China) are taken, by A Liquid and B liquid (as described above) mix according to the ratio in table, and pass through tail vein injection.16-24 After hour, mouse peripheral blood serum is taken, detecting wherein I types interferon using enzyme linked immunosorbent assay contains Amount.

Table 5.Mn²⁺It is injected at the production of Immune inducing in vivo interferon

A liquid	B liquid	MnCl₂Usage amount	Peripheral blood serum I type interference cellulose content (U/ML)
				200μL	0μL	0mg/kg	Do not detect
198μL	2μL	1mg/kg	1.46±0.17
				196μL	4μL	2mg/kg	3.39±0.59
190μL	10μL	5mg/kg	10.25±0.77
				180μL	20μL	10mg/kg	12.60±0.68

Embodiment 4.Mn²⁺Directly suppress human immunodeficiency virus (HIV) to infect people's cell

In order to prepare HIV pseudovirus, by HEK293T cell culture in 10cm culture dishes, To~70% degree of converging, then carrier (pLVX si3LTR Luc, in being derived from are transmitted with 10 μ g Institute of microbiology of the academy of sciences of state Takaomi Ishida), 7.5 μ g package carriers (psPAX2, obtain Derived from Addgene) and 5 μ g envelope vectors (VSV-G- vacation types HIV：PMD2.G, obtain From Addgene；CCR5- preferendums HIV：PHIV JRFL env, it is micro- to be derived from the Chinese Academy of Sciences Biological study institute Takaomi Ishida；And CXCR4- preferendums HIV：PRE11 NL43 env, It is derived from Institute of Microorganism, Academia Sinica Takaomi Ishida), use Lipofectamine 2000 (Invitrogen) transfect HEK293T cells.After 6 hours, culture medium is replaced by 10mL DMEM (contains 20%FBS).After 48-72h, the transfectional cell supernatant containing pseudovirus is harvested, Filtered with 0.45 μm of filter.

Prepared pseudovirus is used to infect MAGIC5 cells at once or is stored in -80 DEG C.It is logical Luciferase assay is crossed to determine virus titer.

To infect MAGIC5 cells, the previous day is infected, is inoculated with cell with 30% or so degree of converging In 12 orifice plates.Trigger (prime) cell 4h using the supernatant from THP1 or PBMC, Then itself and CCR5- preferendums, CXCR4- preferendums or VSV-G- vacation types HIV are incubated 6h together. Then, culture medium is replaced by DMEM (containing 10%FBS), cultivated 3 days.Remove culture Base, with 50 μ l 1 × Passive Lysis Buffer (Promega)/hole cell lysis 30 minutes, It is measured using Firefly Luciferase Assay System (Promega).

Take the logarithm the mankind being incubated in the DMEM culture mediums that with the addition of 10%FBS in growth period Cervical cancer tumer line Magic5, A liquid and B liquid (as described above) are mixed according to the ratio in table And it is added in cultivating system.After 24 hours, expression Fluc (firefly is added Luciferase HIV pseudovirus) infects MAGIC5 cells, HIV pseudovirus used difference It is：JRFL (CCR5) type HIV and NL43 (CXCR4) type HIV.After 24 hours, prison Survey by uciferase activity in infected cell, and then judge inhibiting rate (such as table 6 below of virus infection It is shown).

Table 6.Mn²⁺The inhibiting rate that inhibition of HIV infects after processing

A liquid	B liquid	Culture medium	JRFL-HIV inhibiting rates	NL43-HIV inhibiting rates
					10μL	0μL	1mL	0% ± 0.02%	0% ± 0.7%
9μL	1μL	1mL	25.7% ± 0.1%	21.3% ± 0.8%
					8μL	2μL	1mL	60.8% ± 2.3%	44.3% ± 10.9%
5μL	5μL	1mL	89.7% ± 5.9%	65.8% ±
					0μL	10μL	1mL	92.8% ±	89.6% ±

Embodiment 5.Mn²⁺Available for treating tumour

Eight week old SPF level experiments C57BL/6 (buying from Beijing company of dimension tonneau China) are taken, under rib Inject 5x10⁶The mouse lymphoma cell EL4 (Dr.Minghui Zhang, Tsing-Hua University) an of/mouse. 2,4,6 days after inoculated tumour cell respectively.A liquid and B liquid (as described above) according to Ratio mixing in table, carries out intratumor injection.And swollen into 10 after knurl, measurement in 15,20 days The size of knurl, as a result see the table below 7.

Table 7.Mn²⁺HBV titre after processing

Embodiment 6.Mn²⁺Processing inducing cytokine, which produces, depends on cGAS-STING paths

First, the various concentrations MnCl shown in Fig. 7 A₂Solution handles HeLa cells and THP1 is thin After born of the same parents 24 hours, IRF3 phosphorylations and vipoxin (Viperin) production are determined.Specifically, Cell is cracked, the production of IRF3 phosphorylations and vipoxin is determined by western blot.

In order to study Mn²⁺Cell factor caused by processing produces to be worked by which kind of approach, is sent out A person of good sense has obtained the HeLa cells of series of genes knockout using CRISPR/Cas9 systems, then With 500 μM of MnCl₂The HeLa cells of gene knockout, Fig. 7 B show 24 shown in solution processing IRF3 phosphorylations and vipoxin production after hour, as a result show MnCl₂The IRF3 phosphorus of induction Acidifying and the production of vipoxin depend on cGAS, STING, TBK1 and IRF3, and disobey Rely in RIG-I or MAVS.Fig. 7 C are shown with 500 μM of MnCl₂Solution, VACV or SeV The IRF3 phosphorylations of gene deletion shown in processing and the HeLa cells of redemption after 24 hours (pass through Western blot determines), wherein the HeLa cells saved are to use to contain expression people IRF3 respectively, The cDNA sequence (being amplified from THP1 cells) of STING, TBK1 and RIG-I N-terminal The HeLa cells of pcDNA3.1 (Invitrogen) carrier transfection, are as a result shown in and knock out in cell MnCl can be saved by rebuilding cGAS, STING and IRF3₂The IRF3 phosphorylations of induction and adder The production of toxin.

In order to further study Mn²⁺Acted on inside processing, inventor uses CRISPR/Cas9 System has obtained the mouse (being based on C57BL/6J mouse) of series of genes knockout.Fig. 7 D are shown With 500 μM of MnCl₂Solution, VACV or SeV processing WT or shown knock out mice 24 Vipoxin and ISG, which are produced, after hour, in the peritoneal macrophages therefrom obtained (passes through Western Trace determines), as a result equally show MnCl₂The IRF3 phosphorylations of induction and the production of vipoxin Dependent on cGAS, STING, TBK1 and IRF3, and independent of RIG-I or MAVS. Fig. 7 E are shown with 500 μM of MnCl₂Solution, VACV or SeV processing WT or shown clpp genes It is as a result similar to above except the IFN after mouse 24 hours produces (using I-IFN biologicall tests).

Result above fully proves Mn²⁺Played a role by cGAS-STING paths.

Embodiment 7.Mn²⁺Processing directly activates cGAS

In order to further study Mn²⁺The mechanism of action, by total length (1-522) the people cGAS of purifying With MnCl₂, the nucleic acid shown in figure is incubated together.By reaction product and use digitonin The THP1 of (Sigma, D141) permeabilization is mixed 30 minutes, and then cell lysis, lysate is used In the IRF3 phosphorylations of measure cGAMP inductions, in the presence of as a result display only has dsDNA, Mn²⁺ Activate cGAS (as shown in Figure 8 A).Fig. 8 B figures show the people cGAS of total length or mutation with MnCl₂/MgCl₂, a variety of different amounts of dsDNA be incubated together, as a result display and Mg²⁺Compare, Mn²⁺In the presence of cGAS at least 10 are improved to dsDNA sensitiveness⁴Times (10^-2Compared to 10^-6), Wherein mut is the people cGAS that catalysed cationic combination residue is undergone mutation：E225A/D227A.Figure 8C shows people cGAS albumen and 0.5mM MnCl₂Or 5mM MgCl₂, and 10-2 Mg/ml dsDNA are incubated together, are produced using Mono Q ion exchange columns analysis cGAMP, With cGAMP, ATP and GTP as control.These as shown by data, Mn²⁺Considerably improve CGAS hence in so that can draw to DNA sensitiveness under low-down endochylema DNA concentration Send out cGAS enzymatic activity.

On the other hand, inventor compares Mn²⁺And Mg²⁺Effect in stimulating I-IFN to produce, Figure 12 A show Mn²⁺I-IFN is stimulated to produce, but Mg²⁺Almost do not act on.

Inventor have studied Mn²⁺Action site on cGAS, Figure 12 B show Coomassie brilliant blue Purifying total length (1-522), mutant (E225A/D227A) and the truncated mutant (161-522) of dyeing People cGAS, Figure 12 C are shown, are obtained using (161-522) people cGAS and total length cGAS is truncated To similar result.

Figure 12 D are shown in 1mM MnCl₂Or 1mM MgCl₂In the presence of, pass through isothermal Total length the people cGAS and dsDNA of titration calorimetry (ITC) measure combination.

Embodiment 8.Mn²⁺Show strong adjuvanticity

Test (a)

Eight week old SPF level experiments C57BL/6 (buying from Beijing company of dimension tonneau China) are taken, by chicken Ovalbumin antigen (OVA) result is shown, uses Mn²⁺OVA is generated as immunologic adjuvant The antigen specific immune activation of specific CD4+ cells and CD8+ cells, produces substantial amounts of γ Interferon and interleukin 2.

Specifically, at the 0th day and the 10th day with OVA (10 μ g), OVA+10 μ g MnCl₂ Or OVA+30 μ g DMXAA (Selleck, S1537) intramuscular immunisation mouse, at the 17th day Serum and splenocyte are taken, determining OVA specific IgGs 1 (passing through ELISA) using serum produces, As a result as shown in Figure 9 A.As shown by data Mn²⁺The production of antibody is drastically increased as immunologic adjuvant It is raw, and it is better than known STING activator DMXAA.

By above-mentioned splenocyte with 1x10⁶/ hole is inoculated in 24 orifice plates, the OVA synthesized with 10 μ g/ml Peptide H-2Kb (SIINFEKL) or I-Ab (ISQAVHAAHAEINEAGR) or control Peptide (FAPGNYPAL) stimulates splenocyte, takes supernatant to determine IFN γ by ELISA after 24 hours (Liankebio, EK2801) and IL-2 (eBioscience, #88-7024-22) secrete, as a result such as Shown in Fig. 9 B and 9C.As shown by data Mn²⁺Specific CD4+ is greatly enhanced as immunologic adjuvant The antigen specific immune of cell and CD8+ cells activates.

Test (b)

Eight week old SPF level experiments C57BL/6 (buying from Beijing company of dimension tonneau China) are taken, will 10 μ g chicken egg whites antigens (OVA) and A liquid and B liquid (as described above) are according to the ratio in table Example mixing, passes through intramuscular injection；7-14 days after injecting for the first time, carry out second and inject, injection Method and metering are identical with injecting for the first time.28 days after injecting for the first time, mouse is put to death, takes periphery Blood and separated plasma, the content of anti ova antibody in mice plasma is detected using enzyme linked immunosorbent assay, As a result it is as shown in table 8 below.

Table 8.Mn²⁺The generation of processing enhancing antibody

A liquid	B liquid	MnCl2 usage amounts	Anti ova antibody tormation amount (AU/mL)
				200μL	0μL	0mg/kg	10.3±12.3
198μL	2μL	1mg/kg	597±77.3
				196μL	4μL	2mg/kg	1105±157.3
190μL	10μL	5mg/kg	1900±290.9
				180μL	20μL	10mg/kg	2789±587.9

Embodiment 9.Mn²⁺Produced by CCL3 and suppress HIV

Use Mn²⁺THP1 cells (as shown in Figure 10) are handled, Figure 10 A and B show I-IFN (A figures) and CCL3 (B figures) secrete.As a result have unexpectedly shown that, compared with other stimulations, When the amount that stimulation I-IFN is produced is identical, Mn²⁺Processing can result in more CCL3 secretions (about It is high 2-8 times).This result prompts Mn²⁺Processing may be stimulated by CCL3 suppresses HIV's Infection.

Using come the Mn that hangs oneself²⁺The supernatant of the THP1 cells of processing triggers MAGIC5 cells 4 small When, then using CCR5- preferendums (CCR5-tropic), CXCR4- preferendums or VSV-G- False type (pseudotyped) HIV-Luc is viral (preparation of pseudovirus is as described in example 4 above) Infection cell.After 3 days, luciferase is determined, shows virus infection (as illustrated in figure 10 c).Knot Fruit shows, Mn²⁺Processing significantly inhibits CCR5- preferendums (CCR5-tropic) virus really Infection, and for CXCR4- preferendums or VSV-G- vacations type (pseudotyped) HIV-Luc For virus, inhibition level is light many.

Figure 10 D and E, which are shown, uses Mn²⁺I-IFN (D figures) and CCL3 after processing PBMC cells (E figures) is secreted, and tests as described in the experiment (Figure 10 A and B) of THP1 cells above, no With being only that using the PBMC from healthy adult, Mn is used²⁺Handle PBMC cells 18 or 36 Hour, then determine I-IFN and CCL3.Figure 10 F are shown using come the Mn that hangs oneself²⁺Handle PBMC The supernatant of cell triggers MAGIC5 cells to make after 4 hours (remaining is as described in being tested Figure 10 C) Infected with the virus of HIV pseudovirus.Figure 10 G, which are shown, uses Mn²⁺Processing comes from WT or gene The peritoneal macrophages of knock-out mice, CCL3 secretions are determined by ELISA.

Data above shows, in the primary PBMC cells of people, Mn²⁺Processing significantly suppress CCR5- The infection of preferendum (CCR5-tropic) virus, and CXCR4- preferendums or VSV-G- vacation types (pseudotyped) inhibition level of HIV-Luc viruses is then lighter.

Embodiment 10.Mn²⁺Processing does not cause injury of mitochondria

Figure 13 shows Mn²⁺The cGAS activation of induction is not related to injury of mitochondria.In Figure 13 A It is shown, with 20 μM of ABT-263,20 μM of ABT-737 or MnCl₂(50,100,200 μM) Handle THP1 cells 24 hours, pass through double dyeing FCM, the DNA of Annexin V-FITC/PI Gel and western blot measure apoptosis, are as a result shown under the concentration for stimulating IRF3 phosphorylations Mn²⁺It is not apoptosis-induced in THP1 cells, on the contrary, Bcl-2 inhibitor ABT-737/263 but shows Write apoptosis-induced.As shown in Figure 13 B and C, Mn is used²⁺Handle wild type (WT) and Bax-/Bak-/- MEF (is derived from Kevin M Ryan, Beatson Institute for Cancer Research, UK), Casp3 cuttings and vipoxin induction are then determined, these results are shown Mn²⁺Effect is independent of Bak/Bax caused by processing, also not dependent on SOD2.With 500 μM MnCl2 handles the BMDM from C57BL/6J mouse, handles 24 hours, passes through TEM Mitochondria is shown, determines I-IFN productions using supernatant, TEM schemes as illustrated in figure 13d, display For mitochondria without significantly extending or interconnecting, this is common in TFAM deficient cells.With Peritoneal macrophages of the 1 μ g/ml LPS initiations from WT or shown knock out mice 5 hours, Then handled 6 hours as shown in Figure 13 E and F, use supernatant (Sup) and full cell lysate (WCL) inflammation corpusculum (inflammasome) activation is determined, as a result shows Mn²⁺Induction is coming Trigger the strong inflammation corpusculum to activate from the cell of wild-type mice, but from Asc-/- mouse It can not then trigger in cell.Further, Figure 13 F and G are shown, Mn²⁺The inflammation corpusculum of induction Activation is independent of AIM2, SOD1 or NLRP3.Result above shows, Mn²⁺Processing is not led Cause injury of mitochondria, and Mn²⁺The IFN of induction is produced nor caused by injury of mitochondria 's.

Only it is above description preferred embodiment, it is only used as example and implements institute of the present invention without limiting The combination of required feature.The title provided is not intended to the multiple embodiments of the limitation present invention.

All publications and patent referred in the application are incorporated herein by reference.This is not departed from The scope and spirit of invention, a variety of modifications of described method and composition of the invention and variant pair It is obvious in those skilled in the art.Although this is described by specific preferred embodiment Invention, it should be appreciated that the present invention for required protection should not be inadequately confined to these tools Body embodiment.In fact, those are obvious for real for various equivalent modifications The a variety of variants for applying the described pattern of the present invention are intended to the scope for being included in appended claims It is interior.

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Claims

1. bivalent manganese is preparing the use in being used to improve the medicine of inherent immunity and/or adaptive immunity On the way.

2. purposes according to claim 1, wherein the bivalent manganese is by activating cGAS-STING Path improves inherent immunity and/or adaptive immunity.

3. according to the purposes of foregoing any claim, wherein the bivalent manganese is by strengthening endochylema DNA impressions improve inherent immunity and/or adaptive immunity.

4. according to the purposes of foregoing any claim, wherein the bivalent manganese is by improving cGAS Inherent immunity and/or adaptive immunity are improved to DNA sensitiveness.

5. according to the purposes of foregoing any claim, wherein the bivalent manganese is the shape of manganous salt Formula, preferably described manganous salt are selected from：Manganese chloride, manganous bromide, manganese iodide, manganese sulfate, nitric acid Manganese, Manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, hydrobromic acid manganese, manganese tartrate, Fumaric acid manganese, maleic acid manganese, manganese lactate, benzene sulfonic acid manganese, pantothenic acid manganese, Manganese ascorbate and its any Combination.

6. according to the purposes of foregoing any claim, wherein the bivalent manganese is free bivalent manganese The form of ion.

7. bivalent manganese is preparing the purposes in being used to activate the medicine of cGAS-STING paths.

8. purposes of the bivalent manganese in the medicine experienced for strengthening endochylema DNA is prepared.

9. bivalent manganese is being prepared for improving cGAS to the purposes in the medicine of DNA sensitiveness.

10. bivalent manganese prepare be used to treating selected from bacterium infection, viral infection, parasite, itself Purposes in the medicine of the disease of immunity disease and cancer.

11. purposes according to claim 10, wherein the virus is selected from：DNA virus and RNA Virus, preferably described virus are selected from：Herpetoviridae, Rhabdoviridae, filamentous virus section, just Myxovirus section, Paramyxoviridae, coronaviridae, Picornaviridae, hepadnavirus Section, flaviviridae, Papillomaviridae, Poxviridae and Retroviridae, more preferably institute Virus is stated to be selected from：Herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV.

12. purposes according to claim 10, wherein the bacterium is selected from Gram-negative bacteria and leather Lan Shi positive bacterias, preferably described bacterium are selected from streptococcus pneumonia (Streptococcus Pneumoniae), haemophilus influenzae (Haemophilus influenzae), salmonella (Salmonella), diplococcus meningitidis (Meningococcus), MRSE (Staphylococcus epidermidis), staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Friedlander's bacillus (Klebsiella pneumoniae), Acid-producing Klebsiella bacterium (Klebsiella oxytoca), enterobacter cloacae (Enterobacter cloacae), Citrobacter freundii (Citrobacter freundii), Pseudomonas aeruginosa (Pseudomonas ) and Baume acinetobacter calcoaceticus (Acinetobacter baumanni) aeruginosa.

13. purposes according to claim 10, wherein the autoimmune disease is selected from I types sugar Urinate disease, psoriasis, rheumatoid arthritis, systemic loupus erythematosus and multiple sclerosis.

14. purposes according to claim 10, wherein the cancer is selected from oophoroma, lung cancer, stomach Cancer, breast cancer, liver cancer, cancer of pancreas, cutaneum carcinoma, malignant mela noma, head and neck cancer, sarcoma, courage Pipe cancer, carcinoma of urinary bladder, kidney, colon cancer, Chorionic villi of placenta cancer, cervix cancer, carcinoma of testis, uterus Cancer and leukaemia.

15. purposes according to claim 10, wherein the parasite is cytozoon, it is excellent Selection of land is selected from plasmodium, Infection of Toxoplasma Gondii, trypanosome, blood fluke, filaria and Leishmania.

16. purposes of the bivalent manganese in immunologic adjuvant is prepared.

17. purposes according to claim 16, wherein immunologic adjuvant activation T cell activation and / or antibody producing.

18. bivalent manganese is being prepared for the purposes in the medicine of induced chemokine CCL3 productions.

19. purposes according to claim 18, wherein Chemokines CC CL3 production and then suppression HIV.

20. vaccine combination, it is included：

One or more of antigens, and

Bivalent manganese.

21. for the kit of immunity inoculation, it is included：

First container, wherein accommodating one or more of antigens；With

Second container, wherein accommodating bivalent manganese；

Optionally, first container and/or second container also include pharmaceutically acceptable carrier.