CN107412260B - cGAS-STING pathway activators and uses thereof - Google Patents

Abstract

The invention provides a novel activator of a cGAS-STING pathway and application of the activator for activating the cGAS-STING pathway. The invention also provides the use of said activator for improving innate and/or adaptive immunity.

Description

cGAS-STING pathway activators and uses thereof

Technical Field

The invention provides a novel activator of cGAS-STING pathway and application of the activator for activating the cGAS-STING pathway. The invention also provides the use of said activator for improving innate and/or adaptive immunity.

Background

The natural immune system plays an important role in resisting infection, inhibiting tumor growth and the pathogenesis of autoimmune diseases, mainly recognizes components of pathogenic microorganisms and cancer cells through a pattern recognition receptor, starts a downstream signal path, and finally kills the pathogenic microorganisms and infected or cancerized cells on one hand by inducing cytokine expression, and activates an acquired immune system to promote the generation of antibodies and specific T lymphocytes on the other hand, thereby protecting the body from the invasion of the pathogenic microorganisms and the tumors.

Specifically, the innate immune system uses a series of pathogen recognition receptors, called Pattern Recognition Receptors (PRRs), to monitor extracellular danger signals, non-essential and self-essential components of the endosome and cytoplasm, such as LPS, bacterial flagellin, cyclic dinucleotides, CpG DNA, AT-rich DNA, dsDNA, ssDNA, etc. PRR can generate a moderate immune response against pathogens or self tissue damage, and DNA entering the cytoplasm of cells is considered by the body to be a danger signal for pathogen infection, and if cytoplasmic DNA cannot be cleared reasonably, it will accumulate in the cytoplasm and trigger pathological inflammation and autoimmune diseases, such as Systemic Lupus Erythematosus (SLE).

Free DNA present in the cell cytoplasm is recognized by the host's innate immune system as a potential danger signal, but the mechanisms by which the immune system recognizes and clears these danger signals are currently unclear. DNA sensors (DNA sensors) are a bridge for host to sense DNA and immune defense, more than 10 DNA sensors are found at present, and interferon stimulating genes (STING) play an important signal transmission role in sensing cytoplasmic DNA and immune defense as a key linker molecule at the downstream of a DNA sensing path. In particular, a novel DNA receptor, cGAS (cGMP-AMP synthase), has recently been discovered in mammalian cells, a nucleic acid transferase that produces endogenous CDN: 2 '-3' Cyclic AMP-GMP (cGAMP), which in turn activates STING. This pathway, known as the cas-STING pathway, plays an important role in the initiation of the immune response in the body.

Disclosure of Invention

The present inventors have surprisingly discovered a novel class of cGAS-STING activators that are capable of effectively activating the cas-STING pathway, thereby improving innate and/or adaptive immunity. In particular, the cGAS-STING activator is selected from the group consisting of divalent manganese, divalent manganese element, a divalent manganese source, manganese (which is present in the subject as divalent manganese), and manganese element (which is present in the subject as divalent manganese).

In one aspect, the invention provides the use of divalent manganese in the manufacture of a medicament for improving innate and/or adaptive immunity. In some embodiments, the divalent manganese improves innate and/or adaptive immunity by activating the cGAS-STING pathway. In other embodiments, the divalent manganese improves innate and/or adaptive immunity by enhancing cytoplasmic DNA perception. In still other embodiments, the divalent manganese improves innate and/or adaptive immunity by increasing cGAS sensitivity to DNA.

In another aspect, the invention provides the use of a source of divalent manganese in the manufacture of a medicament for improving innate and/or adaptive immunity in a subject. Preferably, the source of divalent manganese in the medicament is present in the subject as divalent manganese. In some embodiments, the divalent manganese improves innate and/or adaptive immunity by activating the cGAS-STING pathway. In other embodiments, the divalent manganese improves innate and/or adaptive immunity by enhancing cytoplasmic DNA perception. In still other embodiments, the divalent manganese improves innate and/or adaptive immunity by increasing cGAS sensitivity to DNA.

In another aspect, the invention provides the use of elemental manganese in the manufacture of a medicament for improving innate and/or adaptive immunity in a subject, wherein the elemental manganese in the medicament is present in the subject as divalent manganese. In some embodiments, the manganese improves innate and/or adaptive immunity by activating the cGAS-STING pathway. In other embodiments, the manganese improves innate and/or adaptive immunity by enhancing cytoplasmic DNA perception. In still other embodiments, the manganese improves innate and/or adaptive immunity by increasing cGAS sensitivity to DNA.

In one aspect, the present invention provides a method of improving innate and/or adaptive immunity in a subject comprising administering to the subject divalent manganese or a prodrug thereof.

In another aspect, the invention provides a method of improving innate and/or adaptive immunity in a subject comprising administering to the subject a source of divalent manganese. Preferably, the source of divalent manganese is present in the subject in the form of divalent manganese or is converted to the form of divalent manganese.

In another aspect, the present invention provides a method of improving innate and/or adaptive immunity in a subject comprising administering to the subject an element of manganese, wherein the element of manganese is present in or converted to a form of divalent manganese in the subject.

In one aspect, the present invention provides divalent manganese or a prodrug thereof, which improves innate and/or adaptive immunity in a subject.

In another aspect, the invention provides a source of divalent manganese that improves innate and/or adaptive immunity in a subject. Preferably, the source of divalent manganese is present in the subject in the form of divalent manganese or is converted to the form of divalent manganese.

In another aspect, the present invention provides an element of manganese that improves innate and/or adaptive immunity in a subject, wherein the element of manganese is present in or converted to a form of divalent manganese in the subject.

In some embodiments, the divalent manganese is in the form of a divalent manganese salt, preferably the divalent manganese salt is selected from the group consisting of: manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, manganese hydrobromide, manganese tartrate, manganese fumarate, manganese maleate, manganese lactate, manganese benzenesulfonate, manganese pantothenate, manganese ascorbate, and any combination thereof.

In other embodiments, the divalent manganese is in the form of free divalent manganese ions.

In yet another aspect, the invention provides the use of divalent manganese, a source of divalent manganese, or an element of manganese in the manufacture of a medicament for activating the cGAS-STING pathway.

In a further aspect, the invention provides the use of divalent manganese, a source of divalent manganese or an element of manganese in the manufacture of a medicament for enhancing perception of cytoplasmic DNA. Preferably, divalent manganese enhances cytoplasmic DNA perception by at least 10, 50, 100, 500, 1000, 5000, or 10 compared to when divalent manganese is absent⁴And (4) doubling.

In a further aspect, the invention provides the use of divalent manganese, a source of divalent manganese or an element of manganese in the manufacture of a medicament for increasing cGAS sensitivity to DNA. In particular, increased sensitivity of cGAS to DNA means that cGAS is activated in the presence of lower levels of DNA (e.g., cytoplasmic DNA) than in the absence of divalent manganese. Preferably, the sensitivity of cGAS to DNA is increased by at least 10, 50, 100, 500, 1000, 5000, or 10 compared to when divalent manganese is absent⁴Multiple, or cGAS as low as 10^-7、10^-6、10^-5Or 10^-4Activated at dsDNA concentration of mg/ml.

In a further aspect, the invention provides the use of divalent manganese, a source of divalent manganese or an element of manganese in the manufacture of a medicament for the treatment of a disease selected from bacterial infection, viral infection, parasite, autoimmune disease and cancer.

In some embodiments, the virus is selected from: DNA viruses and RNA viruses, preferably said viruses are selected from: herpesviridae, rhabdoviridae, filoviridae, orthomyxoviridae, paramyxoviridae, coronaviridae, picornaviridae, hepadnaviridae, flaviviridae, papilloma viridae, poxviridae, and retroviridae, more preferably the virus is selected from the group consisting of: herpes simplex virus, vesicular stomatitis virus, vaccinia virus, HIV and HBV.

In some embodiments, the bacterium is selected from gram-negative and gram-positive bacteria, preferably the bacterium is selected from the group consisting of Streptococcus pneumoniae (Streptococcus pneumoniae), Haemophilus influenzae (Haemophilus influenzae), Salmonella (Salmonella), diplococcus meningitides (Meningococcus), Staphylococcus epidermidis (Staphylococcus epidermidis), Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Klebsiella pneumoniae (Klebsiella pneumoniae), Klebsiella oxytoca (Klebsiella oxytoca), Enterobacter cloacae (Enterobacter cloacae), Citrobacter freundii (Citrobacter freundiii), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Acinetobacter baumannii (Acinetobacter baumannii).

In some embodiments, the autoimmune disease is selected from type I diabetes, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis.

In some embodiments, the cancer is selected from ovarian cancer, lung cancer, gastric 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.

In some embodiments, the parasite is an intracellular parasite, preferably selected from the group consisting of plasmodium, toxoplasma, trypanosoma, schistosoma, filarial and leishmania.

In yet another aspect, the invention provides the use of divalent manganese, a source of divalent manganese, or an element of manganese in the preparation of an immune adjuvant. In some embodiments, the immune adjuvant activates T cell activation and/or antibody production. Preferably, the immune adjuvant is used in a vaccine composition for the treatment of a disease selected from the group consisting of a bacterial infection, a viral infection, a parasite, an autoimmune disease and cancer.

In a further aspect, the invention provides the use of divalent manganese, a source of divalent manganese or an element of manganese in the preparation of a vaccine composition, wherein the vaccine composition further comprises one or more antigens. In some embodiments, the divalent manganese, source of divalent manganese, or elemental manganese in the vaccine composition activates T cell activation and/or antibody production. Preferably, the vaccine composition is for use in the treatment of a disease selected from the group consisting of a bacterial infection, a viral infection, a parasite, an autoimmune disease and cancer.

In yet another aspect, the invention provides the use of divalent manganese, a source of divalent manganese, or an element of manganese in the manufacture of a medicament for inducing the production of the chemokine CCL 3. In some embodiments, the production of the chemokine CCL3 further inhibits HIV infection.

In yet another aspect, the present invention provides a vaccine composition comprising: one or more antigens, and divalent manganese. Preferably, the vaccine composition of the present invention further comprises a pharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a kit for immunization comprising: a first container containing one or more antigens therein; and a second container in which divalent manganese is contained. Preferably, the first container and/or the second container further comprise a pharmaceutically acceptable carrier. In particular, the kit is used for one or more purposes of the present invention.

In some embodiments, the antigen used in the present invention is selected from: viral or bacterial or parasitic antigens, for example, hepatitis A, B, C, D and E3 viruses, HIV, herpes viruses types 1, 2, 6 and 7, cytomegalovirus, varicella zoster, papilloma virus, EB virus, influenza virus, parainfluenza virus, adenovirus, bunyavirus (hantavirus), coxsackievirus, picornavirus, rotavirus, respiratory syncytial virus, poxvirus, rhinovirus, rubella virus, papovaviruses, mumps virus and measles virus, mycobacteria causing tuberculosis and leprosy, pneumococci, aerobic gram negative bacilli, mycoplasma, staphylococcal infection, streptococcal infection, salmonella and chlamydia, helicobacter pylori, malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, schistosomiasis and filariasis.

In some embodiments, the divalent manganese source, the manganese element, and/or the antigen in the present invention are in effective amounts.

Preferably, the source of divalent manganese and/or the element of manganese is present in the subject in the form of divalent manganese or is converted to the form of divalent manganese.

In yet another aspect, the present invention provides a method for activating the cGAS-STING pathway in a subject, comprising administering to the subject divalent manganese, a source of divalent manganese, and/or an element of manganese. Furthermore, the present invention provides a method for in vitro activation of the cGAS-STING pathway in a cell comprising applying divalent manganese to said cell, preferably said method is of non-therapeutic interest.

In yet another aspect, the invention provides a method for enhancing perception of cytoplasmic DNA in a subject comprising administering to the subject divalent manganese, a source of divalent manganese, and/or an element of manganese. Furthermore, the invention provides a method for enhancing the perception of cytoplasmic DNA in a cell in vitro comprising applying divalent manganese to said cell, preferably said method is of non-therapeutic interest.

In yet another aspect, the present invention provides a method for increasing cGAS sensitivity to DNA in a subject comprising administering to the subject divalent manganese, a source of divalent manganese, and/or an element of manganese. Furthermore, the present invention provides a method for increasing cGAS sensitivity to DNA in vitro comprising applying divalent manganese to said cells, preferably said method is of non-therapeutic purpose.

In yet another aspect, the invention provides a method for treating a disease selected from the group consisting of a bacterial infection, a viral infection, a parasite, an autoimmune disease, and cancer in a subject, comprising administering to the subject divalent manganese, a source of divalent manganese, or an element of manganese.

In yet another aspect, the invention provides a method for activating T cell activation and/or antibody production in a subject, comprising administering to the subject divalent manganese, a source of divalent manganese, or an element of manganese.

In yet another aspect, the present invention provides a method for inducing production of chemokine CCL3 in a subject, comprising administering to the subject divalent manganese, a source of divalent manganese, or an element of manganese.

In a further aspect, the present invention provides bivalent manganese, a bivalent manganese source and/or an element of manganese for use in activating the cGAS-STING pathway, enhancing cytoplasmic DNA perception, increasing cGAS sensitivity to DNA, treating a disease selected from the group consisting of bacterial infection, viral infection, parasite, autoimmune disease and cancer, activating T cell activation and/or antibody production, and/or inducing chemokine CCL3 production in a subject.

Drawings

The foregoing and other aspects of the invention will become apparent from the following detailed description of the invention and the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, it being understood, however, that the invention is not limited to the specific embodiments disclosed.

FIG. 1 shows Mn²⁺The pretreatment confers resistance to viral infection to the host cell. Wherein 100. mu.M MnCl is used₂THP1 cells were pretreated for 12 hours and then infected with HSV-1-GFP, VSV-GFP or NDVGFP. After 24 hours, the cells were photographed (left) and measured using flow cytometry (right).

FIG. 2 shows Mn²⁺Dose dependence of antiviral activity of the pretreatment. Wherein MnCl is used in the indicated concentration₂Or dmxaa (dmx) for 24 hours in THP1 cells, followed by infection of the cells with the indicated GFP-viruses for 24 hours, after which GFP expression was measured by Western blot.

FIG. 3 shows Mn²⁺Dose dependence of antiviral activity of the pretreatment. Wherein MnCl is used in the indicated concentration₂Or dmxaa (dmx) for 24 hours in THP1 cells, then infecting the cells with the indicated GFP-viruses for 24 hours, after which the Median Fluorescence Intensity (MFI) distribution of virus-GFP, where MnCl would not be used, was measured by flow cytometry₂The MFI of treated THP1 cells was normalized to 1.

FIG. 4 shows Mn injected intravenously²⁺The mice were given virus resistance. Wherein 2mg/kg of MnCl is used₂Mice were injected intravenously and 24 hours later, they were challenged with VSV, VACV or HSV-1 and monitored for survival.

FIG. 5 showsIndicating intravenous Mn ²⁺The mice were given virus resistance. Wherein 2mg/kg of MnCl is used₂Mice were injected intravenously, 24 hours later, challenged with VSV, VACV or HSV-1, lungs and spleen were taken four days after infection, homogenized, and viral titers were measured by plaque assay (plaque assay), where data are expressed as mean ± SEM, with similar results obtained in at least 3 independent experiments.

FIG. 6 shows Mn²⁺Effect of treatment on gene expression. Wherein, the A picture shows the use of MnCl₂THP1 cells were treated for 12 hours, with VACV, IFN β or media (control), RNA extracted and sequenced. By calculating log₂(treated RPKM)/(control RPKM)) to obtain a heat map of gene expression; b shows Mn²⁺Treatment induced I-IFN production with 200. mu.M MnCl₂THP1 cells were treated and 2, 12, 24 hours later, the cells containing MnCl were discarded₂The medium was replaced with fresh medium, NW indicated that MnCl was not discarded₂A culture medium; c-diagram shows the induction of I-IFN production using different manganese salt treatments with MnCl at the concentrations indicated₂，Mn(OAc)₂Or Mn (OAc)₃THP1 cells were treated for 24 hours and then supernatants were analyzed using an I-IFN bioassay; panel D shows intravenous injection of MnCl in mice₂Inducing I-IFN production using 0, 2 or 5mg/kg MnCl in PBS ₂The solution was injected intravenously into mice, sera were separated and analyzed using I-IFN bioassay; e Panel shows intravenous injection of MnCl in mice₂Organ distribution of post-ISG using 0, 2 or 5mg/kg MnCl in PBS₂The solution was injected intravenously into mice, organs were removed and ISG was measured by Western blot; f diagram shows the use of different concentrations of MnCl₂I-IFN production and ISG expression of PBMCs in healthy adult donors after 36 hours of treatment, I-IFN production was analyzed using an I-IFN bioassay, and ISG expression was determined by Western blotting; g shows the use of 200. mu.M MnCl₂I-IFN production and ISG expression of PBMCs in healthy adult donors after different time periods were treated, I-IFN production was analyzed using an I-IFN bioassay, and ISG expression was determined by Western blotting. Data are presented as mean ± SD.

FIG. 7 shows Mn²⁺Treatment induces cytokine production dependenceIn the cGAS-STING pathway. Panel A shows the use of different concentrations of MnCl₂IRF3 phosphorylation and viper toxin (Viperin) production 24 hours after solution treatment of HeLa and THP1 cells; panel B shows the use of 500. mu.M MnCl₂IRF3 phosphorylation and viper toxin production 24 hours after the solution treatment of HeLa cells with the gene knockout shown; panel C shows the use of 500. mu.M MnCl₂IRF3 phosphorylation 24 hours after treatment of HeLa cells lacking and rescued the indicated genes with solution, VACV or SeV (as determined by Western blot); panel D shows the use of 500. mu.M MnCl ₂Viper toxin and ISG production (by Western blot assay) in peritoneal macrophages taken from WT or indicated knockout mice 24 hours after treatment with solution, VACV or SeV; E-Panel shows IFN production (using I-IFN bioassay) after treatment in D. Data are expressed as mean ± SD, with similar results obtained in at least 3 independent experiments.

FIG. 8 shows Mn²⁺cGAS is directly activated. Panel A shows purified full-Length (1-522) human cGAS with MnCl₂The nucleic acids shown in the figure were incubated together, the reaction was performed using digitonin-permeabilized THP1, the cells were lysed, and the lysates were used to determine cGAMP-induced phosphorylation of IRF 3; panel B shows full-length or mutated human cGAS with MnCl₂/MgCl₂A plurality of different amounts of dsDNA; panel C shows human cGAS protein with 0.5mM MnCl₂Or 5mM MgCl₂And 10-2mg/ml dsDNA, using Mono Q ion exchange column for cGAMP production, using cGAMP, ATP and GTP as controls. Similar results were obtained in at least 3 independent experiments.

FIG. 9 shows Mn²⁺Strong adjuvant activity of (1). Panel a shows mice immunized intramuscularly with OVA (10 μ g), OVA +10 μ g MnCl2 or OVA +30 μ g DMXAA on days 0 and 10, sera and spleen cells were taken on day 17 and OVA-specific IgG1 (by ELISA), AU: OD450 in absorbance units, data expressed as mean + -SD, from three independent experiments spleen leukocytes in A were stimulated with OVA peptide H-2Kb or I-Ab, supernatants were taken for IFN γ (panel B) and IL-2 (panel C) secretion by ELISA, and data expressed as mean + -SD.

FIG. 10 shows Mn²⁺HIV infection was inhibited by CCL3 production.(panels A and B) THP1 cells were treated as indicated in the figure and I-IFN (panel A) and CCL3 (panel B) secretion were determined. (C diagram) use of Mn from warp²⁺Supernatants of treated THP1 cells primed MAGIC5 cells for 4 hours, and then infected with CCR5-tropic (CCR5-tropic), CXCR 4-tropic, or VSV-G-pseudotyped (pseudotyped) HIV-Luc virus. After 3 days luciferase was assayed showing viral infection. The luciferase activity values of the control supernatant primed MAGIC5 cells were normalized to 1. (Panels D and E) experiments were carried out as shown in (Panels A and B) with the only difference that PBMC from healthy adults were used, with Mn²⁺PBMC cells were treated for 18 or 36 hours and then assayed for I-IFN and CCL 3. The F plot is similar to the C plot, except that Mn from warp is used²⁺Supernatants from treated PBMC cells primed MAGIC5 cells. (panel G) peritoneal macrophages from WT or knockout mice were treated as indicated in the figure and CCL3 secretion was determined by ELISA. Data are presented as mean ± SD.

FIG. 11 shows Mn²⁺And DMXAA activates mouse peritoneal macrophages. (graph A) using Mn in the amount shown²⁺And DMXAA treated mouse peritoneal macrophages for 36, 48 hours, followed by assay of viper toxin.

FIG. 12 shows Mn ²⁺cGAS is directly activated. Panel A shows MnCl in the amounts indicated₂/MgCl₂THP1 cells were treated for 24 hours, and the supernatant was collected and assayed for I-IFN. Panel B shows purified full-length (1-522), mutant (E225A/D227A) and truncation mutant (161-522) human cGAS stained with Coomassie Brilliant blue. Panel C shows the use of truncated (161-522) human cGAS and MnCl₂/MgCl₂A variety of different amounts of dsDNA were incubated together, similar to fig. 8B. Panel D shows MnCl at 1mM₂Or 1mM MgCl₂Binding of full-length human cGAS to dsDNA was determined by Isothermal Titration Calorimetry (ITC) in the presence.

FIG. 13 shows Mn²⁺Induced cGAS activation does not involve mitochondrial damage. (Panel A) using 20. mu.M ABT-263, 20. mu.M ABT-737 or MnCl₂THP1 cells were treated (50, 100, 200. mu.M) for 24 hours and apoptosis was determined by Annexin V-FITC/PI double staining FCM, DNA gel and Western blot. (panels B and C) Wild Type (WT) and Bax-/-Bak-/-MEF were treated as indicated and Casp3 cleavage and viper toxin induction were then assayed. (FIG. D)) BMDM from C57BL/6J mice was treated with 500. mu.M MnCl2, mitochondria were visualized by TEM, and supernatant was used to determine I-IFN production. (FIGS. E and F) peritoneal macrophages from WT or the indicated knock-out mice were primed with 1. mu.g/ml LPS for 5 hours and then treated as indicated for 6 hours. Inflammatory corpuscle (inflamosome) activation was determined using supernatant (Sup) and Whole Cell Lysate (WCL). Data are presented as mean ± SD.

Detailed Description

Definition of

The term "innate immunity" as used herein refers to the natural immune defense function developed by the body during phylogenetic development and evolution, i.e., the non-specific defense function that is already present after birth, also known as non-specific immunity (non-specific immunity). Innate immunity involves a variety of cells and molecules such as macrophages, natural killer cells, complements, 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, and the like, CXC chemokines such as CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, and the like, C chemokines, CX3C chemokines), lysozyme, and the like.

The term "adaptive immunity", also known as acquired immunity or specific immunity, as used herein, refers to the specific immunity developed by the body against an antigen following stimulation by the antigen molecule, which involves both cellular and humoral immunity.

The term "adjuvant" as used herein refers to an agent that does not constitute a specific antigen, but enhances the intensity and duration of an immune response to a co-administered antigen.

The term "manganous salt", as used herein, may be a hydrochloride, hydrobromide, sulfate, nitrate, phosphate, tartrate, fumarate, maleate, lactate, benzenesulfonate, pantothenate, ascorbate, etc., or any combination thereof. Preferably, the salt is a pharmaceutically acceptable salt.

Terms such as "comprising," "including," "containing," and "including" as used herein are not intended to be limiting. Further, unless otherwise specified, "or," or "means" and/or.

It should also be noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly and clearly dictates otherwise. And if a particular value is referred to, at least that value is included, unless the context clearly dictates otherwise.

Where values represent approximations, it will be understood that the particular values form another embodiment. As used herein, "about X" (wherein X is a number) means + -10% (inclusive) of the recited values. All ranges, if any, are inclusive and combinable.

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, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, and the like. The type of pharmaceutically acceptable carrier used depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The compositions according to the invention may contain lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring substances, flavoring substances and/or aromatic substances and the like as additives.

The pharmaceutical composition according to the invention may be administered by any suitable route, e.g. orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

The term "administering" as used herein means providing a substance to a subject in a pharmacologically useful manner.

As used herein, "pharmaceutically effective amount" and "effective amount" refer to a dosage sufficient to show its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the subject's own condition and severity. Prescription of treatment (e.g., decisions on dosage, etc.) is ultimately the responsibility of and depends on general practitioners and other physicians, often taking into account the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the physician.

The term "subject" as used herein means animals, including warm-blooded mammals, such as humans and primates; birds; domestic or farm animals, such as cats, dogs, sheep, goats, cattle, horses and pigs; laboratory animals such as mice, rats and guinea pigs; fish; a reptile; zoo animals and wild animals, etc.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Unless otherwise indicated, any component, element, attribute, or step disclosed with respect to one embodiment of the present methods and products can be applied to any other method and product disclosed herein.

Each patent, patent application, cited publication, or description in this document of this disclosure is incorporated by reference in its entirety.

The invention is further defined in the following examples. It will be understood that these examples are given by way of illustration only and are not intended to limit the scope of the present invention. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Materials and methods

Antibodies and reagents

The antibody sources were as follows: anti-GFP (Sungene Biotech, KM8009), anti-GAPDH (Santa Cruz, sc-25778), anti-cGAS (Santa Cruz, sc-245831), anti-p-IRF 3(Epitomics, 2562-1), anti-pKK α/β (Cell signaling, #2078S), anti-IkB α (Cell signaling, #4814), anti-TBK 1(Santa Cruz, sc-52957, Cell signaling, #3504), anti-STING (Proteintetech, 19851-1-AP; Abgent, AP97 9747B), anti-RIG-I (Cell signaling, #3743S), anti-MAVS (Santa Cruz, sc-166583), anti-pase-3 (Santa Cruz, san-271759). Other antibodies were prepared and used by the methods described in the prior art (7, 34, 35), viper toxin (Viperin) (human 75-361aa, mouse 243-360aa), ISG54 (human 6-285aa, mouse 3-289aa), IRF3 (human full length), Lamin A (human full length), Casp1/p20 (mouse 121-296aa), IL1 β/p17 (mouse 118-269aa) and ASC (mouse full length) cDNA was inserted into pET-21b vector (Novagen) and expressed in E.coli BL21(DE 3). The recombinant protein was purified by Ni-NTA affinity column and then injected into mice or rabbits to generate antiserum.

All chemicals were purchased from Sigma-Aldrich (st. louis, MO), unless otherwise noted. Mouse CCL3 ELISA Kit (eBioscience, #88-56013-22), human CCL3 ELISA Kit (Liankibio, 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, sense sequence: 5-tacagatctactagtgatctatgactgatctgtacatgatctaca-3), dsRNA/ssRNA (Dharmacon, sense sequence: 5-uacagaucuacuagugaucuaugacugaucuguacaugaucuaca-3). The dsDNA and dsRNA are annealed in equimolar amounts between the sense and antisense sequences.

Plasmids

The Flag-tagged human cGAS expression vector was awarded by dr. zhijian chen (ut south western Medical center). pET-21b-hcGAS is a gift from Xiaoodong Su (Peking University). pU6-sgRNA and pcDNA3.1-CAS9 were gifted by Jianzhong Jeff Xi (Peking University). pLVX si3LTR Luc, pHIV JRFL env and pRE11 NL43env were gifted by Takaomi Ishida (Institute of Microbiology, CAS). psPAX2 and pmd2.g were obtained from Addgene. pGL3-Basic vector and pRL-SV40-Renilla were obtained from Promega. The N-terminal cDNAs encoding human IRF3, STING, TBK1 and RIG-I were amplified from THP1 cells and cloned into pcDNA3.1 (Invitrogen). All constructs were confirmed by sequencing.

Cells

HEK293T, L929-ISRE, 2fTGH and its derived mutants (U3A, U4A, U5A and 2A), 2fTGH-ISRE, HeLa and its derived knock-outs, iBMDM, BHK21, MAGIC5 cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), 5. mu.g/ml penicillin and 10. mu.g/ml streptomycin. THP1 was cultured in RPMI-1640(Gibco) medium supplemented with 10% FBS (Gibco). Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from peripheral blood of healthy human adult volunteers by continuous centrifugation using Histopaque-1077(Sigma, 10771) and cultured in RPMI-1640 medium supplemented with 5% FBS. Wild-type and knockout MEFs were prepared from day 15 embryos and cultured in DMEM supplemented with 10% FBS. Peritoneal macrophages were harvested from mice 5 days after induction of thioglycolate (BD, Sparks, MD) injection and cultured in DMEM supplemented with 5% FBS. Bone marrow-derived macrophages (BMDM) were collected from femur and tibia and cultured in DMEM conditioned medium supplemented with 20% FBS and 30% L929 for 5-7 days.

Wild-type iBMDM cells of C57BL/6 mouse origin (Kathermine Fitzgerald, University of Massachusetts Medical School), Tlr4-/-iBMDM cells (Aihao Ding, Weill Cornell Medical School), Tbk1-/-MEFs (Wen-Chen Yeh, Amgen), MAGIC5(Takaomi Ishida, Institute of Microbiology, CAS), 2fTGH and its derived mutants (George Stark, Cleveland Clinic) and Bax-/-Bak-/-cells (Kevin M Ryan, Beatson Institute for Cancer Research, UK) are derived as shown.

Mouse

cGAS-/-, Sting-/-and cGAS-/-Sting-/-mice were obtained from the hybridization of cGAS and Sting founder mice (fountain mice) obtained by cytosolic injection of Cas9 mRNA (100 ng/. mu.l) and gRNA (50 ng/. mu.l) into C57BL/6J fertilized eggs. Cas9 mRNA and single guide (singleguide) RNA (gRNA) were transcribed in vitro by mMESSAGE mMACHINE T7 Ultra (Ambion, am 1345). Sod1-/- (#002972) and Sod2-/- (#002973) mice were purchased from Jackson Laboratory. Nlrp3-/-, Asc-/-and Aim 2-/-were awarded by Vishva Dixit (Genentech Inc, USA). Mavs-/-mice were obtained from Zhijian Chen (UT South Western Medical Center), Irf 3-/-and Irf3-/-Irf 7-/-mice were obtained from Tadatsugu Taniguchi (The University of Tokyo).

All mice were fed under sterile conditions to the university of Beijing Laboratory animal center according to the NIH guidelines for the administration of Laboratory Animals (National Institute of Health Guide for Care and Use of Laboratory Animals).

Type I IFN (Type I-IFN) bioassay

Type I IFN concentrations were determined as described previously (36). Briefly, IFN-sensitive luciferase vectors were constructed by cloning IFN-stimulated response elements (ISRE) into pGL3-Basic vector (Promega), and then stably transfected into 2fTGH or L929 cells. 2fTGH-ISRE or L929-ISRE cells were seeded into 96-well plates and incubated with human or mouse cell culture supernatants, respectively. Recombinant human and mouse IFN β (R & D Systems) were used as standards. After 4 hours, the cells were lysed and assayed by the Luciferase Reporter Assay System (Promega).

Luciferase reporter assay

Luciferase reporter assays were performed as described previously (7). Briefly, the-1076 to-1, -800 to-1, -700 to-1 and-601 to-1 of the human CCL3 promoter region, and-155 to-1 of the mouse IFN β promoter region were cloned into pGL3-Basic vector (Promega). HEK293T cells at 1X10⁵Per well density was inoculated into 24 well plates, transfected with the expression vector, using pGL3-Basic derived luciferase reporter vector and pRL-SV40-Renilla (Promega) as internal controls. After 24 hours, the activity of the Reporter gene was measured using a Dual-Luciferase Assay System (Dual-Luciferase Reporter Assay System).

Viral infection

Herpes simplex virus 1-GFP (HSV-1-GFP, Kos strain, from Chinese academic of Medical Sciences, Beijing), vesicular stomatitis virus-GFP (VSV-GFP, from Rongbin Zhou, University of Science and Technology of China), newcastle disease Virus-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 strain from Min Fang, Institute of Microbiology, CAS; or Western Reserve-Vvt7 strain from Meilin Jin, Huazhong Agricultural University), herpes simplex virus 1(HSV-1, wild-type F strain from Hongbing Shu, Wuhan University; F strain from Bernard Roizman, The University of Chicago) and vesicular stomatitis virus (VSV, Ind strain) were given by The same assignee as above. Viral titers were determined by plaque assay using BHK 21.

Virus resistance assay: cells were treated with MnCl2 or primed with supernatant and infected with HSV-1-GFP, VSV-GFP and NDV-GFP at an MOI of 0.5. After 24 hours, flow cytometry was performed by FACSCalibur instrument (BD Biosciences) to determine virus-GFP of different virus infected cells. FACS data was analyzed using FlowJo software.

Cell stimulation: cells were infected with SeV (MOI of 0.1), VACV (Western Reserve-Vvt7 strain, MOI of 0.1), HSV-1(F strain, MOI of 0.1) and NDV-GFP (MOI of 1) for 1 hour, rinsed and cultured in fresh medium.

Mouse survival experiments: with HSV-1 (wild type F strain, 1.4X 10)⁷pfu/mouse) and VSV (8X 10)⁸pfu/mouse) intravenous infection of 8-12 week mice, or with VACV (Western Reserve strain, 1X 10)⁷pfu/mouse) infected mice 8-12 weeks intranasally.

And (3) plaque determination: BHK21 cells were incubated with homogenates (serial dilutions in serum-free DMEM) from infected mouse organs for 2 hours. The medium was then replaced with 0.5% methylcellulose in serum-free DMEM. After 60 hours, fixation was performed with 0.5% (vol/vol) glutaraldehyde and staining was performed with 1% (wt/vol) crystal violet (dissolved in 70% ethanol). Plaques were counted to calculate viral titers in plaque forming units.

Protein expression and purification

Full-length (1-522) and truncated (161-522) human cGAS (hcGAS) were subcloned into the pET-21b vector. His6-hcGAS was expressed in E.coli BL21(DE3) and induced overnight at 18 ℃ using 0.3mM isopropyl beta-D-1-thiogalactoside (IPTG). Cells were lysed in lysis buffer (25mM Tris-HCl, 500mM NaCl, pH 7.5) and cell debris was removed by centrifugation (20,000rpm, 1 h). The supernatant was filtered through a 0.2 μm low protein binding membrane (Pall Corporation). The clarified supernatant was applied to a HisTrap HP column (GE Healthcare) and eluted with a gradient of 0 to 300mM imidazole in lysis buffer.

Proteins were treated overnight with 1U/. mu.l Benzonase (Sigma) to remove DNA and RNA. 2mM EDTA was added to remove divalent cations. Thereafter, the proteins were gel filtered using a Superdex 200 column (GE Healthcare) equilibrated with lysis buffer. Finally, the purified protein was concentrated to 10mg/ml using an Amicon Ultra-410K centrifugal filter (Merck Milipore).

cGAS Activity assay

The purified recombinant full-length, truncated or mutant hcGAS was incubated with 40 μ l of a reaction solution containing the following components at 37 ℃ for 90 min: 1 μ M 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 MgCl ₂. The reaction solution was heated at 99 ℃ to denature the protein, and centrifuged at 16,000g for 10 minutes. The supernatant was mixed with 10. mu.g/ml digitonin 6 × 10⁵THP1 cells were incubated at 37 degrees celsius for 30 minutes, then the cells were lysed and Western blotted to determine cGAMP-induced phosphorylation of IRF 3. In addition, the supernatant was analyzed using Mono Q ion exchange column (GE Healthcare) equilibrated with working buffer (50mM Tris-HCl, pH 8.5), eluting with a gradient of NaCl from 0 to 500 mM.

Isothermal Titration Calorimetry (ITC)

Dissociation components were determined by ITC using iTC200 equipment (GE Healthcare) at 25 degrees celsius. Purification of proteins and ISD by gel filtration chromatography with or without 1mM MnCl₂Or MgCl₂Was eluted in the lysis buffer (25mM Tris-HCl, 500mM NaCl, pH 7.5). ISD (50 μ M in syringe, 18 × 2 μ l injection) and hcGAS (5 μ M in reaction well) were run using the following parameters: reference offset (reference offset)10 μ cal/s, injector speed 500rpm, pre-injection delay 180s, and 5s recording interval. Data were analyzed using Origin 7 (Microcal).

RT-PCR and real-time PCR analysis

Total RNA was isolated using TRIzol reagent (Invitrogen) according to the instructions. 1 microgram of total RNA was converted to cDNA using random primers and Superscript III reverse transcriptase (Invitrogen). PCR was performed using gene-specific primers. The RT-PCR products were gel electrophoresed on a 1.5% agarose gel and visualized by EB staining.

Quantitative real-time PCR was performed in the LightCycler 96 System (Roche) using Sybr green, and data are expressed as mRNA accumulation index (2)^ΔΔCt)。

Apoptosis assay

Apoptosis analysis was performed by annexin V-Fluorescein Isothiocyanate (FITC) and Propidium Iodide (PI) apoptosis assay kit (Solarbio, CA1020) as indicated. Briefly, cells were resuspended in 500. mu.l binding buffer (which contained an additional 5. mu.l Annexin V-FITC and 5. mu.L propidium iodide) and incubated for 15 minutes at room temperature without direct light. The supernatant was discarded and the cells were washed with PBS. Then, the cells were resuspended in 400. mu.l PBS. Samples were analyzed by flow cytometry and data was analyzed using FlowJo. Annexin V positive and PI negative populations indicate early apoptotic cells, while annexin V and PI positive populations indicate apoptotic cells.

DNA fragmentation assay: will 10⁵THP1 cells were resuspended in 500. mu.l buffer D (100mM Tris-HCl pH 8.0, 5mM EDTA, 0.2M NaCl, 0.4% SDS, 0.2mg/ml proteinase K) and incubated overnight at 37 ℃. The protein in the DNA was removed using phenol and phenol chloroform (1: 1) and precipitated with 2 volumes of ethanol. The DNA pellet was resuspended in 15. mu.l TE (pH 8.0) containing 1. mu.g/. mu.l RNase. After incubation for 2 hours at 37 ℃, the DNA was loaded on a 1% agarose gel, electrophoresed, visualized by EB staining and short wave uv irradiation.

Immunization with ovalbumin

Mice were immunized as described previously and the assay was performed (3, 37, 38). Briefly, 10 μ g OVA alone or in combination with 10 μ g MnCl in PBS was used on day 0 and day 10₂Or 30 μ g of DMXAA together intramuscularly in mice. Serum and splenocytes were taken on day 17 for determination of OVA-specific IgG1 and T, respectivelyA cellular response.

OVA-specific IgG1 assay: diluted sera from immunized mice were incubated on ELISA plates coated with 100. mu.g/ml OVA. After washing, bound IgG1 was detected using HRP-conjugated antibody mouse IgG1(eBioscience, # 18-4015-82). The plate was then incubated with the substrate TMB (eBioscience) with 1M H₃PO₄The reaction was stopped and then the absorbance was measured. OVA-specific IgG1(abcam, ab17293) was used as a standard.

OVA-specific T cell responses: splenocytes were seeded in 24-well plates, 1 × 10⁶Wells were stimulated with 10. mu.g/ml of synthetic OVA peptide, I-Ab (ISQAHAAHAEINEAGR), H-2Kb (SIINFEKL), or control peptide (FAPGNYPAL). After 24 hours, cell culture supernatants were harvested and IFN-. gamma. (Liankebio, EK2801) or IL-2(eBioscience, #88-7024-22) concentrations were determined by ELISA.

Pseudotype HIV-1(HIV-Luc) production, infection and detection

Experiments (33, 39-41) were performed as described previously. Briefly, HEK293T cells in a 10cm dish with confluency of-70% were transfected with Lipofectamine 2000(Invitrogen) using 10. mu.g of the delivery vector (pLVX si3LTR Luc), 7.5. mu.g of the packaging vector (psPAX2) and 5. mu.g of the envelope vector (VSV-G-pseudotype HIV pMD2.G, CCR 5-tropic HIV pHIV JRFL env, CXCR 4-tropic HIV pRE11NL43 env). After 6 hours, the medium was changed to 10mL of DMEM containing 20% FBS. After 48-72 hours, the transfected cell supernatant containing pseudotyped virus was harvested and filtered using a 0.45 syringe filter. The pseudotyped virus was used immediately to infect MAGIC5 cells, or frozen at-80 ℃. Viral titers were measured by luciferase assay infected with MAGIC5 cells. To infect MAGIC5, cells were seeded at-30% confluence in 12-well plates the day before infection. Cells were primed with supernatant from THP1 or PBMC and then incubated with CCR 5-tropic, CXCR 4-tropic, or VSV-G-pseudotyped HIV for 6 h. Then, the medium was changed to DMEM containing 10% FBS and cultured for 3 days. The medium was removed and the cells were lysed using 50. mu.l of 1 × Passive Lysis Buffer (Promega)/well for 30 minutes and measured using the Firefly Luciferase Assay System (Promega).

Transmission Electron Microscope (TEM)

The cells were washed twice with 0.1M phosphate buffer (pH7.4) and then fixed with 2% paraformaldehyde/2.5% glutaraldehyde in the same buffer for 2 hours and postfixed with 1% OsO4 at room temperature (postfix) for 1 hour. After rinsing several times with phosphate buffer and distilled water, the cells were incubated in 0.1% tannic acid (in phosphate buffer) for 30 minutes. Cells were rinsed with distilled water and stained in 2% uranyl acetate for 1 hour. Washed again in distilled water, dehydrated in ethanol and embedded in SPI-Pon 812 resin (SPI Supplies, PA, USA). Ultrathin (75nm thick) sections were stained with uranyl acetate and lead citrate and observed under a Tecnai G220 TWIN transmission electron microscope at 120kV acceleration voltage. The images were processed with an Eagle (4k x 4k) digital camera (FEI, Oregon, USA).

Statistical analysis

Data were analyzed using the t-test. Survival curves were compared using the Mantel-Cox test.

Examples

Example 1 Mn²⁺The treatment confers to the host cell resistance to viral infection

Experiment (a)

With 100. mu.M MnCl₂THP1 cells cultured in RPMI-1640 medium (Gibco) supplemented with 10% FBS were pretreated for 12 hours, and then infected with HSV-1-GFP (obtained from the institute of medical sciences, China), VSV-GFP (obtained from Rongbin zhou, China university of science) or NDV-GFP (obtained from Chen Wang, institute of biochemistry and cell biology, China) having an MOI of 0.5. After 24 hours, cells were photographed and measured using flow cytometry (FACSCalibur) and analyzed using FlowJo (as shown in figure 1). The use of Mn is clearly shown in FIG. 1 ²⁺The pretreatment confers resistance to viral infection to the host cell.

As described above, MnCl is used in a concentration shown in FIG. 2 or FIG. 3₂Or dmxaa (dmx) for 24 hours in THP1 cells, followed by infection of the cells for 24 hours with the indicated GFP-viruses, followed by measurement of GFP expression by Western blot and measurement of Median Fluorescence intensity of virus-GFP by flow cytometry (Median Fluorescence IntIntensity, MFI) distribution, in which MnCl will not be used₂MFI normalization of treated THP1 cells to 1, showing Mn²⁺Dose dependence of antiviral activity of the pretreatment.

Experiment (b)

Collecting human cervical cancer cell line HeLa cultured in DMEM (Gibco) medium supplemented with 10% FBS in logarithmic growth phase, and mixing solution A and solution B (solution A component: PBS buffer solution (NaCl137mmol/L, KCl2.7mmol/L, Na)₂HPO₄10mmol/L，KH₂PO₄2mmol/L) or normal saline, pH 7.4. And B, liquid component: MnCl₂100mmol/L) were mixed in the proportions shown in the table and added to the culture system. After 24 hours, a green fluorescent protein-tagged (GFP) virus was added for infection, respectively: herpes simplex virus type I (HSV-1), vesicular stomatitis virus type New Jersey (VSV) and Newcastle Disease Virus (NDV). After 24 hours of virus infection, the virus infection rate (GFP positive rate) was analyzed by flow cytometry, as shown in table 1 below.

TABLE 1 Mn²⁺Treatment significantly reduced viral infection rate

Solution A	Liquid B	Culture medium	Infection rate of HSV-1	VSV infection Rate	Infection rate of NDV
						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％

Experiment (c)

Using Mn in the amount shown in FIG. 11²⁺And DMXAA is used for treating mouse peritoneal macrophages for 36 and 48 hours, and then viper toxin is determined, and the result shows that Mn is displayed²⁺And DMXAA activated mouse peritoneal macrophages (as shown in figure 11A).

Example 2 intravenous injection of Mn²⁺Conferring virus resistance in mice

Experiment (a)

Eight weeks old SPF grade test C57BL/6 (purchased from Wintolite, Beijing) was taken, and solution A and solution B (as described above) were mixed in the proportions shown in the table and injected via the tail vein. After 16-24 hours, herpes simplex virus type I (HSV-1), vesicular stomatitis virus type New Jersey (VSV) and vaccinia virus (VacV) were injected, respectively. After 48 hours, virus titers were measured in mice using the plaque method and virus lethality was monitored.

TABLE 2 intravenous Mn²⁺Viral titer in post-mouse

Solution A	Liquid B	Mncl2Amount of the composition used	HSV-1 titre	VSV titer	VacV titre
						200μL	0μL	0mg/kg
198μL	2μL	1mg/kg
						196μL	4μL	2mg/kg
190μL	10μL	5mg/kg			Decrease by hundreds of times
						180μL	20μL	10mg/kg			Decrease by hundreds of times

TABLE 3 intravenous Mn²⁺Post-mouse lethality

Solution A	B liquid	Mncl2Amount of the composition used	Mortality of HSV-1	Death Rate of VSV	Mortality rate of VacV
						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％

Experiment (b)

Eight weeks old of SPF grade test C57BL/6 (purchased from Wintolite, Beijing) was mixed with 2mg/kg of MnCl ₂Mice were injected intravenously 24 hours later with VSV (8X 10)⁸pfu/mouse), VACV (Western Reserve strain, 1x 10)⁷pfu/mouse) or HSV-1 (wild type F strain, 1.4X10⁷pfu/mouse) mice were challenged and survival of the mice was monitored (as shown in figure 4).

Experiment (c)

Using 2mg/kg of MnCl as described above₂Mice were injected intravenously, and 24 hours later, the mice were challenged with VSV, VACV or HSV-1, and four days after infection, lungs and spleen were taken, homogenized, and then the virus titer was measured by plaque assay (plaque assay), and the results are shown in FIG. 5.

Example 3 Mn²⁺Effect of treatment on downstream Gene expression

Experiment (a)

By 100Mu M MnCl₂THP1 cells were treated for 12 hours, with VACV, IFN β or media (control), RNA extracted and sequenced. By calculating log₂((treated RPKM)/(control RPKM)) gave a heatmap of gene expression (as shown in fig. 6A).

Experiment (b)

With 200. mu.M MnCl₂THP1 cells were treated and 2, 12, 24 hours later, the cells containing MnCl were discarded₂The medium was replaced with fresh medium, NW indicated that MnCl was not discarded₂Media, FIG. 6B shows production of I-IFN.

Experiment (c)

With MnCl at the indicated concentration₂，Mn(OAc)₂Or Mn (OAc)₃THP1 cells were treated for 24 hours and then supernatants were analyzed using an I-IFN bioassay, and FIG. 6C shows that treatment with different manganese salts induced I-IFN production, indicating that both manganese salts were able to induce I-IFN production, while the manganese salt was much less effective.

Experiment (d)

Using 0, 2 or 5mg/kg MnCl in PBS₂The solution was injected intravenously into mice, sera were isolated and analyzed using I-IFN bioassay, the same experiment was performed otherwise, but mice were sacrificed, organs were taken and ISG was determined by Western blot. FIG. 6D shows intravenous injection of MnCl in mice₂Induction of I-IFN production, FIG. 6E shows intravenous injection of MnCl in mice₂Organ distribution of post-ISG, where viper toxin and ISG54 are induced in multiple organs.

Experiment (e)

FIG. 6F shows the use of different concentrations of MnCl₂I-IFN production and ISG expression after 36 h treatment of PBMCs from healthy adult donors (obtained by continuous centrifugation using Histopaque-1077 from Sigma cultured in RPMI-1640 medium containing 5% FBS), I-IFN production was analyzed using I-IFN bioassay and ISG expression was determined by Western blotting. FIG. 6G shows the use of 200. mu.M MnCl₂I-IFN production and ISG expression after different time periods were treated on PBMCs from healthy adult donors. These data show Mn²⁺Treatment of the resulting dose-and time-dependence of I-IFN production and ISG expression, demonstrating Mn²⁺Treatment elicited downstream gene expression in either vitro or in vivo.

Experiment (f)

Logarithmic growth phase of THP1 cells or human primary peripheral blood mononuclear cells (from healthy adult donors) were taken. Solution A and solution B (as described above) were mixed in the proportions shown in the table and added to the culture system. After 16-24 hours, cell culture supernatants were removed and assayed for type I interferon content using ELISA, the results of which are shown in Table 4 below.

TABLE 4 Mn²⁺In vitro treatment induced interferon production

Experiment (g)

Eight weeks old SPF grade test C57BL/6 (purchased from Wintolite, Beijing) was taken, and solution A and solution B (as described above) were mixed in the proportions shown in the table and injected via the tail vein. After 16-24 hours, the peripheral blood serum of the mouse is taken, and the I-type interferon content is detected by using an enzyme-linked immunosorbent assay.

TABLE 5 Mn²⁺Injection induced interferon production in vivo

Solution A	Liquid B	MnCl2Amount of the composition used	Peripheral blood serum type I interferon content (U/ML)
				200μL	0μL	0mg/kg	Not detected out
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

Example 4 Mn²⁺Directly inhibiting Human Immunodeficiency Virus (HIV) infection of human cells

For the preparation of HIV pseudoviruses, HEK293T cells were cultured in 10cm dishes to 70% confluency and then transfected with 10. mu.g of a delivery vector (pLVX si3LTR Luc, obtained from the institute of microbiology, China), 7.5. mu.g of a packaging vector (psPAX2, obtained from Addgene) and 5. mu.g of an envelope vector (VSV-G-pseudotyped HIV: pMD2.G, obtained from Addgene; CCR 5-tropic HIV: pHIV JRFL env, obtained from the institute of microbiology, China, Takaomi Ishida; and CXCR 4-tropic HIV: pRE11 NL43 env, obtained from the institute of microbiology, China), using Lipofectamine 2000(Invitrogen) to obtain HEK293T cells. After 6 hours, the medium was changed to 10mL DMEM (containing 20% FBS). After 48-72h, the supernatant of transfected cells containing pseudovirus was harvested and filtered through a 0.45 μm filter.

The pseudoviruses prepared were used to infect MAGIC5 cells immediately or stored at-80 ℃. Viral titers were determined by luciferase assay.

To infect MAGIC5 cells, cells were seeded at around 30% confluence in 12-well plates the day before infection. Cells were primed (prime) with supernatant from THP1 or PBMC for 4h and then incubated with CCR 5-tropic, CXCR 4-tropic or VSV-G-pseudotyped HIV for 6 h. Then, the medium was changed to DMEM (containing 10% FBS), and cultured for 3 days. The medium was removed and the cells were lysed with 50. mu.l of 1 XPassive Lysis Buffer (Promega)/well for 30 minutes, as determined using the Firefly Luciferase Assay System (Promega).

The human cervical cancer cell line Magic5 cultured in DMEM medium supplemented with 10% FBS in the logarithmic growth phase was taken, and solution a and solution B (as described above) were mixed in the proportions shown in the table and added to the culture system. After 24 hours, the MAGIC5 cells were infected with the addition of a firefly luciferase (firefly luciferase) expressing HIV pseudovirus: HIV of JRFL (CCR5) type and HIV of NL43(CXCR4) type. After 24 hours, luciferase activity in the infected cells was monitored to determine the rate of inhibition of virus infection (as shown in Table 6 below).

TABLE 6 Mn²⁺Inhibition of HIV infection after treatment

Solution A	Liquid B	Culture medium	JRFL-HIV inhibition rate	NL43-HIV inhibition Rate
					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％±

Example 5 Mn²⁺Can be used for treating tumor

An SPF grade test C57BL/6 (purchased from Weitonghua, Beijing)Company), costal injection 5x10⁶Mouse lymphoma cell EL4(dr. minghui Zhang, university of qinghua) of one mouse. 2, 4, 6 days after tumor cell inoculation, respectively. Solution A and solution B (as described above) were mixed in the proportions shown in the table and intratumoral injection was performed. And the size of the tumor was measured 10, 15, and 20 days after the formation of the tumor, and the results are shown in table 7 below.

TABLE 7 Mn²⁺Titer of HBV after treatment

Example 6 Mn²⁺Treatment-induced cytokine production depends on the cGAS-STING pathway

First, MnCl was added at different concentrations as shown in FIG. 7A₂After 24 hours of solution treatment of HeLa cells and THP1 cells, IRF3 phosphorylation and viper toxin (Viperin) production were determined. Specifically, cells were lysed and IRF3 phosphorylation and viper toxin production were determined by Western blot.

To study Mn²⁺Treatment resulting cytokine production by which pathway acted, the inventors obtained a series of gene-knocked-out HeLa cells using the CRISPR/Cas9 system, followed by 500. mu.M MnCl₂The solution treatment of the gene-knocked-out HeLa cells, FIG. 7B shows IRF3 phosphorylation and viper toxin production after 24 hours, and the results show MnCl ₂Induced phosphorylation of IRF3 and production of viper toxin is dependent on cGAS, STING, TBK1 and IRF3, and not on RIG-I or MAVS. FIG. 7C shows the use of 500. mu.M MnCl₂IRF3 phosphorylation (by Western blot) 24 hours after treatment of the indicated gene-deficient and rescued HeLa cells with either solution, VACV or SeV, where the rescued HeLa cells were HeLa cells transfected with pcDNA3.1(Invitrogen) vectors containing cDNA sequences expressing the N-terminus of human IRF3, STING, TBK1 and RIG-I (amplified from THP1 cells), respectively, showed that the reconstitution of cGAS, STING and IRF3 in knock-out cells was able to rescue MnCl₂Induced phosphorylation of IRF3 and production of viper toxin.

To further study Mn²⁺In vivo Effect of treatment, the inventors madeA series of knockout mice (based on C57BL/6J mice) were obtained using the CRISPR/Cas9 system. FIG. 7D shows the use of 500. mu.M MnCl₂After WT or the indicated knockout mice are treated with the solution, VACV or SeV for 24 hours, viper toxin and ISG are produced in peritoneal macrophages (determined by Western blotting) obtained from the WT or the indicated knockout mice, and the result also shows that MnCl₂Induced phosphorylation of IRF3 and production of viper toxin is dependent on cGAS, STING, TBK1 and IRF3, and not on RIG-I or MAVS. FIG. 7E shows the use of 500. mu.M MnCl ₂IFN production (using I-IFN bioassay) 24 hours after treatment of WT or the indicated knockout mice with solution, VACV or SeV was similar to the above.

The above results fully demonstrate Mn²⁺Function through the cGAS-STING pathway.

Example 7 Mn²⁺Treatment of direct activation of cGAS

To further study Mn²⁺The mechanism of action of (1-522) purified full-length human cGAS and MnCl₂Nucleic acids shown in the figure were incubated together. The reaction product was mixed with THP1 permeabilized with digitonin (Sigma, D141) for 30 min, then the cells were lysed and the lysate was used to determine cGAMP-induced phosphorylation of IRF3, which showed Mn in the presence of dsDNA only²⁺cGAS was activated (as shown in fig. 8A). FIG. 8B is a graph showing full-length or mutant human cGAS and MnCl₂/MgCl₂Multiple different amounts of dsDNA incubated together, the results showed Mg²⁺In contrast, Mn²⁺Increased sensitivity of cGAS to dsDNA in the presence of at least 10⁴Multiple (10)^-2Compared with 10^-6) Wherein mut is human cGAS which catalyzes the mutation of a cation-binding residue: E225A/D227A. FIG. 8C shows human cGAS protein with 0.5mM MnCl₂Or 5mM MgCl₂And 10-2mg/ml dsDNA, using Mono Q ion exchange column for cGAMP production, using cGAMP, ATP and GTP as controls. These data indicate that Mn is present ²⁺Sensitivity of cGAS to DNA is significantly improved and thus enables the enzymatic activity of cGAS to be triggered at very low cytoplasmic DNA concentrations.

On the other hand, the inventors compared Mn²⁺And Mg²⁺Effect in stimulating I-IFN productionFIG. 12A shows Mn²⁺Stimulation of I-IFN production, but Mg²⁺There is little effect.

The inventors have studied Mn²⁺At the site of action on cGAS, FIG. 12B shows Coomassie brilliant blue stained purified full-length (1-522), mutant (E225A/D227A) and truncation mutant (161-522) human cGAS, and FIG. 12C shows that similar results were obtained for human cGAS using truncation (161-522) and full-length cGAS.

FIG. 12D shows MnCl at 1mM₂Or 1mM MgCl₂Binding of full-length human cGAS to dsDNA in the presence, as determined by Isothermal Titration Calorimetry (ITC).

Example 8 Mn²⁺Show strong adjuvant activity

Experiment (a)

The results of ovalbumin antigen (OVA) obtained from the eight-week-old SPF-grade test C57BL/6 (purchased from Wintolite, Beijing) were shown to be Mn²⁺The antigen-specific immune activation of specific CD4+ cells and CD8+ cells is generated on OVA as an immune adjuvant, and a large amount of gamma interferon and interleukin 2 are generated.

Specifically, OVA (10. mu.g), OVA + 10. mu.g MnCl were used on days 0 and 10₂Alternatively OVA + 30. mu.g DMXAA (Selleck, S1537) was immunized intramuscularly in mice, sera and splenocytes taken on day 17 were assayed for production of OVA-specific IgG1 (by ELISA) using sera, the results are shown in FIG. 9A. The data show Mn ²⁺The application of the polypeptide as an immunological adjuvant greatly improves the production of antibodies and is stronger than the known STING activator DMXAA.

The spleen cells were washed at 1 × 10⁶Each well was inoculated in a 24-well plate, spleen cells were stimulated with 10. mu.g/ml of the synthesized OVA peptide H-2Kb (SIINFEKL) or I-Ab (ISQAHAAHAEINEAGR) or control peptide (FAPGNYPAL), and 24 hours later supernatants were assayed for secretion of IFN γ (Liankebio, EK2801) and IL-2(eBioscience, #88-7024-22) by ELISA, as shown in FIGS. 9B and 9C. The data show Mn²⁺As an immune adjuvant, the antigen-specific immune activation of specific CD4+ cells and CD8+ cells was greatly improved.

Experiment (b)

An eight-week-old SPF-grade test C57BL/6 (purchased from Wintolite, Beijing) was prepared by mixing 10 μ g of ovalbumin antigen (OVA) with solution A and solution B (as described above) in the proportions shown in the table and intramuscular injection; 7-14 days after the first injection, a second injection was made, with the same method and dosage as the first injection. 28 days after the first injection, the mice were sacrificed, peripheral blood was taken and plasma was separated, and the content of anti-OVA antibody in the plasma of the mice was measured using an enzyme-linked immunosorbent assay, and the results are shown in Table 8 below.

TABLE 8 Mn²⁺Treatment enhances antibody production

Solution A	Liquid B	MnCl2 usage amount	anti-OVA antibody production 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

Example 9 Mn²⁺Inhibition of HIV infection by CCL3 production

Using Mn²⁺THP1 cells were treated (as shown in figure 10), and figures 10A and B show I-IFN (panel a) and CCL3 (panel B) secretion. The results show, surprisingly, that Mn is present in the same amount that stimulates I-IFN production compared to other stimuli²⁺Treatment resulted in more secretion of CCL3 (approximately 2-8 fold higher). This result suggests Mn²⁺Treatment may inhibit HIV infection by CCL3 stimulation.

Using Mn from the warp²⁺Supernatants of treated THP1 cells primed MAGIC5 cells for 4 hours, and then infected cells with CCR 5-tropism (CCR5-tropic), CXCR 4-tropism, or VSV-G-pseudotyped (pseudovirus preparation as described in example 4) HIV-Luc virus. After 3 days, luciferase was assayed, showing viral infection (as shown in fig. 10C). The results show that Mn²⁺Treatment did significantly inhibit infection by the CCR5-tropic (CCR5-tropic) virus, whereas the extent of inhibition was much less for the CXCR 4-tropic or VSV-G-pseudotyped (pseudotyped) HIV-Luc virus.

FIGS. 10D and E show the use of Mn²⁺I-IFN (panel D) and CCL3 (panel E) secretion following PBMC cell treatment, experiments were as described above in experiments with THP1 cells (FIGS. 10A and B), except that PBMC from healthy adults were used, with Mn ²⁺PBMC cells were treated for 18 or 36 hours and then assayed for I-IFN and CCL 3. FIG. 10F shows the use of Mn from warp ²⁺4 hours after treatment of supernatant from PBMC cells to prime MAGIC5 cells (the remainder were as described in FIG. 10C), viral infectivity by HIV pseudovirus was measuredAnd (6) dyeing. FIG. 10G shows the use of Mn²⁺Peritoneal macrophages from WT or knockout mice were treated and CCL3 secretion was determined by ELISA.

The above data indicate that Mn is present in human primary PBMC cells²⁺Treatment significantly inhibited infection by the CCR5-tropic (CCR5-tropic) virus, whereas CXCR 4-tropic or VSV-G-pseudotyped (pseudotyped) HIV-Luc virus inhibited to a lesser extent.

Example 10 Mn²⁺The treatment does not cause mitochondrial damage

FIG. 13 shows Mn²⁺Induced cGAS activation does not involve mitochondrial damage. As shown in FIG. 13A, with 20. mu.M ABT-263, 20. mu.M ABT-737 or MnCl₂THP1 cells were treated (50, 100, 200. mu.M) for 24 hours and apoptosis was determined by Annexin V-FITC/PI double staining of FCM, DNA gel and Western blot, showing Mn at a concentration that stimulates phosphorylation of IRF3²⁺No apoptosis was induced in THP1 cells, in contrast, the Bcl-2 inhibitor, ABT-737/263, significantly induced apoptosis. As shown in FIGS. 13B and C, with Mn²⁺Wild Type (WT) and Bax-/Bak-/-MEF (obtained from Kevin M Ryan, Beatson Institute for Cancer Research, UK) were treated and cleavage by Casp3 and induction by viper toxin were then determined, and these results showed Mn ²⁺The effect produced by the treatment was independent of Bak/Bax and independent of SOD 2. BMDM from C57BL/6J mice was treated with 500 μ M MnCl2 for 24 hours, mitochondria were visualized by TEM, and supernatant was used to determine I-IFN production, TEM images as shown in fig. 13D, showing no significant elongation or interconnection of mitochondria, as is common in TFAM-deficient cells. Peritoneal macrophages from WT or the indicated knock-out mice were primed with 1. mu.g/ml LPS for 5 hours, then treated as shown in FIGS. 13E and F for 6 hours, and inflammasome (inflamseveral) activation was measured using supernatant (Sup) and Whole Cell Lysate (WCL), showing Mn²⁺Induction elicited strong inflammatory corpuscle activation in cells from wild type mice, but not in cells from Asc-/-mice. Further, FIGS. 13F and G show that Mn²⁺Induced inflammasome activation was independent of AIM2, SOD1 or NLRP 3. The above results show that Mn²⁺The treatment does not cause mitochondrial damage, and Mn²⁺Induction ofNor is IFN production due to mitochondrial damage.

The foregoing description is of the preferred embodiments only, which are by way of example only and do not limit the combination of features necessary to practice the invention. The headings provided are not meant to limit the various embodiments of the invention.

All publications and patents mentioned in this application are herein incorporated by reference. Various modifications and variations of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

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Claims (7)

1. A method for activating cGAS-STING pathway in a cell in vitro comprising administering divalent manganese to the cell.

2. The method according to claim 1, wherein said administering enhances perception of cytoplasmic DNA in the cell.

3. The method according to claim 1, wherein said administering increases cGAS sensitivity to DNA in the cell.

4. The method according to claim 1, wherein said administering induces the production of the chemokine CCL3 in the cell.

5. A method according to any one of claims 1 to 4 wherein the divalent manganese is in the form of a divalent manganese salt.

6. The method according to claim 5, wherein the divalent manganese salt is selected from the group consisting of: manganese chloride, manganese bromide, manganese iodide, manganese sulfate, manganese nitrate, manganese perchlorate, manganese acetate, manganese carbonate, manganese borate, manganese phosphate, manganese hydrobromide, manganese tartrate, manganese fumarate, manganese maleate, manganese lactate, manganese benzenesulfonate, manganese pantothenate, manganese ascorbate, and any combination thereof.

7. A method according to any one of claims 1 to 4 wherein the divalent manganese is in the form of free divalent manganese ions.

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