Interferon-inducing oligonucleotide double helix and method of use

Abstract

This invention provides compositions and methods for novel broad-spectrum therapies that inhibit infections caused by many different types of viruses and pathogens. [Solution] A composition and method for inducing type I interferon production are provided. The provided composition comprises an immunostimulatory oligonucleotide bihedra containing a 5'-terminal monophosphate-CUGA-3' sequence. The composition comprising the provided immunostimulatory oligonucleotide bihedra can be used to treat interferon-responsive diseases or disorders.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits under Section 119(e) of the United States Patent Act as granted by U.S. Provisional Application No. 63 / 029,199, filed on 22 May 2020, and U.S. Provisional Application No. 63 / 082,742, filed on 24 September 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Government support This invention was made with government support under HL141797 issued by the National Institutes of Health and W911NF-12-2-0036 and W911NF-16-C-0050 issued by the United States Army. The United States Government has certain rights to this invention.

[0003] Technical field The techniques described herein relate to compositions and methods for immunostimulation. [Background technology]

[0004] background Pathogenic infections trigger complex regulatory systems of innate and adaptive immune responses designed to defend against pathogens in the host organism. One of many responses to pathogen invasion, such as viral, bacterial, fungal, or parasitic infections, is the induction of interferon (IFN) production, a pleomorphic group of cytokines that play a crucial role in the human immune response by "interfering" pathogen activity, such as viral replication. The increasing emergence of pandemic viruses such as influenza, MERS, SARS, and now SARS-CoV-2 necessitates the development of new broad-spectrum therapies that inhibit infections by many different types of viruses and pathogens. [Overview of the project]

[0005] overview The compositions and methods described herein relate, in part, to the discovery of oligonucleotide bihedra that induce interferon production.

[0006] In one aspect, this specification describes immunostimulatory oligonucleotide double helixes containing SEQ ID NO:1 at the 5' end.

[0007] In this aspect or any other aspect, the oligonucleotide double helix is ​​RNA.

[0008] In this aspect or any other aspect, in another aspect, the oligonucleotide double chain contains a 5'-monophosphate group.

[0009] In this aspect or any other aspect, there is no modification to the 5' terminal sequence (SEQ ID NO:1).

[0010] In this aspect or any other aspect, in another aspect, the oligonucleotide double helix is ​​at least 20 nucleic acid bases long.

[0011] In this aspect, or in another aspect of any other aspect, the oligonucleotide double helix is ​​a double-stranded RNA.

[0012] In this aspect or in another aspect of any other aspect, the oligonucleotide duplex is sufficient to induce interferon (IFN) production in cells that come into contact with the duplex.

[0013] In this aspect, or in another aspect of any other aspect, the oligonucleotide bihedra activate the RIG-I-IRF3 pathway.

[0014] In this aspect or any other aspect, the oligonucleotide duplex reduces the viral titer in cells or cell populations that come into contact with the duplex.

[0015] In another aspect of this or any other aspect, the oligonucleotide duplex increases STAT1 and STAT2 in cells contacted by the duplex.

[0016] The immunostimulatory oligonucleotide duplexes described herein can be used to treat or assist in the treatment of any disease or disorder in which induction of an interferon response can be beneficial. Such diseases or disorders include viral infections, as well as infections by bacterial, fungal or parasitic pathogens, and cancers and autoimmune diseases in which interferon induction is beneficial. Accordingly, disclosed herein are methods of treating viral, bacterial, fungal or parasitic infections, comprising the step of administering to a subject in need thereof an immunostimulatory oligonucleotide duplex described herein. Similarly, disclosed herein are methods of treating cancer or autoimmune diseases, comprising the step of administering to a subject in need thereof an immunostimulatory oligonucleotide duplex described herein.

[0017] In another aspect, described herein is a method of inducing an antiviral response in a subject, comprising the step of administering to a subject in need thereof an immunostimulatory oligonucleotide duplex described herein.

[0018] In one aspect of this or any other aspect, the subject in need thereof has a viral infection or is at risk of having a viral infection.

[0019] In another aspect of this or any other aspect, the method further comprises the step of diagnosing the subject as having a viral infection or being at risk of having a viral infection, prior to the step of administering.

[0020] In another aspect of this or any other aspect, the method further comprises the step of receiving the results of an assay diagnosing the subject as having a viral infection or being at risk of having a viral infection, prior to the step of administering.

[0021] In another aspect of this or any other aspect, the viral infection is caused by a virus selected from the group consisting of John Cunningham virus, measles virus, lymphocytic choriomeningitis virus, alphavirus, rabies virus, rhinovirus, parainfluenza virus, respiratory syncytial virus, herpes simplex virus, herpes simplex type 1, herpes simplex type 2, human herpesvirus 6, adenovirus, cytomegalovirus, Epstein - Barr virus, mumps virus, influenza A virus, influenza B virus, coronavirus, SARS coronavirus, SARS - CoV - 2 virus, Coxsackievirus A, Coxsackievirus B, poliovirus, HTLV - 1, hepatitis A, B, C, D, and E viruses, varicella - zoster virus, smallpox virus, molluscum contagiosum, human papillomavirus, parvovirus B19, rubella virus, human immunodeficiency virus, rotavirus, norovirus, astrovirus, Ebola virus, Marburg virus, dengue virus (DENV), and Zika virus.

[0022] In another aspect of this or any other aspect, the viral infection is an infection of a tissue selected from the group consisting of central nervous system tissue, eye tissue, upper respiratory system tissue, lower respiratory system tissue, lung tissue, kidney tissue, bladder tissue, spleen tissue, heart tissue, gastrointestinal tissue, epidermal tissue, reproductive tissue, nasal tissue, laryngeal tissue, tracheal tissue, bronchial tissue, oral tissue, blood tissue, and muscle tissue.

[0023] In another aspect of this or any other aspect, the administration is systemic.

[0024] In another aspect of this or any other aspect, the administration is local to the site of infection.

[0025] In another aspect of this or any other aspect, the method further comprises the step of administering at least one additional therapeutic agent.

[0026] In this aspect or any other aspect, in another aspect, the at least one additional therapeutic agent is an antiviral therapeutic agent.

[0027] In another aspect, this specification describes a method for treating an influenza infection in a subject, comprising the step of administering an immunostimulant oligonucleotide double chain described herein to a subject having an influenza infection.

[0028] In this aspect or in any other aspect, the influenza infection is either an influenza A infection or an influenza B infection.

[0029] In this aspect or any other aspect, the method further includes the step of administering at least one additional antiviral therapeutic agent.

[0030] In another aspect, this specification describes a method for treating coronavirus disease in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-strand described herein to a subject having coronavirus disease.

[0031] In this aspect or in any other aspect, coronavirus disease is COVID-19.

[0032] In this aspect or any other aspect, the method further includes the step of administering at least one additional antiviral therapeutic agent.

[0033] In this aspect or any other aspect, the method further includes the step of administering plasma obtained from a subject who has recovered from coronavirus disease.

[0034] In another aspect, this specification describes a method for increasing the potency of an antiviral therapeutic agent, comprising the step of administering an immunostimulant oligonucleotide double-strand described herein and at least one antiviral therapeutic agent.

[0035] In this aspect or in any other aspect, antiviral drugs include abacavir, acyclovir (Acyclovir, Aciclovir), adefovir, amantadine, ampligen, amprenavir (agenese), amodiaquin, apirimod, arbidol, atazanavir, atripra, atovaquone, paravir, baloxavir marboxil (Xofluza®), biktarvy, boceprevir (Victrelis®), cidofovir, clofazimine, clomiphene, clofazamine, cobicistat (Tybos t(registered trademark), Combivir (fixed-dose drug), Daclatasvir (Daklinza(registered trademark)), Darunavir, Delavirdine, Descovy, Didanosine, Docosanol, Dolutegravir, Doravirine (Pifeltro(registered trademark)), Ecoliebel, Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine (Intelence(registered trademark)), Famciclovir, Favipiravir, Fenofibrate, Homivirsen, Fosamprenavir, Foscarnet, Phosphonet, Fusion inhibitors Ganciclovir (Cytovene®), ivacitabine, ibalizumab (Trogarzo®), idoxuridine, imiquimod, immunovir, indinavir, inosine, integrase inhibitors, type I interferon, type II interferon, type III interferon, interferon, ivermectin, lamivudine, rasaloside, letermovir (Prevymis®), lopinavir, roviride, mannose-binding lectin, maraviroc, methisazone, moloxidine, nafamostat, nelfinavir, nevirapin Nexavir (registered trademark), nilotinib, nitazoxanide, Norvir, nucleoside analog, oseltamivir (Tamiflu (registered trademark)), pazopanib, pegylated interferon alfa-2a, pegylated interferon alfa-2b, penciclovir, peramivir (Rapivab (registered trademark)), preconalil, podophyllotoxin, protease inhibitors (pharmacology), pionarizine, pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant (registered trademark)), rimantadine, ritonavir,The following drugs are selected from the group consisting of saquinavir, simeprevir (Olysio®), sofosbuvir, stabuzine, synergistic enhancer (antiretroviral drug), tafenoquin, telaprevir, terbivudine (Tyzeka®), tenofovir alafenamide, tenofovir disoproxil, tenofovir, toremifene, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir (Valtrex), valganciclovir, bermurafenib, venetoclax, bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza®), and zidovudine.

[0036] In this aspect or any other aspect, the immunostimulatory oligonucleotide duplex and the at least one antiviral drug are administered substantially simultaneously.

[0037] In this aspect or any other aspect, the immunostimulatory oligonucleotide duplex and the at least one antiviral drug are administered at different time points.

[0038] In another aspect, this specification describes a pharmaceutical composition comprising an immunostimulant oligonucleotide double helix described herein and a pharmaceutically acceptable carrier.

[0039] In one aspect of this or any other aspect, the composition is formulated for airway administration. In another aspect of this or any other aspect, the composition is formulated for aerosol administration, nebulizer administration, or tracheal lavage administration.

[0040] In another aspect, this specification describes a pharmaceutical composition comprising an immunostimulatory oligonucleotide double-strand described herein and at least one antiviral therapeutic agent.

[0041] In this aspect or any other aspect, the composition is formulated for intravenous, intramuscular, intraperitoneal, subcutaneous, or intrathecal administration.

[0042] In another aspect, this specification describes a method for inducing interferon (IFN) production, comprising administering to a subject in need thereof an immunostimulatory oligonucleotide double-strand as described herein or a pharmaceutical composition comprising such a double-strand as described herein, thereby increasing IFN production after administration.

[0043] In this aspect or in any other aspect, IFN production is the production of type I IFN, type II IFN, or type III IFN.

[0044] In this aspect or in another aspect of any other aspect, IFN production is the production of type I IFNs, including one or more of IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω.

[0045] In this aspect, or in another aspect of any other aspect, type II IFN is IFN-γ.

[0046] In this aspect, or in another aspect of any other aspect, increased IFN production increases the cell's resistance to viral infection.

[0047] In this aspect or any other aspect, the subject requiring this is either having an IFN-related disease or at risk of having an IFN-related disease.

[0048] In this aspect or any other aspect, the method further includes a step of diagnosing a subject having or being at risk of having an IFN-related disease prior to the administration step.

[0049] In this aspect or any other aspect, the method further includes a step of receiving the results of an assay that diagnoses the subject having or being at risk of having an IFN-related disease, prior to the administration step.

[0050] In this aspect, or in another aspect of any other aspect, IFN-related disease is a disease characterized by reduced IFN levels compared to normal levels.

[0051] In this aspect, or in another aspect of any other aspect, IFN-related disorders are disorders characterized by reduced levels of type I IFN compared to normal levels.

[0052] In this aspect or another aspect of any other aspect, IFN-related diseases are selected from the group consisting of viral infectious diseases, bacterial infectious diseases, fungal infectious diseases, parasitic infectious diseases, cancer, and autoimmune diseases.

[0053] In this aspect or any other aspect, the method further includes the step of administering at least one additional therapeutic agent.

[0054] In this aspect or any other aspect, in another aspect, the at least one additional therapeutic agent is an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, an anticancer agent, or an anti-autoimmune agent.

[0055] In another aspect, this specification describes a composition comprising an immunostimulant oligonucleotide double-chain described herein and at least one antibacterial therapeutic agent. In one aspect of this aspect, the composition further comprises a pharmaceutically acceptable carrier.

[0056] In another aspect, this specification describes a composition comprising an immunostimulatory oligonucleotide double-strand described herein and at least one antifungal therapeutic agent. In one aspect of this specification, the composition further comprises a pharmaceutically acceptable carrier.

[0057] In another aspect, this specification describes a composition comprising an immunostimulant oligonucleotide double-strand described herein and at least one antiparasitic therapeutic agent. In one aspect of this specification, the composition further comprises a pharmaceutically acceptable carrier.

[0058] In another aspect, this specification describes a composition comprising an immunostimulant oligonucleotide double-strand described herein and at least one anticancer drug. In one aspect of this aspect, the composition further comprises a pharmaceutically acceptable carrier.

[0059] In another aspect, this specification describes a composition comprising an immunostimulatory oligonucleotide double-strand described herein and at least one therapeutic agent for the treatment of an autoimmune disease. In one aspect of this aspect, the composition further comprises a pharmaceutically acceptable carrier.

[0060] In another aspect, this specification describes immunostimulatory oligonucleotide duplexes described herein, conjugated to an antigen or nucleic acid sequence encoding an antigen, for use, for example, as a vaccine.

[0061] In another aspect, this specification describes compositions comprising an immunostimulatory oligonucleotide duplex described herein and a vaccine.

[0062] In another aspect, this specification describes compositions comprising the immunostimulant oligonucleotide double chain described herein and nanoparticles.

[0063] In another aspect, this specification describes nanoparticles comprising immunostimulatory oligonucleotide double chains as described herein. In one embodiment of either aspect, the nanoparticles are lipid nanoparticles.

[0064] In another aspect, this specification describes a method for administering a vaccine, comprising the step of administering an immunostimulatory oligonucleotide duplex described herein to a subject in need thereof. In this aspect or in any other aspect, the immunostimulatory oligonucleotide duplex is administered together with an antigen or a nucleic acid sequence encoding an antigen. In another aspect of this aspect or in any other aspect, the antigen or nucleic acid encoding an antigen is conjugated to the immunostimulatory oligonucleotide duplex.

[0065] In another aspect, this specification describes a method for increasing the efficacy of a vaccine, comprising the step of administering an immunostimulatory oligonucleotide duplex described herein to a subject in need thereof. In one aspect of this aspect or any other aspect, the immunostimulatory oligonucleotide duplex is administered together with an antigen or a nucleic acid sequence encoding an antigen. In another aspect of this aspect or any other aspect, the antigen or nucleic acid encoding an antigen is conjugated to the immunostimulatory oligonucleotide duplex.

[0066] Definition: As used herein, “oligonucleotide double helix” encompasses two separate ribonucleic acid strands that form a double helix by hybridizing through the formation of complementary base pairs under physiologically reasonable temperature and ionic strength conditions. The term oligonucleotide double helix also encompasses single strands containing self-complementary sequences that enable hybridization for double helix formation under similar conditions. A double helix formed from a single strand may include a hairpin structure in which it folds back at a few unhybridized nucleotides at the transition from one strand to the other, or a hairpin-loop or stem-loop structure in which it includes a more obvious loop of unhybridized nucleotides between the hybridized sequences. The immunostimulant oligonucleotide double helix described herein will have a duplexed length of 20 nucleotides or more without a single-stranded overhang (generally GG or a modified form thereof). The minimum length of the double-stranded sequence, 20 nucleotides including the 5'-terminal monophosphate-CUGA-3' duplexed sequence, was determined for the immunostimulatory activity of the oligonucleotide double-stranded sequences described herein. However, within the remaining minimum 16-nucleotide length of the double-stranded sequence, some degree of mismatch is permissible, for example, that at least 11 of the remaining 16 nucleotides must be complementary. For example, at least 11 of the 16 nucleotides, at least 12 of the 16 nucleotides, at least 13 of the 16 nucleotides, at least 14 of the 16 nucleotides, at least 15 of the 16 nucleotides, or all of the remaining 16 nucleotides may be complementary. If there is one or more mismatches, it is expected that the mismatches will be better tolerated if they are located within the 20-nucleotide sequence forming the double-stranded sequence, i.e., if the stretches of the nucleotides at both ends are perfectly complementary. Furthermore, if there are two or more mismatches in the sequence, consecutive mismatches are expected to be less desirable.If there is one or more mismatches, it is conceivable that a relatively high GC content in the remaining nucleotides could help offset any relative disadvantages of the mismatches. The same principle would likely apply to mismatches where the double-stranded region is longer than 20 nucleotides.

[0067] As used herein, the term “RNA” refers to ribonucleic acid, which, as typically transcribed in nature, includes the purine nucleobases adenine and guanine and the pyrimidine nucleobases cytosine and uracil. The RNA oligonucleotides described herein may include modifications to the nucleobases or the ribose-phosphate ester backbone, for example, to enhance stability or resistance to degradation. Examples of such modifications are discussed herein or are known in the art. In any aspect described herein, the modification is not the removal of the 2' hydroxyl that distinguishes RNA from deoxyribonucleic acid.

[0068] As used herein, the expression “an oligonucleotide duplex contains a 5' monophosphate group” means that the monophosphate is located on the 5' terminal carbon of the sequence 5'-CUGA-3' contained in the immunostimulant oligonucleotide duplex described herein, or on an analogue or modified form thereof.

[0069] The terms “increase,” “enhance,” or “activate” are used herein to mean a reproducible and statistically significant increase. In some embodiments, the terms “increase,” “enhance,” or “activate” can mean an increase of at least 10% compared to a baseline level, e.g., at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% (including 100%), or any increase between 10% and 100% compared to a baseline level, or at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a suitable control, such as a 20-fold increase, a 30-fold increase, a 40-fold increase, a 50-fold increase, a 6-fold increase, a 75-fold increase, a 100-fold increase, or any increase between 2 times and 10 times or more. In relation to the marker, "increase" refers to a statistically significant increase of that level that is reproducible.

[0070] The terms “decrease,” “reduction,” “reduction,” or “inhibition” are used herein to describe a statistically significant reduction. In some embodiments, “decrease,” “reduction,” “reduction,” or “inhibition” typically mean a reduction of at least 10% compared to a suitable control (e.g., no given treatment), and may include reductions of, for example, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or greater. As used herein, “reduction” or “inhibition” does not encompass complete inhibition or complete reduction compared to a baseline level. “Complete inhibition” is 100% inhibition compared to a suitable control.

[0071] As used herein, “reference level” means a normal, otherwise unaffected population of cells or tissue (e.g., a biological sample obtained from a healthy subject, or a biological sample obtained from such subject at a prior point in time, e.g., a biological sample obtained from a patient before diagnosis of interferon-mediated disease, or a biological sample that has not been in contact with any of the compositions disclosed herein).

[0072] As used herein, “appropriate control” means untreated, otherwise identical cells or populations (for example, patients who were not administered the active ingredient described herein, or patients who were administered only a portion of the composition described herein compared to non-control cells).

[0073] As used herein, the terms “inducing interferon production” or “increasing interferon production” mean that interferon production increases by at least threefold after administration of the immunostimulatory oligonucleotide duplex described herein, or after contact of cells, populations of cells, tissues, or organisms with such immunostimulatory oligonucleotide duplex. In some embodiments, the increase in interferon production can be at least fourfold, at least fivefold, at least tenfold, at least fifteenfold, at least twentyfold, or more. Interferon production can be measured, for example, by immunoassays (e.g., ELISA, immunoprecipitation, etc.), biological reporter assays, or other assays known in the art.

[0074] As used herein, “interferon-related disease or disorder” or “interferon-associated disease or disorder” refers to a disease or disorder that can be treated by administering interferon or by inducing interferon production.

[0075] As used herein, the term “reduce a viral titer” or “reduces viral titer” means that the number of infectious viral particles in a sample such as serum, blood, or tissue sample, or in a cell culture supernatant, is reduced by at least 10% by treatment of a subject or cell culture with the immunostimulatory oligonucleotide double helix described herein.

[0076] As used herein, the terms “to treat,” “to treat,” “to treat,” or “amelioration” refer to therapeutic treatments aimed at reversing, mitigating, improving, inhibiting, slowing, or halting the progression or severity of a condition associated with a disease or disorder. The term “to treat” encompasses reducing or mitigating at least one adverse effect or symptom of an infection-related condition, disease, or disorder. A treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is “effective” if the progression of the disease is reduced or halted. That is, “treatment” encompasses not only improvement of symptoms or markers but also the halt or at least slowing of the progression or worsening of symptoms that would have been expected without treatment. Beneficial or desirable clinical outcomes include, but are not limited to, the relief of one or more symptoms, whether detectable or undetectable; a reduction in the severity of the disease; stabilization of the disease state (i.e., no worsening); delay or slowing of disease progression; improvement or palliation of the disease state; and remission (whether partial or complete), whether detectable or undetectable. The term "treatment" for a disease also includes the alleviation of the symptoms or side effects of the disease (including reactive treatment).

[0077] As used herein, “prevention” or “prevention” refers to any methodology that, as a result of the action of that methodology, prevents the disease condition from occurring (for example, the administration of a vaccine that prevents infection or disease caused by a certain pathogen, but is not limited thereto). In one aspect, prevention may also be understood to mean that the disease is not established to the same extent as it would occur in an untreated control. Therefore, prevention of disease includes reducing the likelihood that a subject may develop the disease compared to an untreated subject (for example, a subject not treated with the methods or compositions described herein).

[0078] The terms "statistically significant" or "significant" refer to statistical significance, generally meaning a difference of two standard deviations (2SD) or more.

[0079] As used herein, the terms “comprising” or “comprises” (including) are used in relation to compositions, methods, and their respective components that are essential to the Method or Composition, but the inclusion of elements not specified, whether essential or not, is also permitted.

[0080] The singular terms “a,” “an,” and “the” encompass multiple referents unless the context makes it clear otherwise. Similarly, the word “or” encompasses “and” unless the context makes it clear otherwise. In the implementation or testing of this disclosure, methods and materials similar to or equivalent to those described herein may be used, but suitable methods and materials are described below. The abbreviation “eg” is derived from the Latin “exempli gratia” and is used herein to indicate non-limiting examples. Thus, the abbreviation “eg” is synonymous with “for example.” [Brief explanation of the drawing]

[0081] [Figure 1]Figures 1A and 1B illustrate the discovery of double-stranded RNA, which reduced influenza virus infection after treatment. (Figure 1A) A549 cells were transfected with double-stranded RNA (IDT Inc). After 24 hours, the cells were infected with influenza A / WSN / 33(H1N1) virus (MOI=0.01). The supernatant was collected for detection of viral titer by plaque formation assay. Scrambled DisRNA was used as a control. (Figure 1B) Human airway tips were transfected with double-stranded RNA 1 (IDT Inc). After 24 hours, the cells were infected with influenza A / WSN / 33(H1N1) virus (MOI=0.01). Samples were collected for detection of viral NP genes by RT-qPCR. Scrambled double-stranded RNA was used as a control. [Figure 2] Figure 2 illustrates that different versions of RNA containing common sequences can increase the IRF-IFN pathway in A549 cells. A549 Dual® (InvivoGen®) cells (10,000 cells / well) were seeded in 96-well plates and transfected with the indicated RNAs for 48 hours. Luciferase activity, representing the level of IRF-IFN pathway activation, was then measured using the QUANTI-Luc® kit according to the manufacturer's instructions. The OD value of the scrambled RNA group was set to 1. Six replicates were performed for each sample. [Figure 3] Figures 3A-3D show that double-stranded RNA1 is a positive regulator of the type I interferon (IFN-1) pathway. (Figure 3A) Volcano plot of differentially expressed genes (DEGs) from RNA-seq after treatment with double-stranded RNA1. (Figure 3B) GO enrichment analysis of DEGs. (Figure 3C) Volcano plot of differentially expressed proteins from TMT mass spectrometry after treatment with double-stranded RNA1. (Figure 3D) GO enrichment analysis of differentially expressed proteins. [Figure 4]Figure 4 shows that double-stranded RNA-1 specifically increases STAT1 and STAT2, which are specific to the IFN pathway. A549 cells were transfected with double-stranded RNA-1 and cultured for 48 hours, after which cell samples were collected for qPCR detection of the indicated genes. [Figure 5] Figure 5 shows that IRF3 knockout eliminated the effect of double-stranded RNA1 on the IFN-1 pathway. To knock down DGCR5, wild-type HAP1 cells, IRF7 knockout HAP1 cells, or IRF3 knockout HAP1 cells were transfected with double-stranded RNA1 (IDT Inc). After 48 hours, cells were collected for RT-qPCR detection of IFN-1 pathway genes, including STAT1, IL4L1, TRAIL, IFFI6, and IFN-β1. Scrambled double-stranded RNA was used as a control. [Figure 6] Figure 6 shows that double-stranded RNA-1 induces IFN production by affecting the phosphorylation of IRF3. A549 cells were transfected with double-stranded RNA-1 and cultured for 48 hours, after which IRF3 mRNA levels were detected by qPCR or IRF3 phosphorylation levels were detected by immunofluorescence staining. [Figure 7] Figure 7 is a table of RNA oligonucleotides examined in Example 1. [Figure 8] Figure 8 shows that dsRNA-4 increases IFN-β production. Differentiated human primary airway epithelial cells, human primary alveolar epithelial cells, or human primary pulmonary microvascular endothelial cells (HMVECs) were transfected with either a negative control (NC) or dsRNA-4. After 48 hours, qPCR was performed to measure IFN-β production. [Figure 9]Figure 9 shows that dsRNA-1 and dsRNA-2 induced high levels of IFN-beta and inhibited native SARS-CoV-2 by approximately 104-fold. ACE2-expressing A549 cells were transfected with the indicated dsRNAs. 24 hours after transfection, ACE2-A549 cells were infected with SARS-CoV-2 at an MOI of 0.05 for 48 hours. Cells were collected in Trizol®, and total RNA was isolated and treated with DNAse-I using the Zymo RNA Miniprep Kit® according to the manufacturer's protocol. qPCR was performed to detect the gene levels of the indicated dsRNAs. The gene level of low-dose dsRNA was set to 1. [Figure 10-1] Figures 10A–10D show the evaluation of novel immunostimulatory RNAs. Figure 10A shows A549 cells transfected with RNA-A, RNA-B, or scrambled double-stranded RNA control and infected with influenza A / WSN / 33(H1N1) virus (MOI=0.01) 24 hours later. The titer of progeny viruses in the culture supernatant collected 48 hours after infection was determined by quantifying plaque-forming units (PFUs). Data are shown as a percentage of viral infection measured in cells treated with control RNA (data shown are mean ± standard deviation; N=3; ***, P<0.001). Figure 10B shows qPCR analysis of IFN-β and IFN-α RNA levels in cells 48 hours after transfecting A549 cells with RNA-A, RNA-B, or scrambled dsRNA control (N=3). [Figure 10-2]Figures 10A–10D show the evaluation of novel immunostimulatory RNAs. Figure 10C shows the RNA-mediated IFN production kinetics measured using the Quanti-Luc assay in wild-type A549-Dual cells transfected with RNA-A, RNA-B, or scrambled RNA control. OD values ​​from cells transfected with scrambled RNA control were subtracted as background (N=6). Figure 10D shows the dose-dependent induction of IFN by RNA-A and RNA-B in A549-Dual cells compared to the scrambled RNA control, measured 48 hours after transfection (control OD values ​​subtracted as background; N=6). [Figure 11] Figures 11A and 11B show profiling of the effects of RNA-B by RNA-seq and TMT mass spectrometry. A549 cells were transfected with RNA-B or scrambled RNA control, and cell lysates were collected at 48 hours and analyzed by RNA-seq (Figure 11A) or TMT mass spectrometry (Figure 11B). Differentially expressed genes (DEGs) or differentially expressed proteins are shown in Volcano plots (top), and GO enrichment analysis was performed for DEGs (bottom) (N=3). Differentially expressed proteins were plotted (top) and GO enrichment analysis was performed (bottom) (N=3). [Figure 12A] Figures 12A and 12B are heatmaps showing the effect of immunostimulatory RNA on IFN pathway-related gene levels. DEGs from RNA-seq (Figure 12A) and differentially expressed proteins from TMT mass spectrometry (Figure 12B), shown in Figures 1B and 11, are represented here as heatmaps (the gene level targeting scrambled RNA is set to 1; N=3). [Figure 12B] See the explanation in Figure 12A. [Figure 13]Figures 13A and 13B show RNA-induced gene expression associated with the type I interferon pathway. Figure 13A is a Venn diagram showing that differentially expressed ISGs from RNA-A TMT mass spectrometry belong to type I or type II interferon-stimulating genes. Figure 13B is a heatmap of qPCR results showing that RNA-I preferentially activates the type I interferon pathway. A549 cells were transfected with RNA-A or scrambled dsRNA control, collected at 48 hours, and analyzed by qPCR (expression levels normalized to GAPDH; gene levels induced by RNA control set to 1; N=3). [Figure 14] Figure 14 shows a summary of the RNA oligonucleotide sequences examined in Examples 2-5. [Figure 15] Figure 15 summarizes the characteristics of immunostimulatory RNA. [Figure 16] Figure 16 shows a comparison of the immunostimulatory activity of the different RNAs described in Figure 14. A549-Dual cells were transfected with the indicated double-stranded RNAs for 48 hours, and then IFN pathway activation was measured by quantifying luciferase reporter activity. Data are shown as a fold change relative to the scrambled RNA control (N=6). [Figure 17-1]Figures 17A–17F illustrate that immunostimulatory RNA induces IFN-I production via the RIG-I-IRF3 pathway. Figure 17A shows IFN-β mRNA levels quantified by qPCR after transfecting wild-type (WT) HAP1 cells, IRF3 knockout HAP1 cells, or IRF7 knockout HAP1 cells with RNA-A or scrambled RNA control for 48 hours. Data are shown as a fold change relative to the scrambled RNA control (N=3). Note that IRF3 knockdown completely abolished the IFN-β response. Figure 17B shows IRF3 mRNA levels measured in A549 cells transfected with immunostimulatory RNA-D or scrambled RNA control, determined by qPCR 48 hours after transfection (data are shown as a fold change relative to control RNA; N=3). Figure 17C shows total IRF3 protein and phosphorylated IRF3 detected 48 hours after transfection by Western blot analysis in A549 cells transfected with RNA-D or scrambled RNA control (GAPDH was used as a loading control). Figure 17D shows an immunofluorescence microscopy image of the distribution of phosphorylated IRF3 48 hours after transfection in A549 cells transfected with RNA-D or scrambled RNA control (arrowheads indicate nuclei expressing phosphorylated IRF3). Figure 17E shows the quantification of IFN-β expression levels using Quanti-Luc assay or qPCR 48 hours after transfection with immunostimulatory RNA-D or scrambled RNA control in wild-type (WT) A549-Dual cells, RIG-I knockout A549-Dual cells, MDA5 knockout A549-Dual cells, or TLR3 knockout A549 cells (data are shown as a magnification change relative to scrambled RNA control; N=6). Note that RIG-I knockout eliminated the IFN-β inducing ability of immunostimulatory RNA. [Figure 17-2]Figures 17A-17F illustrate that immunostimulatory RNA induces IFN-I production via the RIG-I-IRF3 pathway. Figure 17F: SPR characterization of binding affinity between cellular RNA sensors (RIG-I, MDA5, and TLR3) and RNA-1 immobilized on a streptavidin (SA) sensor chip. Equilibrium dissociation constant (KD), association rate constant (Ka), and dissociation rate constant (Kd) are plotted on the graph. [Figure 18] Figure 18 shows that IRF3 knockout eliminated the ability of immunostimulatory RNA to induce IFN-I pathway-related genes. Wild-type (WT) HAP1 cells, IRF3 knockout HAP1 cells, or IRF7 knockout HAP1 cells were transfected with RNA-A or scrambled RNA controls, and STAT1, IL4L1, TRAIL, and IFI6 mRNA levels were quantified by qPCR 48 hours after transfection. Data are expressed as fold change relative to the RNA control (N=3). [Figure 19] Figure 19 shows that RIG-I knockout eliminated the immunostimulatory RNA-induced effect on IFN-β. Wild-type (WT) A549-Dual cells, RIG-I knockout A549-Dual cells, MDA5 knockout A549-Dual cells, or TLR3 knockout A549 cells were transfected with RNA-A, RNA-B, RNA-C, or scrambled RNA controls. 48 hours after transfection, IFN-β mRNA levels were detected by the Quanti-Luc assay in WT, RIG-I KO, and MDA5 KO A549-Dual cells, and by qPCR in TLR3 KO A549 cells. Data are shown as a fold change relative to the scrambled RNA control (N=6). [Figure 20-1]Figures 20A–20D show that immunostimulatory RNA induces IFN-β production in differentiated human lung epithelial and endothelial cells in the organ chip, exhibiting broad-spectrum inhibition of infection by influenza H3N2, SARS-CoV-2, SARS-CoV-1, MERS-CoV, and HCoV-NL63. Figure 20A: Schematic cross-section of a human lung-on-chip that faithfully reproduces the physiological function and pathophysiology of the human lung. Figure 20B: Human lung airway and alveolar chips were transfected with RNA-1 or scrambled RNA control by perfusion through both channels of the chip, and epithelial and endothelial cells were collected after 48 hours for detection of IFN-β mRNA by qPCR (data are expressed as a magnification change relative to the RNA control; N=3; *, p<0.05; ***, p<0.001). Figure 20C: Effect of treatment with RNA-1 or scrambled control in human lung airway or alveolar chips infected with influenza A / HK / 8 / 68(H3N2)(MOI=0.1). Viral load was determined by quantifying viral NP genes by qPCR in lysates 48 hours after infection. Results are shown as a fold change relative to RNA control; N=3; *, p<0.05. [Figure 20-2]Figures 20A–20D show that immunostimulatory RNA induces IFN-β production in differentiated human lung epithelial and endothelial cells in organ chips, exhibiting broad-spectrum inhibition of infection by influenza H3N2, SARS-CoV-2, SARS-CoV-1, MERS-CoV, and HCoV-NL63. Figure 20D: Treatment with immunostimulatory double-stranded RNA resulted in potent inhibition of multiple potential pandemic viruses, including SARS-CoV-2. Cells shown were treated with RNA-1, RNA-2, or scrambled controls and infected with influenza A / HK / 8 / 68 (H3N2) (MOI=0.1), SARS-CoV-2 (MOI=0.05), SARS-CoV-1 (MOI=0.01), MERS-CoV (MOI=0.01), and HCoV-NL63 (MOI=0.002), respectively. 48 hours after infection, viral load was determined by quantifying the viral NP gene for H3N2 and the N gene for SARS-CoV-2 and HCoV-NL63 in cell lysates using qPCR, and then quantifying the viral titer by plaque assay. All results are shown as a fold change relative to the RNA control; N=3; *, p<0.05; ***, p<0.001. [Figure 21] Figure 21 shows immunostimulatory RNA-mediated production of IFN in A549 cells overexpressing ACE2. IFN-β and ISG15 levels were detected by qPCR 48 hours after transfection in cells transfected with RNA-A, RNA-B, or scrambled dsRNA controls. Levels of IFN-β or ISG15 induced by the scrambled dsRNA control were set to 1. Data are shown as a fold change relative to the control (N=3). [Figure 22]Figures 22A and 22B show that immunostimulatory RNA is a more potent IFN-β inducer than poly(I:C) and 5'ppp-dsRNA, but does not induce the production of pro-inflammatory cytokines. Figure 22A: Comparison of titers of IFN-β-inducing RNA-1, poly(I:C), and 5'ppp-dsRNA at the same concentration (2.8 μg / mL) in A549 cells 48 hours after transfection. Data are shown as a multiplier change relative to the scrambled RNA control (N=3). Figure 22B: Heatmap comparing changes in inflammatory gene expression induced by poly(I:C) or RNA-1 and RNA-2 in A549 cells 48 hours after transfection. [Figure 23] Figure 23 shows that immunostimulatory RNA can inhibit influenza infection in human lung epithelial cells. [Figure 24] Figure 24 shows that immunostimulatory RNA can inhibit infection with the common cold coronavirus in monkey kidney cells. [Figure 25] Figure 25 shows the inhibition of SARS-CoV-2 virus by RNA-A and RNA-B in human lung epithelial cells overexpressing ACE2. [Figure 26] Figure 26 shows that immunostimulatory RNA inhibits SARS-CoV-2 infection in vivo. Induction of type I interferon by double-stranded dsRNA administered on days -1, 0, and +1 was sufficient to significantly reduce viral load in hamsters. [Figure 27] Figure 27 illustrates motif 1, which includes motifs 1 and 2 of the immunostimulatory RNA double helix described herein. [Figure 28]Figures 28A–28C illustrate the inhibition of in vivo natural SARS-CoV-2 infection. Figure 28A: Reduction in viral load in the lungs of hamsters prophylactically treated with RNA-1 (20 ug in PBS) administered intranasally one day before, on the day of infection, and one day after infection, measured by qPCR for subgenomic RNA encoding the SARS-CoV-2 N protein (left; *, p=0.030) or by quantification of viral titer by plaque assay (right; *, p=0.032). Figure 28B: Reduction in viral load in hamster lungs (*p=0.01) produced by intranasal administration of RNA-1 (20ug) once daily for two days, starting one day after intranasal administration of SARS-CoV-2 virus (103pFU), and measured one day later by qPCR for subgenomic RNA encoding the SARS-CoV-2 N protein. Figure 28C: Low-magnification (left) and high-magnification (right) H&E stained tissue images of lungs from B treated with delivery vehicle alone (top) or vehicle containing RNA-1 (bottom), starting one day after infection (left bar, 2.5 mm; right bar, 100 μm). [Figure 29] Figures 29A and 29B show profiling of the effects of RNA-2 by RNA-seq and TMT mass spectrometry. A549 cells were transfected with RNA-2 or scrambled RNA control. Cell lysates were collected at 48 hours and analyzed by RNA-seq (Figure 29A) or TMT mass spectrometry (Figure 29B). Differentially expressed genes (DEGs) or differentially expressed proteins are shown in Volcano plots (top), and GO enrichment analysis was performed for DEGs (bottom) (N=3). Differentially expressed proteins were plotted (top) and GO enrichment analysis was performed (bottom) (N=3). [Figure 30] Figure 30 shows the subgenome N transcript for RNA-1 compared to the vehicle control. The levels are compared to the actin-loading control. [Modes for carrying out the invention]

[0082] Detailed explanation The compositions and methods described herein relate, in part, to the discovery of immunomodulatory / immunostimulatory oligonucleotide RNA duplexes that induce interferon (IFN) production. The immunostimulatory oligonucleotide duplexes described herein have the ability to induce robust innate immune responses and inhibit or treat diseases that can be treated with interferon, or diseases in which increased interferon is beneficial, such as, but not limited to, viral infections, bacterial infections, fungal infections and / or parasitic infections, cancer, and autoimmune diseases.

[0083] The following are considerations to enable those skilled in the art to manufacture and use this technology.

[0084] Interferons (IFNs) are a group of multifaceted cytokines produced and released by immune cells as part of the innate immune response to infection. IFNs have been used as therapeutic agents to treat autoimmune diseases (e.g., multiple sclerosis and lupus), many types of cancer, and viral infections. For example, see Paolicelli, D., Direnzo, V., & Trojano, M. (2009), Review of interferon beta-1b in the treatment of early and relapsing multiple sclerosis. Biologics:targets&therapy, 3, 369-376; Tamura T, Yanai H, Savitsky D, Taniguchi T, The IRF family transcription factors in immunity and oncogenesis. Annu Rev Immunol. (2008); McNab F, Mayer-Barber K, Sher A, Wack A, O'Garra A, Type I interferons in infectious disease. Nat Rev Immunol. (2015). Each of these references is incorporated herein by reference in its entirety.

[0085] The immunomodulatory effects of interferon (IFN) are exerted across a wide range of cell types that express interferon polypeptide receptors. The downstream effects of interferon enable the regulation of the immune system by activating signaling and activator-transcription (STAT) complexes and other signaling molecules. STAT is a family of transcription factors that regulate the expression of several immune system genes. Interferon signaling pathways are well known in the art. For example, see Muller U, et al. Functional role of type I and type II interferons in antiviral defense. Science (1994), Honda et al. Immunity, 25, 349-360 (2006), Marchetti M, et al. Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors. Mol Biol Cell (2006), Lee and Ashkar, Front. Immunol., 2018, and Platanias LC. Mechanisms of type-I-and type-II-interferon-mediated signalling. Nat Rev Immunol. (2005) 5:375-86. Each of these references is incorporated herein by reference in its entirety.

[0086] The induction of interferon (IFN) production plays a crucial role in the human immune response by "interfering" viral replication. Induction of IFN gene expression can lead to increased cellular resistance to infections, including viral infections, by activating immune cells (e.g., natural killer cells and macrophages) and increasing the expression of major histocompatibility complex (MHC) antigens, thereby upregulating antigen presentation and enhancing host defense. There are several types of IFN genes and proteins, which in humans are typically classified into three classes: type I IFNs (IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), type II IFNs (IFN-γ), and type III IFNs. All three classes of IFNs are involved in the fight against infection and the regulation of the immune system.

[0087] The regulation of IFN expression is complex and intricately controlled by interferon regulatory factors (IRFs). IRFs are a family of transcription factors involved in many aspects of the immune response, including the development and differentiation of immune cells and the regulation of responses to pathogens. The functional roles and signaling pathways of IRFs are well known in the art, e.g., Jefferries, Front. Immunol., 2019 and Bustamante et al. Clinical Immunology, 5 th See ed. (2019). These references are incorporated herein by reference in their entirety. IRF3, one such IRF, is a positive regulator of type I interferon gene induction. IRF3 is an intracellular polypeptide activated downstream of the intracellular RNA sensor, pattern recognition receptor RIG-I. In particular, IRF3 inhibits IL-12β and TGF-β, while directly inducing the expression of IFN-β and cytokines such as CXCL10, RANTES, ISG56, IL-12p35, IL-23, and IL-15, in addition to type I IFN.

[0088] The interferon pathway is involved in pathogenic infections caused by viruses, bacteria, fungi, and parasites, as well as in many diseases, including cancer and autoimmune diseases. Since increased interferon production is often part of the natural response to infection, treatments that further promote such production can help in the fight against infection. In other examples, in some viral infections, particularly those caused by SARS-CoV-2 coronavirus, the body's interferon response is not activated or is suppressed compared to that seen with other viruses or pathogens, so treatments that promote interferon production can help in the fight against infection. Therefore, the immunostimulatory oligonucleotide double-stranded compounds described herein can be used to prevent, mitigate, and / or treat diseases in which interferon-containing or interferon-promoting agents are beneficial or can be treated with such agents.

[0089] Immunomodulatory oligonucleotide double-strand composition The immunomodulatory oligonucleotide duplex disclosed herein is characterized in that the 5' terminal sequence 5'-CUGA-3' (SEQ ID NO:1) forms a complex with its complement 5'-UCAG-3', and the complement contains a 3'GG overhang. Therefore, this immunostimulatory oligonucleotide duplex contains the following sequence: TIFF2026123077000002.tif16128 Underlined part -3' overhang; N = G, A, C, or U, or any modified version thereof; Complementary base for N'=N Bold - Complementary parts of SEQ ID NO:1 and SEQ ID NO:2;

[0090] The immunostimulatory oligonucleotide double helix disclosed herein comprises a double-stranded RNA with a 5'-monophosphate on the 5'-CUGA-3' side and a minimum double-stranded portion length of 20 nucleotides (see also Figure 27). The sequence configuration of the double helix on the 3' side of the 5'-monophosphate-CUGA-3' sequence is not critical for interferon induction. N16 is the minimum value. However, N (and the corresponding N' complementary sequence) may be longer. As discussed elsewhere in this specification, the double helix can tolerate some degree of mismatch, but mismatch is N 16 :N' 16 The number of nucleic acid bases should be five or less. General rules regarding mismatches when they occur will be discussed elsewhere in this specification.

[0091] In some embodiments of the above aspects, the immunostimulatory oligonucleotide double helix is ​​at least 20 nucleic acid bases long. In some embodiments, the immunostimulatory oligonucleotide double helix has a length of 20-300, 20-250, 20-200, 20-150, 20-100, 20-50, 50-300, 50-250, 50-200, 50-150, or 50-100 nucleotides. In some embodiments, the immunostimulatory oligonucleotide double helix is ​​20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, They have lengths of 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides. These lengths do not include a 3'GG overhang that is not double-stranded on the bottom strand.

[0092] In some embodiments, the immunostimulatory oligonucleotide double helix described herein can be conjugated to an antigen or biomolecule. In some embodiments, the immunostimulatory oligonucleotide double helix described herein further comprises a linker. The linker described herein can be used for conjugation of the oligonucleotide sequence to the antigen-coding sequence of an antigen.

[0093] Modification / Replacement The RNA oligonucleotide double-strand sequences described herein may include modified nucleotides, including modifications to the nucleic acid base portion and / or the sugar-phosphate ester backbone portion, insofar as the modified nucleotides allow base pairing with appropriate nucleotides on the opposite strand, and such modifications (one or more) allow for enhanced interferon production by the resulting double-strand molecule, as measured, for example, by methods known in the art or by the methods described herein. Such modifications can alter the stability of the double-strand, for example, by reducing its susceptibility to enzymatic or chemical degradation, or can alter (increase or decrease) intramolecular or intermolecular interactions, including base pairing interactions. RNA oligonucleotide double-strand nucleic acid bases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases cytosine (C) and uracil (U) or modified or related forms thereof.

[0094] In one embodiment, the double-stranded sequence contains one or more modified ribonucleotides in the 5'-monophosphate-CUGA-3' sequence or the 5'-UCAGGG-3' sequence. TIFF2026123077000003.tif11128

[0095] In another embodiment, the double hemisphere is N 16 Array or N' 16The sequence contains one or more modified ribonucleotides, or, if the double helix is ​​longer than 20 nucleotides, somewhere else in the double helix. RNA containing such modifications, for example, translation-allowing modifications, are considered likely to be tolerable in relation to the double helix described herein and likely to retain immunostimulatory / interferon-inducing activity. In a given double helix molecule, it is considered possible to modify one or more, two or more, or three or more ribonucleotides, including all four of the ribonucleotide 5'-CUGA-3'. Furthermore, in a given double helix molecule, it is considered possible to modify one or more, two or more, three or more, four or more, or five or more ribonucleotides, including all six of the ribonucleotide 5'-UCAGGG-3'. Furthermore, N 16 Array or N' 16 A sequence may contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more, or up to all, and all of the modifications to the ribonucleotide, including one or more nucleic acid bases or modifications to the ribose-phosphate ester backbone. Similarly, if the N-N' double helix contains more than 16 ribonucleotides, any one of them or any combination thereof, or up to all of them (including all), may contain one or more modifications to the nucleic acid base or ribose-phosphate ester backbone structure.

[0096] Exemplary nucleic acid modifications include, but are not limited to, nucleic acid base modifications, sugar modifications, sugar junction modifications, conjugates (e.g., ligands), and combinations thereof. In one embodiment, the modification does not involve the substitution of ribose sugars by deoxyribose present in deoxyribonucleic acid. Nucleic acid modifications are known in the art. See, for example, US20160367702A1, US20190060458A1l, U.S. Patent No. 8,710,200, and U.S. Patent No. 7,423,142. These documents are incorporated herein by reference in their entirety.

[0097] Exemplary modified nucleic acid bases include thymine (T), inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine, and uracil, such as 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propinyluracil Lucitosine, 6-azouracil, 6-azocytosine and 6-azothimine, 5-uracil (pseuducol), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and 8-substituted guanines, 5-trifluoromethyl and other 5-substituted uracils and 5-substituted cytosine, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines For example, 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazole, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxy These include, but are not limited to, acetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3 carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases.Further purines and pyrimidines are disclosed in U.S. Patent No. 3,687,808, on pages 858-859 of the Concise Encyclopedia of Polymer Science and Engineering (Kroschwitz, J.I., John Wiley & Sons, 1990), and in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

[0098] Exemplary sugar modifications include, but are not limited to, 2'-fluoro, 3'-fluoro, 2'-OMe, 3'-OMe, and acyclic nucleotides such as peptide nucleic acids (PNA), unlocked nucleic acids (UNA), or glycol nucleic acids (GNA).

[0099] In some embodiments, nucleic acid modifications may include substitution or modification of intersaccharide junctions. Exemplary intersaccharide junction modifications include phosphotriesters, methylphosphonates, phosphoramides, phosphorothioates, methylenemethyliminos, thiodiesters, thionocarbamates, siloxanes, N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5'), hydroxylaminos, siloxanes (dialkylsiloxanes), carboxamides, carbonates, carboxymethyls, carbamates, carboxylic acid esters, thioethers, ethylene oxide linkers, sulfides, sulfonates, sulfonamides, sulfonic acid esters, and thioformacetates. (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methylenecarbonylamino (methykenecarbonylamino), methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether While there are other types such as thi(C3'-O-C5'), thioethers (C3'-S-C5'), and thioacetamides (C3'-N(H)-C(=O)-CH2-S-C5', C3'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5', and 3'-NHP(O)(OCH3)-O-5'), they are not limited to these.

[0100] In some embodiments, nucleic acid modifications may include peptide nucleic acids (PNAs), cross-linked nucleic acids (BNAs), morpholinos, located nucleic acids (LNAs), glycol nucleic acids (GNAs), threose nucleic acids (TNAs), or other xeno nucleic acids (XNAs) described in the art.

[0101] In some embodiments, the immunostimulatory oligonucleotide double-strand can take the form of an intramolecular double-strand of a hairpin molecule or an intramolecular double-strand of a hairpin-loop molecule. In such embodiments, the 5'-terminal sequence and the 3'-terminal sequence are self-complementary and share the 5'-N' 16 5-monophosphate-CUGAN hybridized to the 16 16 UCAGGG-3' structure is provided.

[0102] In another aspect of any of the above aspects, the oligonucleotide double-strand described herein includes a linker. For example, the linker can simply be a nucleic acid backbone linkage, such as a phosphodiester linkage. In addition, the nucleic acid linkers can all be the same, or all different, or some can be the same and some can be different.

[0103] In some embodiments of any of the above aspects, the linker or spacer can be selected from the group consisting of a photocleavable linker, a hydrolyzable linker, a redox-cleavable linker, a phosphate ester-based cleavable linker, an acid-cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, and any combination thereof. In some embodiments, the cleavable linker can include a disulfide bond, a tetrazine-trans-cyclooctene group, a sulfhydryl group, a nitrobenzyl group, a nitroindoline group, a bromohydroxycoumarin group, a bromohydroxyquinoline group, a hydroxyphenacyl group, a dimethozybenzoin group, or a combination thereof.

[0104] In some embodiments, the immunostimulatory oligonucleotide double helix described herein is crosslinked such that the complementary strands are covalently linked. Such crosslinking can provide improved double helix stability, for example, so that the terminal 5'-CUGA'3' sequence identified herein is better preserved in its active conformation. In some embodiments, the crosslinking portion may be a chemical functional group. In some embodiments, the chemical functional group is selected from the group consisting of azides, alkynes, tetrazines, DBCOs, thiols, amines, carbonyls, carboxyl groups, and any combination thereof.

[0105] In some embodiments, the immunostimulant oligonucleotide double helix described herein is crosslinked by a photocrosslinking moiety. Examples of photocrosslinking moieties that are not limited to these include 3-cyanovinylcarbazole (CNVK) nucleotide; 5-bromodeoxycytosine; 5-iododeoxycytosine; 5-bromodeoxyuridine (deoxyurdine); 5-iododeoxyuridine; and nucleotides comprising aryl azides (AB-dUMP), benzophenones (BP-dUMP), perfluorinated aryl azides (FAB-dUMP), or diazirines (DB-dUMP).

[0106] In some embodiments, the immunostimulatory oligonucleotide double strands described herein are conjugated to a pharmaceutically acceptable carrier. In another embodiment, the immunostimulatory oligonucleotide double strands described herein are mixed with a pharmaceutically acceptable carrier.

[0107] In any of the embodiments described herein, the immunostimulatory oligonucleotide double helix described herein is conjugated to an antigen or its antigenic fragment or a sequence encoding an antigen or its antigenic fragment.

[0108] In some embodiments, the immunostimulatory oligonucleotide duplexes described herein may be fused to an antigen-coding sequence or may otherwise contain an antigen-coding sequence. Such compositions would contain an antigen-coding single-stranded RNA sequence fused to, or complexed with, an RNA that gives the terminal 5'-monophosphate-CUGA-3' and 5'-UCAGGG-3' duplex / overhang structure shared by the immunostimulatory oligonucleotide duplexes described herein. Introduction of such compositions into cells can result in both antigen production to stimulate an adaptive immune response and simultaneous stimulation of an interferon response.

[0109] Method for preparing oligonucleotide double helices The immunostimulatory oligonucleotide double helix described herein can be prepared by synthetic methods known in the art, including but not limited to chemical synthesis using nucleoside phosphoramidite approaches or in vitro transcription. Methods for chemical synthesis to include modified nucleotides are also known in the art.

[0110] In in vitro transcription, polymerases can be used, but are not limited to bacteriophage polymerases, such as T7 polymerase, T3 polymerase, and SP6 polymerase, viral polymerases, and E. coli RNA polymerase.

[0111] Oligonucleotide chains can be isolated from a sample using RNA extraction and purification methods known in the art. These methods include, but are not limited to, column purification, ethanol precipitation, phenol-chloroform extraction, or acid guanidinium thiocyanate-phenol-chloroform extraction (AGPC). After isolation of single-stranded oligonucleotides, a double-stranded secondary structure can be formed by hybridization and / or annealing of the top and bottom strands.

[0112] As used herein, the terms “hybridize,” “hybridize,” “hybridization,” “annealing,” or “anneal” are interchangeable in relation to the pairing of complementary nucleic acids using any process that forms a hybridization complex by linking nucleic acid strands with a complementary strand by base pairing. In other words, the term “hybridization” refers to the process by which two single-stranded polynucleotides are joined non-covalently to form a double-stranded polynucleotide. The resulting double-stranded polynucleotide is “hybrid” or “double-stranded.” The conditions for forming a hybridized or double-stranded sequence are known to those skilled in the art and generally include normal or near-normal physiological conditions, such as intracellular salt concentrations and temperature. Generally, the hybridization for forming a double-stranded sequence described herein can be carried out on each strand present at substantially equimolar concentrations.

[0113] After synthesis, hybridization, and optionally removal of non-double helical chains, the immunostimulatory oligonucleotide double helices can be characterized by any method known in the art, such as liquid chromatography, mass spectrometry, next-generation sequencing, polymerase chain reaction (PCR), gel electrophoresis, or any other method that reveals the nucleoside sequence, secondary structure, chemical composition, expression, thermodynamics, binding, or function.

[0114] For further characterization of the immunostimulatory oligonucleotide double helices described herein, 5'-monophosphate can be detected, for example, by a splinted ligation assay. See, for example, Shoenberg et al., Nat Chem Biol 3(9)(2007) and Celesnik H et al. Initiation of RNA decay in Escherichia coli by 5' pyrophosphate removal. Mol Cell. 2007;27:79-90. These publications are incorporated herein by reference in their entirety. By carefully optimizing the reaction conditions and comparing ligated RNA with unligated RNA, this assay can provide quantitative data on the amount of RNA with a 5' monophosphate terminus.

[0115] To improve the stability of the oligonucleotide modifications described above and to produce any of them, the immunostimulatory oligonucleotide double helixes described herein may be chemically modified in appropriate ways. As described above, modifications can be made to satisfy the requirements for the stability of the oligonucleotide double helixes against extracellular and intracellular enzymes, and the requirement for the ability to permeate cell membranes for human therapeutic applications. See, for example, Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 544; Milligan, JF; Matteucci, MD; Martin, JCJ Med. Chem. 1993, 36, 1923; Crooke, ST; Lebleu, B. eds. 1993 "Antisense Research and Applications" (CRC Press: Boca Raton, Florida); and Thuong, NT; Helene, C. Angew. Chim. Int. Ed. 1993, 32, 666. Chemical modifications to nucleic acids may include the introduction of heterocyclic bases, phosphate ester backbone modifications, sugar submodifications, and the attachment of conjugated groups.For example, Beaucage, SL; Iyer, RPTetrahedron 1993, 49, 1925; Beaucage, SL; Iyer, RPTetrahedron 1993, 49, 6123; Manoharan, M. "Antisense Technology" 2001, edited by S.T. Crooke (Marcel Dekker, New York); and Manoharan, M. Antisense & Nucleic Acid Development 2002,12,103, Schweitzer,BA;Kool,ETJOrg.Chem.1994,59,7238, Schweitzer,BA;Kool,ETJAm.Chem.Soc.1995,117,1863,Moran,S.Ren,RX-F.Rumney,S.;Kool,E See TJAm.Chem.Soc.1997,119,2056, Guckian, KM;Kool, ETangew.Chem.Int.Ed.Engl.1997,36,2825, and Mattray, TJ;Kool, ETJAm.Chem.Soc.1998,120,6191. For further information, see Fire, A.; Xu, S.; Montgomery, MK; Kostas, SA; Driver, SE; Mello, CC Nature, 1998, 391, 806; Elbashir, SM; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Nature, 2001, 411, 494; McManus, Mt. Sharp, P Nature Reviews Genetics, 2002, 3, 737; Hannon, G. J Nature, 2002, 418, 244; and Roychowdhury, A.; Iankkoon, H.; Hendrickson, CL; Benner, SA Org. Lett. 2004, 6, 489. These references are incorporated herein by reference in their entirety.

[0116] For certain therapeutic purposes, the immunostimulatory oligonucleotide double helices described herein should possess a certain degree of stability in serum to enable distribution and cellular uptake. Long-term maintenance of therapeutic levels of these oligonucleotides in serum has a significant effect on distribution and cellular uptake, and increased serum stability will affect all cells, unlike conjugate groups that target specific cell receptors.

[0117] Chemical modifications may include the addition of ligands, linkers, and antigens. For example, ligands can be improved in terms of stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability, for example, by endocytosis-dependent or endocytosis-independent mechanisms. Oligonucleotides holding peptide (e.g., antigen) conjugates can be prepared using procedures known in the art. See Trufert et al., Tetrahedron 1996, 52, 3005, and Manoharan, "Oligonucleotide Conjugates in Antisense Technology" in *Antisense Drug Technology* (STCrooke, Marcel Dekker, Inc., 2001). These references are incorporated herein by reference, respectively.

[0118] Pharmaceutical composition The methods and oligonucleotide double-strand compositions described herein may further include formulating the immunostimulant oligonucleotide double-strands described herein with a pharmaceutically acceptable carrier.

[0119] In any of the embodiments described herein, the method further comprises the step of formulating an immunostimulatory oligonucleotide double helix with a pharmaceutically acceptable carrier and an antigen or nucleic acid sequence encoding an antigen. Such a formulation provides an adjuvant effect, for example, when administered as a vaccine or in conjunction with a vaccine, by utilizing the immunostimulatory double helix described herein. In any of the embodiments described herein, the method further comprises the step of formulating an immunostimulatory oligonucleotide double helix with a pharmaceutically acceptable carrier, an antigen or nucleic acid sequence encoding an antigen, and a separate adjuvant.

[0120] For the clinical use of the methods and compositions described herein, the administration of the immunostimulatory oligonucleotide duplexes described herein may include formulation into pharmaceutical compositions or pharmaceutical preparations for parenteral administration, e.g., intravenous administration; mucosal administration, e.g., intranasal administration; ocular administration or other modes of administration. In some embodiments, the immunostimulatory oligonucleotide duplexes described herein may be administered together with any pharmaceutically acceptable carrier compound, material, or composition that results in an effective treatment in the subject. Thus, a pharmaceutical preparation for use in the methods described herein may contain the immunostimulatory oligonucleotide duplexes described herein combined with one or more pharmaceutically acceptable components. The expression "pharmaceutically acceptable" means a compound, material, composition, and / or dosage form that, within reasonable medical judgment, is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate or zinc stearate, or steric acid), or solvent encapsulating material, that is involved in maintaining the stability, solubility, or activity of the immunostimulant oligonucleotide double helix described herein. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient. The terms “excipient,” “carrier,” and “pharmaceutically acceptable carrier” are used interchangeably herein.

[0121] The immunostimulatory oligonucleotide double chains described herein can be formulated for administration to a subject in solid, liquid, or gel form, including, for example, (1) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection as a sterile solution or sterile suspension or sustained-release formulation, (2) transdermal administration, (3) transmucosal administration, or (4) administration by bronchoalveolar lavage.

[0122] In some embodiments, the compositions described herein include a particle or polymer-based vehicle. Examples of particle or polymer-based vehicles include, but are not limited to, nanoparticles, microparticles, polymer microspheres, or polymer-drug conjugates.

[0123] In one aspect of any aspect, the compositions described herein further comprise a lipid vehicle. Examples of lipid vehicles include, but are not limited to, liposomes, phospholipids, micelles, lipid emulsions, and lipid-drug conjugates.

[0124] The formulations can be adapted for delivery to the airways, for example, to address respiratory infections. Such formulations can be adapted for delivery as an aerosol, for example, for inhalation. In some embodiments, the compositions described herein are formulated for aerosol administration, nebulizer administration, or tracheal lavage administration. In some embodiments, the compositions are formulated for intravenous, intramuscular, intraperitoneal, subcutaneous, or intrathecal administration.

[0125] For use as an aerosol, the compositions described herein can be prepared in solution or suspension and filled into a pressurized aerosol container together with a suitable propellant, such as a hydrocarbon propellant like propane, butane, or isobutane, and conventional excipients.

[0126] The oligonucleotide double-chain compositions described herein can also be administered in an unpressurized form, such as in a nebulizer or atomizer that transforms a liquid into fine droplets. Preferably, such atomization allows for the controlled generation of small droplets of uniform size from a larger liquid mass. Atomization can be achieved by any suitable means for this purpose, for example, by using one of the many nebulizers known and currently available on the market, such as the AEROMIST® pneumatic nebulizer available from Inhalation Plastic, Inc. in Niles, Illinois.

[0127] When multiple active ingredients are adapted for administration together or individually by nebulizer, they may take the form of aqueous suspensions or aqueous solutions, with or without appropriate pH or tonic adjustments, as unit doses or as multi-dose devices.

[0128] Furthermore, any suitable gas can be used to apply pressure during atomization, and so far, chemically inert gases are preferred. Exemplary gases such as nitrogen, argon, or helium can be used effectively, though not limited to these.

[0129] In some embodiments, the compositions described herein can also be administered directly to the airways in the form of a dry powder. Thus, immunostimulatory oligonucleotide double chains can be administered by inhaler. Exemplary inhalers include metered-dose inhalers and dry powdered inhalers.

[0130] A metered-dose inhaler, or "MDI," is a pressure-resistant canister or container filled with a product such as a pharmaceutical composition dissolved in a liquefied propellant or fine particles suspended in a liquefied propellant. Possible propellants include chlorofluorocarbons, hydrocarbons, or hydrofluoroalkanes. Commonly used propellants are P134a (tetrafluoroethane) and P227 (heptafluoropropane), which can be used alone or in combination. These are optionally used in combination with one or more other propellants and / or one or more surfactants and / or one or more other excipients, such as ethanol, lubricants, antioxidants, and / or stabilizers.

[0131] A dry powder inhaler (i.e., Turbuhaler® (Astra AB)) is a pressurized air source-operated system for producing dry powder particles of pharmaceutical compositions compressed to extremely small volumes.

[0132] Dry powder aerosols for inhalation therapy are generally produced so that their average diameter is primarily within the range of <5 μm. As particle diameter exceeds 3 μm, phagocytosis by macrophages gradually decreases. However, it has also been found that increasing particle size minimizes the probability of particles (with standard mass density) entering the airways and acini due to excessive deposition in the oropharyngeal or nasal cavity.

[0133] Suitable powder compositions include, for example, powder preparations containing immunostimulant oligonucleotide double chains as described herein. These can be mixed with lactose or other inert powders acceptable for intrabronchial administration. The powder compositions can be administered from an aerosol dispenser or placed in a crushable capsule which the patient or clinician inserts into a device, which then punctures the capsule and blows the powder into a steady flow suitable for inhalation. The compositions may contain a propellant, a surfactant, and a co-solvent and can be filled into a conventional aerosol container closed with a suitable throttle valve.

[0134] Aerosols for delivery to the airways have been described, for example, in Adjei, A. and Garren, J. Pharm. Res., 1:565-569 (1990), Zanan, P. and Lamm, J.-WJ Int. J. Pharm., 114:111-115 (1995), and Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract" in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990), Anderson et al., Am. Rev. Respir. Dis., 140:1317-1324 (1989)), and systemic delivery of peptides and proteins is also potentially possible (Patton and Platz, Advanced Drug Delivery Reviews, 8:179-196 (1992)); Timsina et.al.,Int.J.Pharm.,101:1-13(1995), and Tansey,IP,Spray Technol.Market,4:26-29(1994),French,DL,Edwards,DAand Niven,RW,Aerosol Sci.,27:769-783(1996),Visser,J.,Powder Technology 58:1-10 (1989)), Rudt, S. and RHMuller, J. Controlled Release, 22:263-272 (1992), Tabata, Y, and Y. Ikada, Biomed. Mater. (1995), Patton, J. and Platz, R., Adv.Drug Del.Rev.,8:179-196(1992), Bryon,P.,Adv.Drug.Del.Rev.,5:107-132(1990), Patton,JS,et al.,Controlled Release,28:15 79-85(1994), Damms,B.and Bains,W.*Nature Biotechnology* (1996), *Niven, RW, et al., *Pharm. Res.*, 12(9); 1343-1349 (1995), and *Kobayashi, S., et al., *Pharm. Res.*, 13(1): 80-83 (1996). Each of these publications is incorporated herein by reference in its entirety.

[0135] In addition to the chemical modifications of immunostimulatory oligonucleotide double helixes described herein, efforts to improve transmembrane delivery of nucleic acids and oligonucleotides have utilized protein carriers, antibody carriers, liposome delivery systems, electroporation, direct injection, cell fusion, viral vectors, and calcium phosphate-mediated transformation. U.S. Patents 7,423,142 (B2), 7,786,290 (B2), 8,598,139 (B2), 8,808,747 (B2), 10,125,369 (B2), 10,130,649 (B2), and U.S. Patent Application Publication 2018 / 0369419 (A1) (these documents are incorporated herein by reference, respectively) describe formulations for delivering mRNA, siRNA, and dsRNA compositions to the skin, blood, liver, and other target tissues or organs. As just one example, U.S. Patent No. 8,598,139 (B2) describes several examples of nucleic acid-lipid particles for delivery. See, for example, columns 42-48. Since the interferon-inducing molecules disclosed herein also have a double-stranded characteristic, it is clearly conceivable that the double-stranded molecules disclosed herein can be delivered using formulations for delivering siRNA compositions to such tissues.

[0136] In some embodiments, the immunostimulatory oligonucleotide double-stranded compounds described herein are formulated into compositions comprising micelles, amphiphilic carriers, polymers, cyclodextrins, liposomes, and encapsulation devices.

[0137] Microemulsification technology can improve the bioavailability of several lipophilic (water-insoluble) pharmaceuticals. Examples include trimetrine (Dordunoo, SK, et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991) and REV 5901 (Sheen, PC, et al., J Pharm Sci 80(7), 712-714, 1991). In particular, microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system rather than the circulatory system, thus bypassing the liver and preventing the breakdown of the compound in the hepatobiliary circulation.

[0138] The immunostimulatory oligonucleotide double chains described herein can be formulated using amphiphilic carriers. These amphiphilic carriers are obtained from saturated and monounsaturated polyethylene glycolated fatty acid glycers, for example, from various fully or partially hydrogenated vegetable oils. Such oils comprising tri-, di-, and mono-fatty acid glycers and corresponding di- and mono-polyethylene glycol esters of fatty acids would be preferable. Particularly preferred fatty acid compositions include 4-10% capric acid, 3-9% capric acid, 40-50% lauric acid, 14-24% myristic acid, 4-14% palmitic acid, and 5-15% stearic acid. Another useful class of amphiphilic carriers is partially esterified sorbitan and / or sorbitol with saturated or monounsaturated fatty acids (SPAN series) or their corresponding ethoxylated analogs (TWEEN series).

[0139] Commercially available amphiphilic carriers such as the Gelucire series, Labrafil, Labrasol, or Lauroglycol (all manufactured and sold by Gattefosse Corporation in Saint-Prieste, France), PEG-monooleate, PEG-dioleate, PEG-monolaurate and dilaurate, lecithin, and polysorbate 80 (manufactured and sold by several companies worldwide, including in the United States) are particularly noteworthy.

[0140] The immunostimulatory oligonucleotide double chains described herein can be formulated using hydrophilic polymers. These hydrophilic polymers are water-soluble and can be covalently attached to vesicle-forming lipids, and they are accepted in vivo without toxic effects (i.e., biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic acid (also known as polylactide), polyglycolic acid (also known as polyglycolide), polylactic acid-polyglycolic acid copolymer, and polyvinyl alcohol. Other potentially suitable hydrophilic polymers include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatized cellulose such as hydroxymethylcellulose or hydroxyethylcellulose.

[0141] In certain embodiments, the pharmaceutical compositions described herein include biocompatible polymers selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic acids, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and their copolymers, cellulose, polypropylene, polyethylene, polystyrene, polymers of lactic and glycolic acid, polyanhydrides, poly(ortho)esters, polybutyric acid (poly(butic acid)), polyvaleric acid, poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates, and formulations, mixtures, or copolymers thereof.

[0142] In certain embodiments, the pharmaceutical compositions described herein are formulated as liposomes. Liposomes can be prepared by any of the various techniques known in the art. See, for example, U.S. Patent No. 4,235,871, PCT Publication WO96 / 14057, New RRC, "Liposomes: A practical approach" (IRL Press, Oxford (1990), pp. 33–104), and Lasic DD, "Liposomes from physics to applications" (Elsevier Science Publishers BV, Amsterdam, 1993).

[0143] In any of the embodiments described herein, the immunostimulatory oligonucleotide duplex described herein can be conjugated to an antigen or its antigenic fragment and formulated as a vaccine composition. The therapeutic formulation of the immunostimulatory oligonucleotide duplex described herein can be prepared for storage in the form of a lyophilized formulation or aqueous solution by mixing the immunostimulatory oligonucleotide duplex of desired purity with a pharmaceutically acceptable carrier, excipient or stabilizer of any choice (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, e.g., ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkylparabens, e.g., methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues) polypeptides. These include: proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., poly(vinylpyrrolidone); amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, e.g., glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0144] Vaccines or other pharmaceutical compositions comprising the immunostimulatory oligonucleotide double-chain compositions described herein may contain a pharmaceutically acceptable salt, typically sodium chloride, preferably at a near-physiological concentration. Formulations of vaccines or other pharmaceutical compositions described herein may contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration is in the range of 0.1 to 2.0% (typically v / v). Suitable preservatives include those known in the pharmaceutical field. Examples of preservatives are benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben. Formulations of vaccines or other pharmaceutical compositions described herein may contain a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.

[0145] The therapeutic pharmaceutical compositions described herein may also contain two or more active compounds, preferably those having complementary activities that do not adversely affect each other, as required for the specific indication being treated.

[0146] In some embodiments in which a double-chain compound is formulated for use in or in conjunction with a vaccine, the vaccine composition is formulated using the double-chain compound as an adjuvant. In other embodiments, the vaccine composition can be formulated using an immunostimulatory oligonucleotide double-chain compound and an additional adjuvant, for example, one known in the art.

[0147] As used herein in relation to immunization, immune response, and vaccination, the term “adjuvant” refers to any substance that, when used in combination with a specific antigen, produces a more robust immune response than the antigen alone. When incorporated into a vaccine formulation, adjuvants generally act to accelerate, prolong, or enhance the quality of the specific immune response to the vaccine antigen.

[0148] Adjuvants typically enhance antigen-specific immune responses by promoting the accumulation and / or activation of accessory cells or accessory factors, thereby enhancing the efficacy of vaccines, i.e., the efficacy of antigen-containing compositions or antigen-coding compositions used to induce protective immunity against an antigen.

[0149] Adjuvants generally include those that produce a depot effect, immunostimulant adjuvants, and adjuvants that produce a depot effect and stimulate the immune system. Adjuvants that produce a depot effect prolong the exposure of immune cells to an antigen by slowly releasing the antigen in the body. Adjuvants in this class include, but are not limited to, alum (e.g., aluminum hydroxide, aluminum phosphate); emulsion formulations, such as mineral oil, non-mineral oil, water-in-oil or oil-in-water-in-oil emulsions; oil-in-water emulsions, such as the Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); MF-59 (squalene-type emulsion stabilized at span 85 and tween 80; Chiron Corporation, Emeryville, California); and PROVAX® (oil-in-water emulsion containing a stabilized detergent and micelle-forming agent; IDEC Pharmaceuticals Corporation, San Diego, California).

[0150] Immunostimulant adjuvants are adjuvants that cause activation of cells in the immune system. For example, they cause immune cells to produce and secrete cytokines and interferons. Adjuvants in this class include, but are not limited to, saponins purified from the bark of the soapberry tree (Q. saponaria tree), such as QS21 (a glycolipid that elutes as the 21st peak in HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Massachusetts); poly[di(carboxylatofenoxy)phosphazene] (PCPP polymer; Virus Research Institute, USA); lipopolysaccharide derivatives, such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Montana), muramyl dipeptide (MDP; Ribi), and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide analogue of lipid A; OM Pharma SA, Meyran, Switzerland); and leishmania elongation factor (purified leishmania genus protein; Corixa Corporation, Seattle, Washington). This class of adjuvants also includes CpG DNA.

[0151] Adjuvants that produce a depot effect and stimulate the immune system are compounds that possess both of the functions described above. Adjuvants in this class include ISCOM (an immunostimulatory complex containing mixed saponins and lipids that forms virus-sized particles with pores capable of holding antigens; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham Adjuvant System #2, an oil-in-water emulsion containing MPL and QS21; SmithKline Beecham Biologicals [SBB], Lixensar, Belgium); SB-AS4 (SmithKline Beecham Adjuvant System #4, containing alum and MPL; SBB, Belgium); nonionic block copolymers that form micelles, such as CRL1005 (these contain linear chains of hydrophobic polyoxypropylene sandwiched between polyoxyethylenes; Vaxcel, Inc., Norcross, Georgia); and Syntex Adjuvant Formulation (an oil-in-water emulsion containing SAF, Tween 80, and a nonionic block copolymer; Syntex Examples include Chemicals, Inc. (Boulder, Colorado), but the list is not limited to these.

[0152] The active ingredients of the pharmaceutical compositions described herein may be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose microcapsules, gelatin microcapsules, and poly-(methyl methacrylate) microcapsules, respectively; or in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules); or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

[0153] In some embodiments, sustained-release preparations can be used. A preferred example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing the antigen or fragment thereof as described herein, wherein the matrix is ​​in the form of a film or microcapsule or other manufactured product. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), L-glutamic acid and ethyl-L-glutamic acid copolymers, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT (trademark) (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release over more than 100 days, while certain hydrogels release proteins over a shorter period. Once encapsulated, antigens or their fragments remain in the body for extended periods and, as a result of exposure to moisture at 37°C, may denature or aggregate, leading to loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, reasonable strategies can be devised for stabilization. For example, if the aggregation mechanism is found to be intermolecular SS bond formation via thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling moisture content, using appropriate additives, and developing special polymer matrix compositions.

[0154] immunostimulatory activity The immunostimulatory oligonucleotide double-strands, pharmaceutical compositions, and vaccine compositions described herein can be administered to subjects requiring immunostimulation, particularly subjects requiring or likely to benefit from the induction of interferon production. In various embodiments, interferon-inducing activity may have a therapeutic effect alone, in combination with one or more antiinfective agents (e.g., antiviral, antibacterial, antifungal, or antiparasitic agents), one or more anticancer agents, or one or more autoimmune disease treatments.

[0155] Immunostimulatory activity can be determined, for example, by detecting and measuring the levels of cytokine and interferon production in a biological sample (e.g., serum).

[0156] Methods for detecting, measuring, and determining IFN levels in biological samples are known in the art. IFN polypeptide levels can be detected, for example, by immunoassays. ThermoFisher Scientific sells an ELISA-based kit for measuring human interferon-gamma levels—see catalog no. 29-8319-65. IFN gene expression can also be detected. Methods for measuring gene expression are known in the art, such as PCR, microarrays, and immunodetection methods such as Western blotting and immunocytochemistry. For example, quantitative reverse transcription polymerase chain reaction (qPCR) analysis can be performed using commercially available kits and arrays from, for example, Applied Biosystems®—see Applied Biosystems® TaqMan® Array Human Interferon Pathway, catalog no. 4414154. See also de Veer MJ et al. Functional classification of interferon-stimulated genes identified using microarrays. J Leukoc Biol. (2001) 69:912-20. These references are incorporated herein by reference in their entirety.

[0157] Antibodies specific to a group of interferon polypeptides (e.g., IFN-γ) are known in the art and are commercially available, for example from Abcam®, and can be used in immunohistochemistry, immunofluorescence, and Western blotting.

[0158] Interferon levels and activity can also be determined using reporter assays or bioassays. For example, a reporter assay for detecting biologically active type I interferon, which monitors the activation of the ISGF3 pathway, is available from InvovGen®. See, for example, Rees et al. J Immunol Methods, (2018).

[0159] Viral infection assays can also be used to determine the effect of immunostimulatory oligonucleotide duplexes on viral defense. For example, IFN activity can be measured by the level of protection of cell lines from post-viral cell death compared to a suitable control. See, for example, Barber et al. Host defense, viruses and apoptosis. Cell Death Differ 8, 113-126, doi:10.1038 / sj.cdd.4400823 (2001), and Liu, S. et al. Science 347, (2015). These publications are incorporated herein by reference in their entirety.

[0160] In addition, relevant animal models and human in vitro engineered platforms can also be used to directly or indirectly detect interferon production. Any model known in the art can be used. See, for example, Si, L. et al. Human organs-on-chips as tools for repurposing approved drugs as potential influenza and COVID19 therapeutics in viral pandemics. bioRxiv, doi:10.1101 / 2020.04.13.039917 (2020) and Van den Broek MF, Muller U, Huang S, Zinkernagel RM, Aguet M. Immune defence in mice lacking type I and / or type II interferon receptors. Immunol Rev. (1995).

[0161] Providing protection from relevant pathogens involves stimulating the immune system to elicit a more effective immune response than if the subject were naive to the antigen when subsequently exposed to the microorganism, its antigen, or antigenic fragment (e.g., the antigen on or within a live pathogen). Protection may include faster clearance of the pathogen, reduced severity and / or duration of symptoms, and / or absence of disease or symptom onset. Compared to an equivalent untreated control, such reductions are at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 99% or greater, as measured by any standard technique.

[0162] Treatment method The immunostimulatory oligonucleotides described herein can be used to inhibit influenza infection, as well as to treat a wide range of viral, bacterial, fungal, and parasitic pathogen infections, and IFN-related diseases, including cancer and autoimmune diseases.

[0163] A disease or medical condition is considered interferon-related if it is treated by the administration of interferon or the induction of interferon production. In some diseases or disorders, interferon induction is involved as part of the healing or recovery process, while in other diseases or conditions, the pathology is characterized by a deficiency, low production, or absence of interferon, such as IFN, type I IFN, IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω, type II IFN (IFN-γ), and type III IFN.

[0164] This specification describes a method for treating an infection in a subject requiring treatment for infection, the method comprising administering to the subject an immunostimulant oligonucleotide double chain described herein.

[0165] In this aspect or in any aspect, the oligonucleotide duplex is sufficient to induce interferon (IFN) production in cells that come into contact with the duplex. In another aspect, the step of administering the oligonucleotide duplex to a subject in need is sufficient to increase the level or activity of IFN. In yet another aspect, the step of administering the oligonucleotide duplex to a subject in need is sufficient to increase the immune response in the subject. In yet another aspect, the immune response is an antiviral response.

[0166] The immunostimulatory oligonucleotide double-stranded compounds described herein can be used to treat microbial infections. Examples of microorganisms that can cause microbial infections include, but are not limited to, viruses, bacteria, fungi, and parasites.

[0167] In another aspect, microbial infections are chronic. In another aspect, microbial infections are acute. Acute infections are short-term infections lasting less than two weeks, while chronic infections are long-term, lasting longer than two weeks. The methods used to treat acute infections may be the same as those used to treat chronic infections. Conversely, different methods may be used to treat acute and chronic infections.

[0168] In some aspects of the aforementioned aspects, the microbial infection is a systemic infection. As used herein, "systemic infection" refers to an infection that has spread throughout the body, such as an infection present in the bloodstream. Examples of non-systemic infections include bacterial sepsis and endotoxic shock.

[0169] In some embodiments, microbial infections are caused by bacteria. Examples of bacterial infections that can be treated or prevented by administering the immunostimulatory oligonucleotide double-strands described herein include: Aeromonas infection, African tick-bite fever, American tick-bite fever (Rickettsia parkeri infection), Arcanobacterium haemolyticum infection, bacterial hemangioma, Begel (endemic syphilis), blastomycosis-like pyoderma (proliferative pyoderma), bullous pedipalitis, botryomycosis, Briiii-Zinsser disease, brucellosis (Bang disease, Malta fever, undulating fever), bubonic plague, bullous impetigo, cat-scratch disease (cat-scratch fever), English-Wear infection (English-Wear disease) lymphocytic reticuloendotheliosis, subacute regional lymphadenitis), cellulitis, chancroid, soft chancre, ulcus molle, chlamydial infection, chronic lymphangitis, chronic habitual erysipelas, chronic burrowing ulcer (Melenii gangrene), chromobacteriosis warts flatus, cutaneous actinomycosis, cutaneous anthrax infection, cutaneous C. diphtheriae infection (Burke's sore, diphtheric desert sore, septic sore, prairie ulcer), cutaneous group B streptococcal infection, cutaneous Pasteurella haemolytica hemo / ytica) infections, cutaneous Streptococcus iniae (Streptococcus iniae) Iniae infection, gangrenous dermatitis (skin gangrene), pustules, gangrenous pustules, Ehrlichiosis ewingii infection, elephantiasis (elephantiasis nostras), endemic typhus (murine typhus), typhus (epidemic lice-borne typhus), erysipelas (erysipelas, ignis sacer, Saint Anthony's disease)fire), erysipeloid of Rosenbach, erythema annulare, erythrosclerotic erythrorhizon, otitis externa (external otitis, swimmer's ear), paronychia, flea-borne spotted fever, Flinders Island spotted fever, flying squirrel typhus typhus), folliculitis, Fournier's gangrene (penile or scrotal gangrene), furuncle (furuncle), gas gangrene (Clostridium myonecrosis, myonecrosis), glanders (Equinia, farcy, malleus), gonococcal sepsis (arthritis-cutaneous syndrome, disseminated gonococcal infection), gonorrhea, gram-negative folliculitis, gram-negative interdigital infection, inguinal granuloma (Donovan's disease, genital inguinal granuloma, tropical inguinal granuloma, sexually transmitted granuloma, sexually transmitted inguinal granuloma, lupus-like inguinal ulcer formation, serpentine ulcer formation of the inguinal region, ulcerative granuloma of the vulva, ulcerative sclerosing granuloma), green nail syndrome, JK group Corynebacterium sepsis, Haemophilus influenzae (Haemophi / us) Influenzae cellulitis, Helicobacter cellulitis, hospital furuncle, hot bath folliculitis (Pseudomonas aeruginosa folliculitis), human granulocytotropic anaplasmosis, human monocytotropic ehrlichiosis, impetigo, Japanese spotted fever, leptospirosis (Fort Bragg fever, anterior tibial fever, Weil's disease), listeriosis, Ludwigiangina, lupus-like folliculosis, Lyme disease (Afzelius disease, Lyme borreliosis), lymphogranulomatosis of the inguinal region (climatic stump, Duran-Nicholas Fabre disease, lymphogranulomatosis of the inguinal region, poradenitis inguinale), strumous stump bubo), malakoplakia, Mediterranean spotted fever (button fever), meridianus (Whitmore's disease), meningococcal bacteremia, Missouri Lyme disease, mycoplasma infection, necrotizing fasciitis (flesh-eating bacteria syndrome), neonatal toxic shock syndrome-like exanthema, nocardiosis, neonatal edema (noma).Neonatorum, North Asian tick-borne typhus, neonatal ophthalmitis, Oroya fever (Kallion's disease), Pasteurellosis, perianal cellulitis (perineal dermatitis, streptococcal perianal disease), periapical abscess, pinta, atrophic keratolysis (fissured plantar exfoliative dermatitis, fissured plantar keratoma, ringed keratolysis), plague, primary gonococcal dermatitis, Pseudomonas pyoderma, Pseudomonas hot foot syndrome, suppurative paronychia, suppurative myositis, Q fever, Queensland tick-borne typhus, rat-bite fever, recurrent toxin-borne perineal erythema, rhinitis, Rickettsia aescrimannii Aeschlimannii infection, rickettsial pox, Rocky Mountain spotted fever, xiphoid tibia (anterior tibial varus), saddle nose, salmonellosis, scarlet fever, scrub typhus (tsutsugamushi fever), bacterial dysentery, staphylococcal scalded skin syndrome (neonatal pemphigus, Ritter's disease), streptococcal intertrigo, superficial pustular folliculitis (Bockhardt's impetigo, superficial folliculitis), plaque (barber's pruritus, beard folliculitis), syphilitic rash, syphilis (syphilis, lues), tick-borne lymphadenopathy, toxic shock syndrome (streptococcal toxic shock syndrome, streptococcal toxic shock-like syndrome, toxic streptococcal syndrome), trench fever (five-day fever, quintan fever, urban trench fever) Fever, tropical ulcers (Aden's ulcer, tropical skin disease, Malabar ulcer, tropical gangrene), tularemia (deer fly fever, Ohara disease, Pervant Valley plague, rabbit fever), Peruvian wart, Vibrio vulnificus (VibrioVulnificus infection, strawberry tumor (Bouba, Franbezia, Palangi, Pyang), aquarium granuloma (water tank granuloma, swimming pool granuloma), borderline leprotic leprosy, borderline leprosy, borderline tuberculosis, leprosy, Buruli ulcer (Bairnsdale ulcer, Searl ulcer), indurated erythema (Bazan's disease), histiocytic leprosy, leprotic leprosy, leprosy (leprosy), lichenoid lichen (lichenoid cutaneous tuberculosis), lupus vulgaris (lupus tuberculosis), miliary tuberculosis (disseminated tuberculosis, systemic acute cutaneous tuberculosis, disseminated cutaneous tuberculosis), Mycobacterium avium intracellulare Examples of bacterial infections include, but are not limited to, avium-intracel / ulare complex infections, Mycobacterium haemophi / um infections, Mycobacterium kansasii infections, papular gangrenous tuberculosis, primary inoculated tuberculosis (primary cutaneous changes, primary tuberculous changes, tuberculous chancroid), rapidly developing Mycobacterium infections, cutaneous glandular diseases (softening cutaneous tuberculosis), orifice cutaneous tuberculosis (acute ulcerative tuberculosis, orifice tuberculosis), cutaneous verrucous tuberculosis (lupus wartoids, anatomist's nodules, wartoid cutaneous tuberculosis), tuberculous cellulitis, tuberculous granuloma (metastatic tuberculous abscess, metastatic tuberculous ulcer), tuberculous leprosy, and bacterial sexually transmitted infections. Examples of sexually transmitted infections, including microbial infections, that are not limited to chancroid include chlamydia, chlamydia infection, gonorrhea, lymphogranuloma ventricle, Mycoplasma genitalium, nongonococcal urethritis, pelvic inflammatory disease, syphilis, vaginitis, bacterial vaginosis, yeast vaginitis, and yeast infections.

[0170] In another form, microbial infections are fungal infections. Examples of infectious fungi that cause fungal infections for which the combination therapeutic compositions and methods described herein are intended include, but are not limited to, species of the genera Candida; Cryptococcus; Aspergillus; Microsporum; Trichophyton; Epidermophyton; Trichosporon; Tinea versicolor; Tinea corporis; Tinea cruris; Tinea manuum; Tinea pedis; Nail fungus; Tinea facialis; Tinea imbricate; Tinea incognito; Epidermophyton floccosum; Microsporum canis; Microsporum auzenis audouinii); Trichophyton interdigitale; Trichophyton mentagrophytes; Trichophyton tonsurans; Trichophyton schoenleini; Trichophyton rubrum; Hortaea werneckii; Piedraia hortae; Malasserzia furfur; Coccidioides immitis; Coccidioides posadasii; Histoplasma capsulatum; Histoplasma duboisii duboisii); Lacazia loboi; Paracoccidioides brasiliensis; Blastomyces dermatitidis;Sporothrix schenckii; Penicillium marneffei; Candida albicans; Candida glabrata; Candida tropicalis; Candida lusitaniae; Candida jirovecii; Exophiala jeanselmei; Fonsecaea pedrosoi; Fonsecasea compacta; Phialophora verrucosa; Geotrichum candidum; Pseudallescheria boidi Examples include, but are not limited to, *boydii*, *Rhizopus oryzae*, *Muco indicus*, *Absidia corymbifera*, *Synceplasastrum racemosum*, *Basidiobolus ranarum*, *Conidiobolus coronatus*, *Conidiobolus incongruous*, *Cryptococcus neoformans*, *Enterocytozoan bieneusi*, *Encephalitozoon intestinalis*, and *Rhinosporidium seeberi*.

[0171] Examples of disorders / diseases caused by fungal infections or toxins produced during fungal infections, to which the compositions and methods described herein can be applied in various aspects and manner, include, but are not limited to, infections of superficial wounds or burns; infections of mucous membranes; respiratory infections; infections of the eyes, ears, nose or throat; or infections of enteric pathogens. In another aspect, the fungal infection is an infection of soft tissue or skin, such as superficial mycosis; cutaneous mycosis; subcutaneous mycosis; vaginal mycosis; systemic mycosis, or an infected wound or burn.

[0172] Other medically relevant microorganisms are widely described in the literature. For example, see CGA Thomas, *Medical Microbiology*, Bailliere Tindall, UK, 1983, the contents of which are incorporated herein by reference in their entirety. Each of the items in the above list is illustrative and not intended to be limiting.

[0173] Virus infection The immunostimulant oligonucleotide double-stranded compounds described herein can be used to treat viral infections.

[0174] In one aspect, this specification describes a method for inducing an antiviral response in a subject, comprising the step of administering an immunostimulatory oligonucleotide double chain described herein to the subject.

[0175] In another aspect, this specification describes methods for treating viral infections in subjects.

[0176] In some embodiments, viral infection is an infection of tissues selected from the group consisting of central nervous system tissues, eye tissues, upper respiratory system tissues, lower respiratory system tissues, lung tissues, kidney tissues, bladder tissues, spleen tissues, heart tissues, gastrointestinal tissues, epidermal tissues, reproductive tissues, nasal cavity tissues, laryngeal tissues, tracheal tissues, bronchial tissues, oral tissues, blood tissues, and muscle tissues.

[0177] Examples of non-viral infections include respiratory infections of the nose, throat, upper respiratory tract and lungs, e.g., influenza, pneumonia, coronavirus, SARS, COVID-19, bronchiolitis, and laryngotracheobronchitis; gastrointestinal infections, e.g., gastroenteritis, rotavirus, norovirus; liver infections, e.g., hepatitis; nervous system infections, e.g., rabies, West Nile virus, encephalitis, meningitis, and polio; skin infections, e.g., warts, plaques, and chickenpox; placental and fetal viral infections, e.g., Zika virus, rubella virus, and cytomegalovirus; enterovirus, coxsackievirus; echovirus, chikungunya virus, Crimean-Congo hemorrhagic fever virus, Japanese encephalitis virus, Rift Valley fever virus, Ross River virus, jumping disease virus, John Cunningham virus, measles virus, lymphocytic choriomeningitis virus, arbovirus, rhinovirus, Examples include parainfluenza virus, respiratory syncytial virus, herpes simplex virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus 6, adenovirus, cytomegalovirus, Epstein-Barr virus, mumps virus, influenza A virus, influenza B virus, coronavirus, SARS coronavirus, SARS-CoV-2 virus, coxsackievirus type A, coxsackievirus type B, poliovirus, HTLV-1, hepatitis viruses type A, B, C, D, and E, varicella-zoster virus, smallpox virus, molluscum contagiosum, human papillomavirus, parvovirus B19, rubella virus, human immunodeficiency virus, rotavirus, norovirus, astrovirus, Ebola virus, Marburg virus, dengue virus (DENV), and Zika virus.

[0178] Risk factors for having or developing a viral infection include exposure to the virus, exposure to or contact with a person infected with the virus, exposure to a contaminated surface that has come into contact with the virus, contact with a biological sample or bodily fluid from a person infected with the virus, sexual intercourse with a person infected with the virus, sharing of needles, blood transfusions, drug use, and any other risk factors known in the art that transmit the virus from one person to another. The assessment of a person's risk factors can be performed, for example, by a skilled clinician or by the person themselves.

[0179] Identification of microbial infections In one embodiment, a subject is diagnosed with a microbial infection prior to administration of the immunostimulatory oligonucleotide double-stranded compound described herein. In another embodiment, the method includes a step of diagnosing a subject with a viral infection. In yet another embodiment, the method includes a step of receiving the results of an assay that diagnoses a subject with or at risk of having a viral infection prior to the administration step.

[0180] Various laboratory tests performed to establish or confirm the diagnosis of microbial infections and to identify the causative microorganisms are known to those skilled in the art. Common viral infections, such as measles, rubella, and chickenpox, can be diagnosed based on symptoms. Symptoms associated with viral infections vary depending on the type of virus. For example, in the case of upper respiratory tract viral infections, symptoms include, but are not limited to, cough, shortness of breath, fever, and fatigue.

[0181] In the case of infections occurring during epidemics (e.g., COVID-19 and influenza), the presence of other similar cases can help physicians identify a specific infection. Diagnostic testing is important to distinguish between different viruses that cause similar symptoms, such as COVID-19 (SARS-CoV-2) and influenza.

[0182] Culture of microbial species accompanied by antimicrobial susceptibility testing is considered the gold standard clinical test for some microorganisms. Skin or mucous membrane samples can be collected by: 1) dry, sterile cotton-tipped swabs scrubbed from the site of infection; 2) moist swabs taken from mucous membrane surfaces, e.g., inside the mouth; 3) aspirating bodily fluids / pus from skin lesions using a needle and syringe; and 4) skin biopsy, i.e., small samples of skin removed under local anesthesia. Culture of bacteria and other microorganisms is most commonly performed by brushing skin swabs onto sheep blood agar plates and exposing them to different conditions. The species of microorganisms that grow depend on the culture medium used to cultivate the specimen, the incubation temperature, and the amount of oxygen available. For example, obligate aerobic bacteria can only grow in the presence of oxygen, while obligate anaerobic bacteria cannot grow at all in the presence of oxygen.

[0183] Blood tests require a blood sample accessed via a needle through a vein. Examples of tests that are not limited to microbial infections include: 1) complete blood count, in infections the white blood cell count is often elevated with an increase in neutrophils (neutropenia); 2) C-reactive protein (CRP), in severe infections CRP is often elevated to >50; 3) procalcitonin, a marker of systemic sepsis due to bacterial infection; 4) serology, a 10-day interval test to determine the immune response to specific microorganisms; 5) rapid plasma reagin (RPR) test, if syphilis is suspected; and 6) blood culture to detect if there is a high fever >100.4°F. Blood tests can be performed to identify antibodies produced in the presence of a microbial infection.

[0184] Polymerase chain reaction (PCR) isolates and amplifies fragments of microbial DNA from skin, blood, or other samples. The species is identified by comparing the DNA of the sample with DNA from known organisms.

[0185] Treatment of microbial infections Several drug therapies have been developed to treat infections (e.g., bacterial or viral infections). Treatment of infections may include, for example, antibiotic and antiviral drug therapies administered after infection.

[0186] The term “therapeutic substance” is recognized in the art and refers to any chemical part that is biologically, physiologically, or pharmacologically active in a subject, either locally or systemically. Examples of therapeutic substances, also called “drugs,” are listed in well-known references such as the Merck Index, Physicians' Desk Reference, and The Pharmacological Basis of Therapeutic, and include, but are not limited to, pharmacotherapy; vitamins; mineral supplements; substances used to treat, prevent, diagnose, cure or alleviate diseases or illnesses; substances that affect the structure or function of the body; or prodrugs that become biologically active or have increased biological activity after being placed in a physiological environment. Various forms of therapeutic substances may be used that can be released from the composition of the present invention into adjacent tissues or bodily fluids when administered to a subject.

[0187] Exemplary therapeutic agents and vaccines for the prevention and treatment of infections include penicillin, ceftriaxone, azithromycin, amoxicillin, doxycycline, cephalexin, ciprofloxacin, clindamycin, metronidazole, azithromycin, sulfamethoxazole, trimethoprim, meningococcal polysaccharide vaccine, tetanus toxoid, cholera vaccine, typhoid vaccine, pneumococcal 7-valent vaccine, pneumococcal 13-valent vaccine, pneumococcal 23-valent vaccine, Haemophilus b conjugate, anthrax vaccine, immunovir, indinavir, inosine, lopinavir, lovaride, maravirox, and nebi Examples include, but are not limited to, lapine, nucleoside analogs, oseltamivir, penciclovir, rimantidine, pyrimidine, saquinavir, stabudine, tenofovir, trizivir, tromantadine, trunkada, valacyclovir, ciramidine, zanamivir, zidovudine, MMR vaccine, DTaP vaccine, hepatitis vaccine, Hib vaccine, HPV vaccine, influenza vaccine, polio vaccine, rotavirus vaccine, shingles vaccine, Tdap vaccine, tetanus vaccine, fluconazole, ketoconazole, amphotericin B, and sulfadoxine / pyrimethamine. Further non-limiting examples include abacavir, acyclovir (Acyclovir, Aciclovir), adefovir, amantadine, ampligen, amprenavir (Agenerase), amodiaquin, apirimod, arbidol, atazanavir, atripra, paravir, baloxavir marboxil (Xofluza®), biktarvy, boceprevir (Victrelis®), cidofovir, clofazimine, clomiphene, and coviphene. Cystat (Tybost®), Combivir (fixed-dose drug), Daclatasvir (Daklinza®), Darunavir, Delavirdine, Descovy, Didanosine, Docosanol, Dolutegravir, Doravirine (Pifeltro®), Ecoliebel, Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine (Intelence®), Famciclovir,Favipiravir, fenofibrate, homivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir (Cytovene®), ivacitabine, ibalizumab (Trogarzo®), idoxuridine, imiquimod, immunovir, indinavir, inosine, integrase inhibitors, type I interferon, type II interferon, type III interferon, interferon Ron, Lamivudine, Letermovir (Prevymis®), Lopinavir, Loviride, Mannose-binding lectin, Maraviroc, Methisazone, Moroxidine, Nafamostat, Nelfinavir, Nevirapine, Nexavir®, Nilotinib, Nitazoxanide, Norvir, Nucleoside analog, Oseltamivir (Tamiflu®), Pazopanib, Peginterferon alpha-2a, Peginterferon Ron-alpha-2b, penciclovir, peramivir (Rapivab®), preconalil, podophyllotoxin, protease inhibitors (pharmacology), pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant®), rimantadine, ritonavir, saquinavir, simeprevir (Olysio®), sofosbuvir, stabuzin, synergistic enhancers (anti-retardyl This includes antiviral drugs such as telaprevir, terbivudine (Tyzeka®), tenofovir alafenamide, tenofovir disoproxil, tenofovir, toremifene, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir (Valtrex), valganciclovir, bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza®), and zidovudine.

[0188] In any of the embodiments described herein, the immunostimulatory oligonucleotide double-strand described herein is used as a monotherapy.

[0189] In any other aspect of the foregoing, the compositions described herein can be used in combination with known compositions and therapies for interferon-mediated diseases (e.g., autoimmune diseases, infections, or cancer). The immunostimulatory oligonucleotide double-stranded compositions described herein can be mixed with, for example, antiviral therapeutic agents or administered as a therapeutic regimen for the treatment of interferon-mediated diseases.

[0190] As used herein, “administered in combination” means that two (or more) different treatments are delivered to the subject during the subject’s treatment for an illness, for example, that two or more treatments are delivered after the subject is diagnosed with the disorder (respiratory disease) until the disorder is cured or eliminated or the treatment is discontinued for other reasons. Non-limiting examples of treatments that can be used in combination with the compositions provided herein include abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir (agenese), amodiaquin, apirimod, arbidol, atazanavir, atripra, atovaquone, paravir, baloxavir marboxil (Xofluza®), viktarvy, boceprevir (Victrel is(registered trademark)), cidofovir, clofazimine, clomiphene, clofazamine, cobicistat (Tybost(registered trademark)), combivir (fixed dose drug), daclatasvir (Daklinza(registered trademark)), darunavir, delavirdin, descovy, didanosine, docosanol, dolutegravir, doravirine (Pifeltro(registered trademark)), ecoliebel, edoxudine, efavirenz, elvitegravir, emtricitabine, enfvirtide, e Ntecavir, etravirine (Intelence®), famciclovir, favipiravir, fenofibrate, homivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir (Cytovene®), ivacitabine, ibalizumab (Trogarzo®), idoxuridine, imiquimod, immunovir, indinavir, inosine, integrase inhibitors, type I interferon, type II interferon Taferon, type III interferon, interferon, ivermectin, lamivudine, rasaloside, letermovir (Prevymis®), lopinavir, roviride, mannose-binding lectin, maraviroc, methisazone, moloxidine, nafamostat, nelfinavir, nevirapine, Nexavir®, nilotinib, nitazoxanide, Norvir, nucleoside analog, oseltamivir (Tamiflu®), pazopanib,Peginterferon alfa-2a, peginterferon alfa-2b, penciclovir, peramivir (Rapivab®), preconalil, podophyllotoxin, protease inhibitors (pharmacology), pionarizine, pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant®), rimantadine, ritonavir, saquinavir, simeprevir (Olysio®), sofosbuvir, stabuzin, synergistic enhancers This includes antiretroviral drugs such as tafenoquin, telaprevir, terbivudine (Tyzeka®), tenofovir alafenamide, tenofovir disoproxil, tenofovir, toremifene, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir (Valtrex), valganciclovir, bermurafenib, venetoclax, bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza®), and zidovudine.

[0191] In some embodiments, the immunostimulatory oligonucleotide double-strand and the at least one antiviral drug are administered substantially simultaneously.

[0192] In some embodiments, the at least one antiviral drug is administered at different time points.

[0193] In some embodiments, the delivery of one treatment is still ongoing when the delivery of the second treatment begins, resulting in some overlap in the duration of administration. This may also be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment is completed before the delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective due to concomitant administration. For example, the second treatment is more effective, and an equivalent effect may be observed with fewer doses of the second treatment, or the second treatment reduces symptoms more strongly than would be observed if the second treatment were administered without the first treatment, or a similar situation may be observed with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters related to the impairment is greater than would be observed if one treatment were administered in the absence of the other treatment. The effects of the two treatments are partially additive, fully additive, or exceed the additive effect. Delivery may be carried out in such a manner that the effect of the delivered first treatment is still detectable when the second treatment is delivered. The compositions described herein and the at least one additional treatment may be administered simultaneously in the same composition or in separate compositions, or sequentially. In the case of sequential administration, the compositions described herein may be administered first and the additional compositions second, or the order of administration may be reversed. The compositions and / or other therapeutic compositions, treatment procedures or therapeutic modalities may be administered during periods when the disorder is active, or during periods of remission or when the disease is less active. The compositions may be administered before, concurrently with, or after another treatment, or during remission of the disorder.

[0194] When administered in combination, the composition and additional active substances or compositions (e.g., a second or third active substance), or all of them, may be administered in amounts or doses higher, lower, or the same as each individual active substance used individually, for example, as monotherapy. In certain embodiments, the amount or dosage administered of the active substance, additional active substances (e.g., a second or third active substance), or all of them, is lower than the amount or dosage of each individual active substance used individually (e.g., at least 20%, at least 30%, at least 40%, or at least 50%). In other embodiments, the amount or dosage of the active substance, additional active substances (e.g., a second or third active substance), or all of them, that produce the desired effect (e.g., treatment of a respiratory disease) is lower than the amount or dosage of each individual active substance required individually to achieve the same therapeutic effect (e.g., at least 20%, at least 30%, at least 40%, or at least 50%).

[0195] The vaccine compositions described herein can be used, for example, to protect or treat a subject against a disease. The terms “immunize” and “vaccinate” are often used interchangeably in the art. However, with respect to the administration of the vaccine compositions described herein to provide protection against a disease, such as an infectious disease caused by an antigen-expressing pathogen, the term “immunize” should be understood to refer to the passive protection conferred by the administered vaccine composition.

[0196] Dosage, dosage, and efficacy The immunostimulatory oligonucleotide double-chain, pharmaceutical composition, or vaccine composition described herein may be formulated, divided into appropriate doses, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific patient being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the vaccine composition, the method of administration, the scheduling of administration, and other factors known to healthcare professionals.

[0197] The therapeutic formulation used for in vivo administration, for example parenteral administration, in the methods described herein can be sterile, which can be readily achieved by filtration with a sterile filtration membrane or by other methods known to those skilled in the art.

[0198] The immunostimulatory oligonucleotide double chains and their compositions described herein can be administered to a subject in need by any suitable route that results in an effective treatment in that subject. The terms “administer” and “introduce” as used herein are interchangeable and refer to introducing a vaccine composition, antigen, or fragment thereof to a subject in a manner or by a route that causes such vaccine composition to be localized at least partially to a desired site, e.g., a site of infection, in order to produce the desired effect. The antigen or fragment thereof or vaccine composition can be administered to a subject by any mode of administration that delivers the vaccine composition systemically or to a desired surface or target, and such modes of administration may include, but are not limited to, injection, infusion, intravenous infusion, and inhalation. Oral administration forms are also possible if they can protect the antigen or fragment thereof or vaccine composition from inactivation in the intestines. "Injection" includes, but is not limited to, injections and infusions into veins, muscles, arteries, spinal cords, intraventricular spaces, capsules, orbits, hearts, skin, abdominal cavity, trachea, subcutaneous, subepidermal, joints, capsules, subarachnoid, spinal cord, cerebrospinal cord, and sternum.

[0199] As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical administration, usually by injection. As used herein, the terms “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” refer to the administration of a therapeutic agent other than direct administration to a target site, tissue, or organ, such as a tumor site, in which the therapeutic agent enters the target circulatory system and is thus subjected to metabolism or other processes. In another embodiment, an antibody or its antigen-binding fragment may be administered locally, for example by direct injection, if the site of injury or infection allows, and the injection may be repeated periodically.

[0200] In some embodiments, the compositions described herein are administered by aerosol administration, nebulizer administration, or tracheal lavage administration. In some embodiments, the compositions are formulated for intravenous, intramuscular, intraperitoneal, subcutaneous, or intrathecal administration.

[0201] As used herein, the term “effective dose” refers to the amount of an immunostimulatory oligonucleotide double-chain composition necessary to alleviate or prevent at least one symptom of an infection, disease, or disorder, and relates to the amount of a pharmacological composition sufficient to produce the desired effect, for example, by reducing the level of pathogenic microorganisms at the site of infection, or by reducing the disease state or any symptoms associated with or caused by the pathogenic microorganism. Therefore, the term “therapeutic effective dose” refers to the amount of the antigen or its fragment or vaccine composition described herein sufficient to achieve a specific effect when administered to a typical subject using the methods disclosed herein. The effective dose as used herein may also include amounts sufficient to delay the onset of disease symptoms, alter the course of disease symptoms (e.g., slow the progression of disease symptoms), or reverse disease symptoms. Therefore, it is not possible to specifically define an exact “effective dose.” However, for any given case, a person skilled in the art can determine an appropriate “effective dose” using only routine experiments.

[0202] The effective dose, toxicity, and therapeutic efficacy can be determined in cell cultures or experimental animals using standard pharmaceutical procedures for determining, for example, the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population). Dosage may vary depending on the dosage form used and the route of administration utilized. The dose-to-therapeutic ratio is the therapeutic factor, which can be expressed as the ratio LD50 / ED50. Compositions and methods exhibiting a large therapeutic factor are preferred. The therapeutically effective dose can first be estimated from a cell culture assay. Alternatively, doses can be formulated in animal models to obtain a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antigen or fragment that achieves 50% maximum inhibition of symptoms) determined in cell cultures or suitable animal models. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effect of any particular dosage can be monitored by appropriate bioassays. Physicians can determine the dosage and adjust it, as needed, to match the observed effect of the treatment.

[0203] The immunostimulatory oligonucleotide duplexes, pharmaceutical compositions, or vaccine compositions described herein can, in some embodiments, be formulated together with one or more additional therapeutic agents currently used, for example, to prevent or treat infection. The effective amount of such other active ingredients depends on the amount of immunostimulatory oligonucleotide duplexes in the formulation, the type of disorder or treatment, and other factors considered above. These are generally used in the same dosage and route of administration as previously used herein, or in about 1 to about 99% of the dosage used to date.

[0204] The dosage ranges of the immunostimulatory oligonucleotide double-stranded compounds, pharmaceutical compositions, or vaccine compositions described herein are potency-dependent and encompass an amount sufficient to produce the desired effect. The dosage should not be so high as to cause unacceptable adverse side effects. Generally, the dosage will vary depending on the patient's age, condition, and sex, and can be determined by those skilled in the art. Individual physicians may also adjust the dosage if there are any complications. In some embodiments, the dosage ranges from 0.001 mg / kg body weight to 100 mg / kg body weight. In some embodiments, the dose range is 5 μg / kg body weight to 100 μg / kg body weight. Alternatively, the dose range can be titrated to maintain serum levels between 1 μg / mL and 1000 μg / mL. For systemic administration, therapeutic doses such as 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or higher may be administered. These doses may be administered by single or multiple individual doses, or by continuous infusion. In the case of repeated administration over several days or longer, treatment continues, depending on the condition, until the infection is treated, for example, by the methods described above or by methods known in the art. However, other drug regimens may also be helpful.

[0205] The duration of treatment using the methods described herein will continue for as long as medically necessary or until the desired therapeutic effect (e.g., as described herein) is achieved. In certain embodiments, administration of the vaccine composition described herein may be continued for 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or for the lifespan of the subject.

[0206] As will be understood by those skilled in the art, a suitable administration regimen for a given composition may include a single dose / immunotherapy or multiple doses / immunotherapy. For example, to provide a sustained protective effect, subsequent doses may be administered repeatedly over a period of time, for example, about two weeks or more, up to the lifetime of the subject. Subsequent doses may be spaced apart after the initial immunization, for example, about two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year.

[0207] The precise dosage used in a formulation depends on the route of administration and should be determined based on the physician's judgment and the individual patient's condition. Ultimately, a practitioner or physician will determine the amount of immunostimulant oligonucleotide double chain or its composition to be administered to a specific target.

[0208] In these methods and in some aspects of all such methods described herein, an immunostimulatory oligonucleotide double chain or a composition thereof is administered in an effective amount to provide short-term protection from or to treat an infection. In some aspects, the infection is a viral infection. As used herein, “short-term protection” means protection from an infection, such as malaria, that lasts for at least about two weeks, at least about one month, at least about six weeks, at least about two months, at least about three months, at least about four months, at least about five months, at least about six months, at least about seven months, at least about eight months, at least about nine months, at least about ten months, at least about eleven months, or at least about twelve months. Such protection may involve repeated administration.

[0209] In these methods and in some embodiments of all such methods described herein, immunostimulatory oligonucleotide double chains or compositions thereof are administered in an effective amount to provide protection from infection or to alleviate symptoms of a persistent infection.

[0210] "Alleviating symptoms of persistent infection" means improving any condition or symptoms associated with persistent infection. Alternatively, alleviating symptoms of persistent infection may involve reducing the infectious microbial (e.g., viral, bacterial, fungal, or parasitic) load in a control compared to such load in an untreated control. Compared to an equivalent untreated control, the degree of such reduction or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, as measured by any standard technique. Preferably, the persistent infection is completely eliminated by detection by any standard method known in the art, at which point treatment of the persistent infection is considered complete.

[0211] Patients being treated for a persistent infection are those diagnosed by a physician as having such a condition. The diagnosis may be made by any appropriate means. Diagnosis and monitoring may involve, for example, detecting levels of microbial load in biological samples (e.g., tissue biopsy, blood test, or urine test), detecting levels of surrogate markers of microbial infection in biological samples, detecting symptoms associated with a persistent infection, or detecting immune cells involved in an immune response specific to a persistent infection (e.g., detection of antigen-specific T cells that are anerious and / or functionally impaired). Patients who have been prevented from developing a persistent infection may or may not have received such a diagnosis. It will be understood by those skilled in the art that these patients may have undergone the same standard tests as described above, or they may have been identified as high-risk without testing due to the presence of one or more risk factors (e.g., family history or exposure to infectious material).

[0212] Any specified patents or other publications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methods described in those publications that may be used in connection with this disclosure. These publications are provided solely because their disclosures were made prior to the filing date of this application. In this regard, nothing should be considered by the inventors as an admission that they are not entitled to precede such disclosures on the grounds of prior disclosure or for any other reason. Any statements regarding the dates of these documents or expressions regarding the contents of these documents are based on information available to the applicant but do not constitute an endorsement of the accuracy of the dates or contents of these documents.

[0213] This disclosure is not limited to, and therefore should be understood to be, a variety of, the specific methods, protocols, and reagents provided herein. The terminology used herein is for the sole purpose of describing specific embodiments and is not intended to limit the scope of this disclosure as defined solely by the claims of this application. The present invention is further illustrated by the following examples, but these examples should not be construed as further limitations.

[0214] This technology may be further described in the following numbered sections. 1. An immunostimulatory oligonucleotide double helix containing SEQ ID NO:1 at its 5' end. 2. An immunostimulatory oligonucleotide double helix of item 1, in which the oligonucleotide double helix is ​​RNA. 3. An immunostimulatory oligonucleotide bihedra of any of the preceding items, wherein the oligonucleotide bihedra contains a 5'-monophosphate group. 4. An immunostimulatory oligonucleotide duplex of any of the preceding items, wherein the oligonucleotide duplex has a length of at least 20 nucleic acid bases. 5. An immunostimulatory oligonucleotide double helix of any of the preceding items, in which the oligonucleotide double helix is ​​a double-stranded RNA. 6. An immunostimulatory oligonucleotide duplex of any of the preceding items, wherein the oligonucleotide duplex is sufficient to induce interferon (IFN) production in cells that come into contact with the duplex. 7. One of the methods described in the preceding section, wherein IFN production is type I IFN production. 8. An immunostimulatory oligonucleotide duplex that activates the RIG-I-IRF3 pathway, or one of the preceding items. 9. An immunostimulatory oligonucleotide duplex of any of the preceding items, wherein the oligonucleotide duplex reduces the viral titer or viral load in cells or populations of cells that come into contact with the duplex. 10. An immunostimulatory oligonucleotide duplex of any of the preceding items, wherein the oligonucleotide duplex increases STAT1 and STAT2 in the cell to which the duplex is in contact. 11. A method for inducing an antiviral response in a subject, comprising the step of administering an immunostimulatory oligonucleotide duplex of any of the preceding items to a subject requiring such response. 12. A method for treating a viral infection in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-strand of any of the preceding items to the subject in need of treatment. 13. The subject requiring the above-mentioned method is either infected with a virus or at risk of becoming infected with a virus. 14. Any of the preceding steps further include a step of diagnosing the subject whether or not they have a viral infection or are at risk of having a viral infection, prior to the step of administration. 15. Any method of the preceding item further includes, prior to the administration step, the step of receiving the results of an assay that diagnoses the subject having a viral infection or being at risk of having a viral infection. 16. Viral infections include John Cunningham virus, measles virus, lymphocytic choriomeningitis virus, arbovirus, rabies virus, rhinovirus, parainfluenza virus, respiratory syncytial virus, herpes simplex virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus 6, adenovirus, cytomegalovirus, Epstein-Barr virus, mumps virus, influenza A virus, influenza B virus, coronavirus, SARS coronavirus, SARS-CoV-2 virus, Any of the preceding methods caused by a virus selected from the group consisting of coxsackievirus type A, coxsackievirus type B, poliovirus, HTLV-1, hepatitis viruses type A, B, C, D, and E, varicella-zoster virus, smallpox virus, molluscum contagiosum, human papillomavirus, parvovirus B19, rubella virus, human immunodeficiency virus, rotavirus, norovirus, astrovirus, Ebola virus, Marburg virus, dengue virus (DENV), and Zika virus. 17. Any of the preceding methods, wherein the viral infection is an infection of tissue selected from the group consisting of central nervous system tissue, eye tissue, upper respiratory system tissue, lower respiratory system tissue, lung tissue, kidney tissue, bladder tissue, spleen tissue, heart tissue, gastrointestinal tissue, epidermal tissue, reproductive tissue, nasal cavity tissue, laryngeal tissue, tracheal tissue, bronchial tissue, oral tissue, blood tissue, and muscle tissue. 18. The aforementioned administration is systemic, or any of the methods described in the preceding items. 19. Any of the preceding methods wherein the administration is localized to the site of viral infection. 20. Any of the preceding methods, further comprising the step of administering at least one additional therapeutic agent. twenty one. One of the preceding methods, in which at least one additional treatment is an antiviral drug. twenty two. A method for treating influenza infection in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-strand of any of the preceding items to a subject having an influenza infection. twenty three. The influenza infection is either a type A influenza infection or a type B influenza infection, according to one of the preceding methods. twenty four. Any of the preceding methods, further comprising the step of administering at least one additional antiviral drug. twenty five. A method for treating coronavirus disease in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-strand of any of the preceding items to a subject having coronavirus disease. 26. The coronavirus disease is COVID-19, according to one of the preceding methods. 27. Any of the preceding methods, further comprising the step of administering at least one additional antiviral drug. 28. Any of the preceding methods, further comprising the step of administering plasma obtained from a subject who has recovered from coronavirus disease. 29. A method for increasing the efficacy of an antiviral drug, comprising the step of administering an immunostimulatory oligonucleotide double-strand of any of the preceding items and at least one antiviral drug. 30. Antiviral drugs include abacavir, acyclovir (Acyclovir, Aciclovir), adefovir, amantadine, ampligen, amprenavir (Agenerase), amodiaquin, apirimod, arbidol, atazanavir, atripra, atovaquone, paravir, baloxavir marboxil (Xofluza®), biktarvy, boceprevir (Victrelis®), cidofovir, clofazimine, clomiphene, clofazamine, cobicistat (Tybost®), Combivir (fixed-dose drug), daclatasvir (Daklinza®), darunavir, delavirdin, descovy, didanosine, docosanol, dolutegravir, doravirine (Pifeltro®), ecoliebel, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine (Intelence®), famciclovir, favipiravir, fenofibrate, homivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir (Cytovene®), Ibasitabine, Ibalizumab (Trogarzo®), Idoxuridine, Imiquimod, Immunovir, Indinavir, Inosine, Integrase inhibitors, Type I interferon, Type II interferon, Type III interferon, Interferon, Ivermectin, Lamivudine, Lasaroside, Letermovir (Prevymis®), Lopinavir, Lobilide, Mannose-binding lectin, Maraviroc, Methisazone, Moroxidine, Nafamostat, Nelfinavir, Nevirapine, Nexav ir(registered trademark), nilotinib, nitazoxanide, Norvir, nucleoside analog, oseltamivir (Tamiflu(registered trademark)), pazopanib, pegylated interferon alfa-2a, pegylated interferon alfa-2b, penciclovir, peramivir (Rapivab(registered trademark)), preconalil, podophyllotoxin, protease inhibitors (pharmacology), pionarizine, pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant(registered trademark)), rimantadine, ritonavir, saquinavir,A method selected from the group consisting of simeprevir (Olysio®), sofosbuvir, stabuzin, synergistic enhancer (antiretroviral drug), tafenoquin, telaprevir, terbivudine (Tyzeka®), tenofovir alafenamide, tenofovir disoproxil, tenofovir, toremifene, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir (Valtrex), valganciclovir, bermurafenib, venetoclax, bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza®), and zidovudine, one of the preceding items. 31. Any of the preceding methods, wherein an immunostimulatory oligonucleotide double-strand and at least one antiviral drug are administered substantially simultaneously. 32. Either of the preceding methods, in which an immunostimulatory oligonucleotide double-strand and at least one antiviral drug are administered at different time points. 33. A pharmaceutical composition comprising an immunostimulatory oligonucleotide double helix of any of the preceding items and a pharmaceutically acceptable carrier. 34. A pharmaceutical composition comprising a double-stranded immunostimulatory oligonucleotide of any of the preceding items and at least one antiviral therapeutic agent. 35. A composition of any of the preceding items, formulated for airway administration. 36. A composition of any of the preceding items, formulated for aerosol administration, nebulizer administration, or tracheal lavage administration. 37. A method for inducing interferon (IFN) production, comprising the step of administering to a subject in need of interferon production an immunostimulatory oligonucleotide double chain of any of the preceding items, or a pharmaceutical composition of any of the preceding items, thereby increasing IFN production after administration. 38. The method described in the preceding item, wherein IFN production is the production of type I IFN, type II IFN, or type III IFN. 39. One of the methods described in the preceding items, where IFN production is the production of type I IFN. 40. The method described in the preceding item, wherein the type I IFN is IFN-α, IFN-β, IFN-ε, IFN-κ, or IFN-ω. 41. Type II IFN is IFN-γ, or any of the methods described in the preceding section. 42. One of the preceding methods states that increased IFN production increases the cell's resistance to viral infection. 43. A method for treating an IFN-related disease, comprising the step of administering an immunostimulatory oligonucleotide double chain of any of the preceding items to a subject in need thereof. 44. The method described above, which determines whether the subject requiring such treatment has an IFN-related disease or is at risk of developing an IFN-related disease. 45. Any of the preceding steps, further comprising the step of diagnosing the subject whether they have an IFN-related disease or are at risk of developing an IFN-related disease, prior to the administration step. 46. Any of the preceding steps further include, prior to the administration step, receiving the results of an assay that diagnoses whether the subject has or is at risk of having an IFN-related disease. 47. One of the preceding methods, in which IFN-related disease is a disease accompanied by reduced IFN levels compared to the reference level. 48. One of the preceding methods, in which the IFN-related disease is a disease accompanied by reduced type I IFN levels compared to the reference level. 49. One of the preceding methods, wherein IFN-related diseases are selected from the group consisting of viral infectious diseases, bacterial infectious diseases, fungal infectious diseases, parasitic infectious diseases, cancer, and autoimmune diseases. 50. Any of the preceding methods, further comprising the step of administering at least one additional therapeutic agent. 51. The method of any of the preceding items, wherein at least one additional treatment is an antiviral, antibacterial, antifungal, antiparasitic, anticancer, or anti-autoimmune drug. 52. A composition comprising an immunostimulatory oligonucleotide double-chain of any of the preceding items and at least one antibacterial therapeutic agent. 53. A composition comprising one of the preceding immunostimulatory oligonucleotide double-strands and at least one antifungal therapeutic agent. 54. A composition comprising one of the preceding immunostimulatory oligonucleotide double hemispheres and at least one antiparasitic therapeutic agent. 55. A composition comprising one of the preceding immunostimulatory oligonucleotide double hemispheres and at least one anticancer drug. 56. A composition comprising an immunostimulatory oligonucleotide double-strand of any of the preceding items and at least one anti-autoimmune drug. 57. A composition of any of the preceding items, further comprising a pharmaceutically acceptable carrier. 58. An immunostimulatory oligonucleotide double helix conjugated to an antigen or vaccine, containing SEQ ID NO:1 at its 5' end. 59. A composition comprising a double-stranded immunostimulatory oligonucleotide of any of the preceding items. 60. A composition comprising a double-stranded immunostimulatory oligonucleotide of any of the preceding items and a vaccine. 61. A composition comprising an immunostimulatory oligonucleotide duplex of any of the preceding items and a nanoparticle. 62. A nanoparticle comprising an immunostimulatory oligonucleotide duplex of any of the preceding items. 63. A composition comprising an immunostimulatory oligonucleotide duplex of any of the preceding items and a nanoparticle. 64. A nanoparticle comprising an immunostimulatory oligonucleotide duplex of any of the preceding items. 65. A composition of any of the preceding items further comprising a pharmaceutically acceptable carrier. 66. A method of vaccination, comprising administering to a subject in need thereof a. an immunostimulatory oligonucleotide duplex of any of the preceding items, b. a composition of any of the preceding items, or c. an immunostimulatory oligonucleotide duplex of any of the preceding items, and a vaccine The method comprising the step of administering. 67. A method of increasing the efficacy of a vaccine, comprising administering to a subject in need thereof a. an immunostimulatory oligonucleotide duplex of any of the preceding items, b. a composition of any of the preceding items, or c. an immunostimulatory oligonucleotide duplex of any of the preceding items, and a vaccine The method comprising the step of administering. 68. A composition of any of the preceding items formulated for intravenous, intramuscular, intraperitoneal, subcutaneous, or intrathecal administration.

Example

[0215] Example 1: Double-stranded RNA interferon inducer therapeutic agent for pathogenic infections and other immune response-related diseases The increasing emergence of potential pandemic viruses such as influenza, MERS, SARS, and now SARS-CoV-2 necessitates the development of new broad-spectrum therapies that inhibit infection by many different types of viruses. The best way to achieve this is to target the general host response to the virus rather than the virus itself. As an example, influenza A virus is a significant human pathogen that causes seasonal epidemics and accidental pandemics, with serious public health and economic impacts. Infection and replication of influenza in host cells is a multi-step process. The virus binds to host surface receptors, enters the cell, and then releases its genome into the cytoplasm. The viral genome is then translocated to the nucleus, where viral transcription and replication occur, and the newly synthesized viral proteins and RNA are assembled into progeny viral particles, which are released into the extracellular environment by budding.

[0216] Following infection by viruses and other types of pathogens (e.g., bacteria, fungi, parasites), the human body triggers a complex regulatory system of innate and adaptive immune responses designed to defend against viruses. One of the many responses to viral entry is the induction of interferon (IFN) production, a multifaceted cytokine that plays a crucial role in the human immune response by "interfering" viral replication. 1Induction of IFN gene expression also leads to increased cellular resistance to viral infection by activating immune cells (e.g., natural killer cells and macrophages) and by increasing the expression of major histocompatibility complex (MHC) antigens, thereby upregulating antigen presentation and enhancing host defense. There are numerous different types of IFN genes and proteins, which in humans are typically classified into three classes: type I IFNs (IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), type II IFNs (IFN-γ), and type III IFNs. All three classes of IFNs are important for fighting viral infection and regulating the immune system. IFNs have also been used as therapeutic agents in the treatment of multiple sclerosis, many types of cancer, and viral infections.

[0217] While increased IFN production can inhibit infections from influenza and many other types of viruses, this potential therapeutic pathway may be particularly useful in treating COVID-19. This is because, compared to responses to influenza A virus and respiratory syncytial virus, the virus that causes COVID-19 (SARS-CoV-2) elicits a weaker response lacking robust induction of a subset of cytokines, including type I and type III IFNs, while continuing to produce other inflammatory cytokines that can lead to cytokine storms, a cause of death in many patients. 2 .

[0218] This specification describes specific double-stranded RNA sequences that, when transfected into human A549 lung epithelial cells, activate the interferon pathway, upregulate type I IFN expression, and reduce influenza A virus infection. The double-stranded RNA sequences identified herein also result in a significant (100-fold) reduction in influenza A virus infection when transfected into human A549 lung epithelial cells.

[0219] Screening for lncRNAs that mediate influenza virus infection This specification describes a CRISPR / Cas9-based screening strategy for identifying lncRNAs that mediate influenza virus infection. Cells containing sgRNAs that knock out lncRNAs, conferring resistance to influenza infection without affecting cell growth, can survive and rapidly proliferate. Enriched lncRNAs were identified using Model-based Analysis of Genome-wide CRISPR / Cas9 knockout (MAGeCK) to prioritize sgRNAs, genes, and pathways in a genome-scale CRISPR / Cas9 knockout screen after deep sequencing. Hundreds of Dicer-Substrate Short Interfering RNAs (DsiRNAs) targeting relevant lncRNA sequences were tested by transfecting them into human A549 lung epithelial cells and then infecting those cells with influenza virus. This analysis revealed that transfection with two of these DsiRNAs, targeting the lncRNAs DGCR5 (double-stranded RNA-1) and LINC00261 (double-stranded RNA-2), suppressed influenza infection by approximately 80% and 98%, respectively (Figure 1A). When the same experiment was performed using an influenza-infected human lung airway chip that more closely mimics the pathophysiology of the human lung airway, approximately 100-fold inhibition of viral titer was observed (Figure 1B). 3~5 .

[0220] siRNAs with a common sequence induced interferon directly, rather than via lncRNA. Importantly, in order to further validate the functions of DGCR5 and LINC00261, additional studies were conducted using multiple DsiRNAs targeting DGCR5 and LINC00261. Surprisingly, it was found that only a subset of siRNAs could both knock down DGCR5 or LINC00261 and induce IFN production, while other siRNAs, although specifically knocking down DGCR5 or LINC00261 as designed, did not induce IFN production. Even more importantly, all of the active siRNAs that induced IFN production contained the same sequence (shown in gray and light gray, Table 1, Figure 7), although they were designed to target different lncRNAs. It is important to note that these double-stranded RNAs contain sequence-coding structures specifically designed not to induce interferon induction, as shown by other researchers. 6 Therefore, the common sequences found in both of these double-stranded RNAs (double-stranded RNA-1 and -2) shown in Table 1 (Figure 7) appear to specifically induce IFN production based on their specific nucleotide sequence composition, rather than by repressing lncRNA expression.

[0221] (Table 1) Double-stranded and single-stranded RNA See also Figure 7 in TIFF2026123077000004.tif131160.

[0222] To explore this hypothesis, we designed different versions of double-stranded and single-stranded RNA (Table 1, Figure 7). We found that retaining either the gray common sequence (double-stranded RNA-4) or both the gray common sequence and the light gray common sequence (double-stranded RNA-6) allowed for similar levels of IFN production to be induced even when the remaining sequences were shuffled (Table 1, Figure 7; Figure 2). This suggests that the gray common sequence is necessary for IFN production induction, while the light gray sequence is not. This is demonstrated by double-stranded RNA-5, which lacks the gray common sequence and does not induce IFN production (Table 1, Figure 7; Figure 2). In addition, since short RNA sequences containing the gray common sequence (double-stranded RNA-3 and double-stranded RNA-7) exhibited different IFN-producing abilities (Table 1, Figure 7; Figure 2), it is suggested that the length of the double-stranded RNA also affects the ability of the gray common sequence to induce IFN production. Single-stranded RNA (single-stranded RNA-8 and single-stranded RNA-9) could not induce IFN production (Table 1, Figure 7; Figure 2), and therefore it was found that the double-stranded structure is important.

[0223] Double-stranded RNA negatively regulates the type I interferon pathway by modulating IRF3. To characterize the mechanism by which these double-stranded RNAs reduce viral infection, transcriptome changes were characterized using RNA-seq. After treatment with double-stranded RNA1, 21 genes showed more than a twofold increase at a threshold p-value of 0.01 (Figure 3A). Gene ontology (GO) enrichment analysis revealed that the biological processes of these genes are related to the type I IFN signaling pathway and the defense response against viral infection (Figure 3B). In parallel, tandem mass tag (TMT) mass spectrometry quantification revealed upregulation of 73 proteins with more than a fourfold increase at a threshold p-value of 0.01 (Figure 3C). GO enrichment analysis also confirmed the association between double-stranded RNA1 treatment and upregulation of the type I IFN pathway (Figure 3D). The fact that double-stranded RNA-1 primarily activates the type I IFN pathway compared to the type II IFN pathway was further verified by qPCR assays (Tables 2-3, see below).

[0224] (Table 2) Effect of double-stranded RNA-1 on IFN-α / β genes TIFF2026123077000005.tif20889

[0225] (Table 3) Effects of double-stranded RNA-1 on genes in the IFN-γ pathway TIFF2026123077000006.tif168128TIFF2026123077000007.tif150128

[0226] In addition, double-stranded RNA-1 can increase only the levels of STAT1 and STAT2 specific to the IFN pathway (Figure 4). These results indicate that treatment with double-stranded RNA-1 can specifically activate the type I IFN pathway, which explains why treatment with double-stranded RNA-1 suppresses influenza infection.

[0227] The effects of double-stranded RNA-1 on the type I IFN system were further investigated in wild-type HAP1 cells, interferon regulator 3 (IRF3) knockout HAP1 cells, and IRF7 knockout HAP1 cells. IRF3 and IRF7 are transcription factors that play extremely important roles in the production and function of interferon-I (IFN-1) during viral infection. 7 Our results showed that knockout of IRF3 eliminates the ability of double-stranded RNA-1 to activate the type I IFN pathway, but knockout of IRF7 does not (Figure 5). Taken together, our results suggest that double-stranded RNA-1 positively regulates the type I IFN pathway via IRF3. Further investigation showed that double-stranded RNA-1 does not affect the expression level of IRF3, but it alters its phosphorylation state (Figure 6). A similar mechanism was observed for double-stranded RNA-2.

[0228] To determine whether double-stranded RNA can increase interferon production in human primary alveolar epithelium, differentiated human primary airway epithelial cells, human primary alveolar epithelial cells, or human primary lung microvascular endothelial cells (HMVECs) were transfected with either a negative control (NC) or dsRNA-4 using the airway chip described in Benam et al. Nature Methods (2016). Following the addition of dsRNA-4, qPCR was performed 48 hours later to measure IFN-β production. dsRNA-4 increased interferon-β production by nearly fourfold compared to the control airway chip (Figure 8). Therefore, dsRNA-4 increases interferon-β production not only in airway and endothelial cells but also in human primary alveolar epithelial cells on the chip.

[0229] Furthermore, the data presented herein show that increased interferon-β production led to a significant reduction of SARS-CoV-2 N mRNA in cells infected with the SARS-CoV-2 virus (Figure 9). "N" in SARS-CoV-2 N mRNA refers to the gene of SARS-CoV-2 that encodes the viral nucleocapsid. When ACE2-expressing A549 cells were transfected with dsRNA-1 and dsRNA-2 at low or high doses, interferon-β production increased variably as a result. The transfected ACE2-A549 cells were infected with SARS-CoV-2 at an MOI of 0.05 24 hours after transfection. 48 hours after infection, the cells were harvested, total RNA was isolated, and qPCR was performed to detect the levels of specific genes. In infected cells, SARS-CoV-2 N mRNA was found to be reduced approximately 10 4 -fold. Therefore, these data confirm that the activation of interferon-β production is effective in reducing, inhibiting or preventing viral infections, particularly SARS-CoV-2 infection.

[0230] Considering that these specific double-stranded RNAs activate the type I IFN pathway, they can be used as broad-spectrum prophylactic and therapeutic agents for IFN-related diseases, including infections by a wide range of viral, bacterial, fungal and parasitic pathogens, as well as cancer and autoimmune diseases, not only inhibiting influenza virus infection as shown in our proof-of-concept studies. As described above, the induction of type I IFN signaling can be particularly useful in treating COVID19 patients in whom this pathway is abnormally suppressed.

[0231] To treat viral infections, IFN pathway-activated double-stranded RNA can be administered directly to the lung epithelium using nanoparticles, liposomes, droplets, or other formulations via aerosol, nebulizer, or tracheal lavage. Alternatively, the double-stranded RNA can be delivered intravenously, subcutaneously, intraperitoneally, or intramuscularly, with or without a drug delivery vehicle. The route of administration and dosage of these double-stranded RNA molecules will need to be optimized depending on the disease being treated.

[0232] Summary of results: This specification describes double-stranded RNAs that share a common sequence and can inhibit viral infection by inducing IFN production.

[0233] Furthermore, it was determined that IRF3 mediates the effects of these double-stranded RNAs on IFN expression.

[0234] Because the IFN-I pathway is involved in many diseases, these RNAs or related molecules containing the same key functional double-stranded polynucleotide sequence could become novel therapeutic agents for interventions in a variety of immune-related diseases that rely on the IFN response, including various types of pathogenic infections caused by bacteria, fungi, or parasites, as well as cancer and autoimmune disorders.

[0235] References: TIFF2026123077000008.tif98160

[0236] Example 2: Inhibition of SARS-CoV-2, HCoV-NL63, and influenza by type I interferon-inducing 5'-monophosphate RNA. The COVID-19 crisis highlighted the need for therapeutic agents capable of inhibiting infection by the SARS-CoV-2 virus and other highly infectious viral variants that could potentially cause future pandemics. This specification provides a novel class of 5'-monophosphate-containing immunostimulatory double-stranded RNAs that inhibit SARS-CoV-2, HCoV-NL63, and influenza virus infections by potently inducing the production of type I interferon (IFN-I), particularly IFN-β, in a wide range of cells, including highly differentiated primary airways, alveolar epithelium, and microvascular endothelium, grown within a microfluidic human organ-on-a-chip. These RNAs possess none of the sequence or structural features of known immunostimulatory RNAs; instead, they require a unique, conserved sequence motif (sense strand: 5'-CUGA-3', antisense strand: 3'-GGGACU-5') and a minimum length of 20 nucleotides for their immunostimulatory activity. Surprisingly, RNA containing this motif, despite containing a 5'-monophosphate, induces IFN-I production by activating the RIG-I / IRF3 pathway. This novel type of immunostimulatory RNA may prove useful in the future as a broad-spectrum prophylactic or therapeutic agent for viral infections and pandemics, including COVID-19, as well as other diseases involving abnormal IFN-I regulation.

[0237] Coronavirus disease 2019 (COVID-19) is a global health crisis caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). There are no approved drugs or vaccines to treat or prevent this disease, and there has been a strenuous search for new forms of therapeutic intervention. Type I and III interferons (IFN-I and IFN-III), produced by host cells when faced with the pathogen, constitute the first line of natural host defense against viral infection. Recognition of viral components by cellular sensors, such as Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA-5), initiates a signaling cascade that induces the secretion of IFN-I / -III and the subsequent upregulation of hundreds of interferon-stimulated genes (ISGs) that mediate the biological and therapeutic effects of this antiviral response (1). Recombinant IFN-I and IFN-III, or their synthetic derivatives, have been explored for the treatment of viral infections, as well as autoimmune diseases and cancer, due to their potent and broad-spectrum effects (2-4). Since SARS-CoV-2 has been shown to interfere with the induction of the IFN-I pathway (5, 6), their potential therapeutic efficacy in COVID-19 patients has also been investigated (4, 7-9). However, recombinant IFNs previously used to treat related pathogenic coronaviruses SARS-CoV and MERS-CoV have been questionable in their ability to reduce viral load, suggesting the need to explore alternative approaches to leverage this innate defense mechanism, including the use of synthetic agonists (4). Furthermore, it has recently been shown that SARS-CoV-2 non-structural protein 1 (nsp1) inhibits the RIG-I-dependent innate immune response that would otherwise facilitate infection clearance (10). Therefore, developing immunostimulatory therapies that activate the host's antiviral IFN response by counteracting the inhibition of the RIG-I pathway could be a promising strategy in combating COVID-19.

[0238] result Discovery of IFN-I pathway-activating immunostimulatory RNA We unexpectedly discovered novel RNA inducers of the IFN-I pathway that exhibit potent broad-spectrum inhibitory activity against SARS-CoV-2 and other viruses. While using >200 small interfering RNA molecules (siRNAs) to identify host genes mediating the human A549 lung epithelial cell response to influenza A / WSN / 33(H1N1) infection, we found that two siRNAs (RNA-A and RNA-B) inhibited H1N1 infection by over 90% (Figure 10A). To explore the mechanism of action of these siRNAs, which target the long non-coding RNAs (lncRNAs) DGCR5 and LINC00261, respectively, we profiled the transcriptome and proteome of A549 cells transfected with RNA-A and RNA-B, using scrambled siRNA as a control (Figures 11A-11B). RNA-seq analysis showed that RNA-A more than doubled the expression of 21 genes (threshold p-value of 0.01) (Figure 11A). Gene Oncology (GO) enrichment analysis revealed that these genes, including MX1, OASL, IFIT1, and ISG15, are involved in the IFN-I signaling pathway and the host defense response to viral infection (Figure 12A, left). In parallel, tandem mass-tagged mass spectrometry (TMT mass spectrometry) quantification showed more than four-fold upregulation (threshold p-value of 0.01) of 73 proteins, including IL4I1, TNFSF10, XAF1, IFI6, and IFIT3 (Figure 12B). GO enrichment analysis of these upregulated proteins also confirmed the association between RNA-A treatment and induction of the IFN-I pathway (Figure 13A). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays independently verified that RNA-A preferentially activates the IFN-I pathway compared to type II or type III IFN pathways (Figure 13B), and IFN-β was induced to much higher levels (>1000-fold) than IFN-α (Figure 10B). Similar patterns of gene and protein expression were observed for RNA-B as well (Figure 10B and Figures 11-12).

[0239] Interestingly, further investigation of the function of the lncRNAs they target by performing studies with additional siRNAs revealed that knockdown of DGCR5 or LINC00261 by these other siRNAs did not induce IFN production. This was surprising because, since the inception of RNA interference technology, it has been known that short double-stranded siRNAs induce IFN-I (11, 12), and subsequent designs of these molecules, including those used in our studies, have been optimized to avoid this effect and potential immunomodulatory side effects (13). siRNAs synthesized by phage polymerase with a 5'-triphosphate end can induce potent induction of interferon α and β (12), and siRNAs containing 9 nucleotides (5'-GUCCUUCAA-3') at the 3' end can induce IFN-α via TLR-7 (14), but the double-stranded RNAs described herein do not have either of these structures. The RNAs described herein also contain 5'-monophosphate, which is present in host RNA, and this actively suppresses IFN-I activation via RIG-I signaling by double-stranded RNA containing 5'-diphosphate or 5'-triphosphate (15). Therefore, these data suggest that these two special RNAs (RNA-A and RNA-B), which were found to be potent IFN-I inducers, may correspond to novel immunostimulatory RNAs.

[0240] To further explore this, we evaluated IFN-I production induced by these two putative immunostimulant RNAs using the A549-Dual™ IFN reporter cell line (16), which stably expresses a luciferase gene driven by a promoter containing an IFN-stimulated response element. These studies revealed that both RNA-A and RNA-B initiated induction of IFN production as early as 6 hours after transfection, consistent with IFN-I being an early response gene in innate immunity, and high levels of IFN expression persisted for at least 24–48 hours (Figure 10C). Dose-dependent induction of IFN production by these double-stranded RNAs was also observed in the nM region (Figure 10D). Taken together, these results confirm that the two identified siRNAs are indeed immunostimulant RNAs that specifically upregulate a strong IFN-I response.

[0241] Novel immunostimulatory RNA motifs sensed by RIG-I Active RNA-1 and -2 are chemically synthesized 27-mer RNA double helixes containing a 5'-monophosphate and a single 2-nucleotide 3' overhang on the antisense strand (Figure 14, Table 4 below). Their sequence and structural features do not match any existing immunostimulatory RNA molecules (Figure 15), suggesting that previously unknown elements must be responsible for this immunological activity.

[0242] (Table 4) RNA double-strand sequences TIFF2026123077000009.tif23684TIFF2026123077000010.tif49128

[0243] Surprisingly, despite being designed to target different host genes, sequence alignment revealed that RNA-A and RNA-B contain two identical motifs: one at its 5' end (motif-1; sense strand: 5'-CUGA-3', antisense strand: 3'-GGGACU-5') and the other in its central region (motif-2; sense strand: 5'-ACUG-3', antisense strand: 3'-UGAC-5') (Figure 14). Since both RNAs were potent inducers of IFN-β, we hypothesized that these common motifs might mediate their immunostimulatory activity, although this does not align with any specific theory.

[0244] To verify this, IFN-I production induced by 18 different RNA-A sequence variants (Figure 14) was systematically investigated using IFN reporter-expressing cell lines. Maintaining motifs 1 and 2 while replacing the remaining nucleotides with random sequences (RNA-C vs. -A and -B) did not affect the immunostimulatory activity of double-stranded RNA (Figures 16 and 14). Further substitution of motif 2 with random sequences also did not affect its immunostimulatory activity (RNA-D vs. RNA-C), suggesting that motif 1, not motif 2, is responsible for double-stranded RNA-mediated IFN-I production. This was demonstrated by the complete disappearance of RNA immunostimulatory activity when motif 1 was replaced or deleted while the remaining nucleotides were kept unchanged (RNA-E and -F vs. -A).

[0245] To determine the minimum sequence of motif-1 responsible for immunostimulatory activity, mutants were created by deleting or substituting nucleotides in motif-1. Deletion or substitution of the two overhanging bases GG at the 3' end of the antisense strand resulted in the loss of their immunostimulatory activity (RNA-G and -H vs. -A). Even when the remaining bases of motif-1 were altered while retaining the overhanging bases GG, the immunostimulatory activity was significantly reduced (RNA-J and -K), or even completely lost (RNA-I vs. -A). These data confirm that motif-1 is necessary for IFN-I induction and that its immunostimulatory potency is sensitive to sequence substitutions or deletions. The effect of RNA length on motif-1-mediated IFN production was further evaluated by gradually truncating the bases from the 3' end of RNA-A. As the number of bases removed increased, immunostimulatory activity gradually decreased (RNA-L and -M vs. -A), and when more than 8 bases were removed from the 3' end of RNA-A, activity was completely lost (RNA-N, -O, and -P). Therefore, the minimum length of this novel form of immunostimulatory RNA required for INF induction is 20 bases (antisense strand). In addition, since neither a single sense strand nor a single antisense strand of RNA-A induced IFN production (RNA-Q and -R), it was shown that a double-stranded RNA structure is required for its immunostimulatory activity. Since the chemically synthesized RNA contains a 5'-hydroxyl group, we also tested whether the addition of 5'-monophosphate affects interferon-inducing activity. This is important because 5'-monophosphate is present in host RNA and actively suppresses IFN-I activation by RIG-I signaling induced by double-stranded RNA containing 5'-di or triphosphate. However, when analyzed by qPCR, RNA-1 containing 5'-monophosphate was found to induce IFN-β expression to a level similar to that of RNA-1 containing 5'-hydroxyl (Figure 2B). This suggests that while 5'-monophosphate in these short double-stranded RNAs is not necessary for their effects, it does not interfere with them either.

[0246] The transcription factors interferon regulators 3 (IRF3) and 7 (IRF7) play crucial roles in IFN-I production (17, 18). Using IRF3 knockout (KO) cells and IRF7 KO cells, it was found that loss of IRF3 completely eliminates the ability of RNA-A to induce IFN-β (Figure 17A) and downstream ISGs including STAT1, IL4L1, TRAIL, and IFI6 (Figure 18), but loss of IRF7 does not. IRF3 is the master and primary transcriptional activator of IFN-I, and its IFN-I induction involves a cascade of events including phosphorylation, dimerization, and nuclear translocation of IRF3 (19, 20). To mitigate the potential interference from host gene knockdown by RNA-A developed as siRNA, further mechanistic studies were conducted using RNA-D, which contains similarly active immunostimulatory motifs but does not target (silence) any host genes. RNA-D did not affect IRF3 mRNA levels or total IRF3 protein levels (Figures 17B-17C), but it increased IRF3 phosphorylation (Figure 17C), which is essential for IRF3 transcriptional activity (17), and subsequently, IRF3's translocation to the nucleus (19, 20), where it acts as a transcription factor that induces IFN-I expression (Figure 17D).

[0247] This short double-stranded RNA containing a unique immunostimulatory motif binds directly to RIG-I. RIG-I, MDA5, and TLR3 are major RNA-recognizing sensors located upstream of IRF3 (21). To determine which of these detects our novel double-stranded RNA, RNA-mediated production of IFN-I was quantified in RIG-I, MDA5, or TLR3 knockout cells. RIG-I knockout completely suppressed the ability of RNA-D (Figure 17E) and RNA-A, -B, and -C (Figure 19) to induce IFN-I, while loss of MDA5 or TLR3 did not affect RNA-mediated IFN-I production (Figures 17E and 19). Importantly, surface plasmon resonance (SPR) analysis revealed that RNA-1 directly interacts with the RIG-I cell RNA sensor, rather than with MDA5 or TLR3 (Figure 3F). Therefore, the small double-stranded RNAs containing the novel immunostimulatory motifs described above stimulate IFN-I production by specifically utilizing the RIG-I / IRF3 pathway, even if they contain 5'-monophosphate, which has been previously suggested to antagonize rather than stimulate RIG-I-dependent activation of IFN production (15).

[0248] Finally, these novel immunostimulatory RNAs exhibited a far more potent induction of IFN-β production (>2,000 times) than poly(I:C), a commonly used pathogen recognition receptor (PRR) agonist, or compared to double-stranded RNAs containing 5'-triphosphate known to be activating RIG-I ligands (Figure S6A). More importantly, in contrast to poly(I:C), RNA-seq analysis revealed that these novel immunostimulatory RNAs did not induce the expression of a wide range of inflammation-related genes (Figure S6B). This is clinically significant because, while poly(I:C) can induce an IFN response, its potential use in patients is limited due to complex toxicity associated with the induction of more systemic inflammatory responses.

[0249] Broad-spectrum inhibition of multiple coronaviruses and influenza A viruses To explore the potential physiological and clinical significance of these novel RNAs that exhibited immunostimulatory activity in established cell lines, we investigated whether they could induce an IFN-I response in a human lung airway and alveolar chip microfluidic culture device covered with human primary lung epithelium grown at the gas-liquid interface in close proximity to lung microvascular endothelium cultured under dynamic fluid flow, which has been demonstrated to faithfully reproduce the physiological function and pathophysiology of the human lung at the organ level (22-24). When RNA-A was transfected into human airway and alveolar epithelial cells through the air channels of the human lung chip (Figure 20A), a 4- to 12-fold increase in IFN-β expression was observed compared to the scrambled double-stranded RNA control. In addition, RNA-A treatment induced robust (>40-fold) IFN-β expression in human primary lung endothelium on-chip (Figure 20A) when introduced into the chip's vascular channels.

[0250] Building on the initial finding that RNA-A and -B inhibit H1N1 infection (Figure 1A) and the known antiviral function of IFN-I (25), we then explored the universality of these effects. First, we examined the potential of these IFN-I-inducible RNAs to inhibit infection by influenza A / HK / 8 / 68 (H3N2) virus. Cells were transfected with RNA one day prior to infection. Subsequently, with the emergence of the COVID-19 pandemic, we expanded this study by performing similar research using SARS-CoV-2 and related coronaviruses HCoV-NL63. Analysis of viral mRNA by qPCR revealed that treatment with immunostimulatory RNA significantly suppressed infection by influenza H3N2 virus in A549 cells (>95% inhibition) and in human alveolar chips covered with primary alveolar epithelium bordering pulmonary microvascular endothelium (approximately 80% inhibition), similar to the results obtained with influenza H1N1 virus in A549 cells (Figure 10A) (Figure 20B).

[0251] These same double-stranded RNAs inhibited HCoV-NL63 by >90% in LLC-MK2 cells (Figure 20B), and impressively, they were even more potent inhibitors of SARS-CoV-2 infection, reducing viral load by more than 10,000 times (>99.99%) in ACE2 receptor-overexpressing A549 cells (Figures 20B and 21). This is consistent with observations that SARS-CoV-2 regulates IFN-I signaling in a different way and fails to induce its expression in contrast to these other viruses (6). Importantly, RNA-seq analysis revealed that these immunostimulatory RNAs did not induce the expression of a wide range of inflammation-related genes, in contrast to the commonly used pathogen-recognition receptor (PRR) agonist poly(I:C) (Figure 22). This is significant. This is because, although poly(I:C) can induce an IFN response, its potential clinical use is limited due to complex toxicity associated with the induction of a more systemic inflammatory response (26, 27).

[0252] Based on the potent inhibitory activity against SARS-CoV-2 observed in vitro, RNA-1 was next evaluated in a hamster COVID-19 model. RNA-1 was dissolved in phosphate-buffered saline (PBS), and animals were subjected to SARS-CoV-2 virus (10 2 RNA-1 was administered intranasally to PFU (Prophylaxis Fluid) one day before intranasal infection, on the day of infection, and one day after infection. Two days after viral challenge, SARS-CoV-2 virus N transcripts were measured in the lungs of these hamsters. RNA-1 prophylaxis resulted in a significant reduction in viral load, both by RT-qPCR (p=0.030) and by quantification of viral titer using a plaque assay (p=0.032), indicating that infection was effectively prevented (Figure 5A). In addition, RNA-1 in the culture medium was added to the viral infection (10 3Similar inhibition of viral infection was measured when RNA-1 was administered in therapeutic mode by daily intranasal delivery via vehicle over two days, starting one day after PFU (Figure 5B), followed by lung analysis by RT-PCR (Figure 5B). Most importantly, histological analysis of these lungs revealed that the reduction in viral load resulting from RNA-1 treatment initiated one day after infection led to a significant decrease in immune cell infiltration into the alveolar spaces, which were completely obstructed and filled with cells and exudate in control infected lungs treated with vehicle alone (Figure 5C).

[0253] Consideration In this study, a novel type of short, overhanged double-stranded RNA containing a 5'-monophosphate and a unique sequence motif elicited potent stimulation of IFN-I signaling in broad-spectrum human cells, with IFN-β induction being particularly efficient compared to IFN-α. This contrasts with previously described immunostimulatory RNAs containing 5'-di or triphosphates that primarily induce IFN-α or other inflammatory cytokines (28). By systematically investigating the effects of sequence and length of these RNAs on IFN-I induction, it was determined that these double-stranded RNAs require a minimum length of 20 nucleotides, in addition to a conserved overhanged immunostimulatory motif (sense strand: 5'-CUGA-3', antisense strand: 3'-GGGACU-5') and a 5'-monophosphate terminus, to exhibit their immunostimulatory activity. Despite previous studies showing that double-stranded RNAs with 5'-monophosphate antagonize IFN signaling by RNAs with 5'-di or triphosphate (15, 29), investigations into the mechanism revealed that these novel immunostimulatory RNAs specifically activate the RIG-I / IRF3 pathway. In addition, the observed RNA-mediated IFN-I production resulted in significant inhibition of infection by multiple human respiratory viruses, including influenza H1N1 and H3N2, as well as coronaviruses HCoV-NL63 and SARS-CoV-2. Notably, these novel immunostimulatory RNAs reduced SARS-CoV-2 viral load by more than 10,000-fold. These findings suggest that these IFN-I-inducible immunostimulatory RNAs offer potential broad-spectrum protection against a wide range of respiratory viruses that may emerge in the future, as well as novel preventive or therapeutic strategies for the current COVID-19 pandemic.

[0254] Based on the overlapping sequences of two RNAs exhibiting potent IFN-β inducing activity, a conserved overhanging immunostimulatory motif was identified as containing the sense strand 5'-CUGA-3' and the antisense strand 3'-GGGACU-5' along with 5'-monophosphate. Since no immunostimulatory activity was observed in RNAs containing this motif in the central region or at the 3' end, it is suggested that its location at the 5' end is necessary for immunostimulation. This finding is consistent with studies (15) showing that RIG-I recognizes the 5' end of double-stranded RNA, but our immunostimulatory RNAs contain 5'-monophosphate along with the overhang and exhibit sequence-dependent activation by RIG-I, so they do not belong to any category of immunostimulatory RNAs known to date (Figure 15), and therefore represent a new type of immunostimulatory RNA.

[0255] The findings demonstrated above have also led to the identification of a new form of cellular recognition of RNA by cytoplasmic RNA sensors. At least four signaling pathways, including RIG-I, MDA5, TLR3, and TLR7 / 8, have been shown to recognize immunostimulatory RNA molecules and induce the production of IFN-I and pro-inflammatory cytokines (Figure 15). MDA5 recognizes long RNA molecules (approximately 0.5–7 kb in length) (30), TLR3 detects double-stranded RNA molecules of at least 40–50 bp in length within endosomes (31), and TLR7 and TLR8 detect GU-rich short single-stranded RNA as well as small artificial molecules, such as nucleoside analogs and imidazoquinolines (32). RIG-I is a central component of the mammalian innate immune system, which detects pathogen-associated RNA molecules and induces a rapid antiviral immune response. Previous studies have shown that RIG-I recognizes long dsRNAs (300–1,000 bp in length), small self-RNAs produced by RNase L, or short blunt-ended double-stranded RNAs with 5'-di or triphosphates (28, 33–39). As mentioned above, RIG-I is thought to be antagonized by RNA containing 5'-monophosphate (15), and another study has shown that almost any type of 5' or 3' overhang can interfere with RIG-I binding and eliminate signaling (33). In contrast, potent sequence-dependent activation of RIG-I by short double-stranded RNAs with 5'-monophosphate overhangs was observed. This represents an entirely novel form of RNA recognition by RIG-I.

[0256] While siRNA-mediated immune stimulation is undesirable for some gene silencing applications, it can be beneficial in other gene silencing applications, such as the treatment of viral infections or cancer. This increases the potential to provide even stronger titers by designing siRNAs that possess both RNAi and immune stimulating activity. The motif identified in our research is suitable for this purpose because it is located at the 5' end of RNA and can therefore be coupled with sequences targeting viral mRNA or other infection-associated host genes without impairing RNAi activity. The IFN response constitutes the primary first line of defense against viruses, and these infectious pathogens, including SARS-CoV-2, have evolved various strategies to suppress this response (5, 6). In particular, transcriptome analyses of human cultured cells infected with SARS-CoV-2 and COVID-19 patients have revealed that SARS-CoV-2 infection produces a unique inflammatory response in which chemokine and pro-inflammatory cytokine production is stimulated, while IFN-I, IFN-III, and related ISG responses are extremely low (5, 6). This imbalance may contribute to increased morbidity and mortality in late-stage COVID-19 patients. As there are no approved antiviral therapies or vaccines for this emerging respiratory virus, type I and type III IFNs are being evaluated for their efficacy in preclinical models and clinical trials (4, 8, 9, 40). Since IFN pretreatment has been shown to dramatically reduce viral titers, inducing an IFN-I response suggests that it could be a potentially effective approach for the prevention or early treatment of SARS-CoV-2 infection (41, 42). Recently, tripartite therapy with IFN-β, lopinavir, ritonavir, and ribavirin has also been reported to shorten the viral shedding period and hospitalization length in patients with mild or moderate COVID-19 (7). However, since IFN-β treatment generally requires systemic administration by injection, the level of therapeutic drug delivered may be limited by systemic toxicity, which makes it difficult to use as a prophylactic treatment.

[0257] Consistent with these observations, the results provided herein demonstrate that pretreatment with IFN-I-inducible RNA results in a dramatic reduction in infection by SARS-CoV-2, HCoV-NL63, and influenza viruses. Importantly, our immunostimulatory RNAs specifically activate the RIG-I / IFN-I pathway but are not recognized by other cellular RNA sensors such as MDA5 or TLR3. This is intriguing because recent studies have shown that SARS-CoV-2 inhibits RIG-I signaling and infection elimination by nsp1 expression (10), and therefore our results demonstrate that these novel double-stranded RNAs can overcome this inhibition, at least in cultured human lung epithelium and endothelial cells maintained in organ chip cultures that have been previously shown to reproduce the physiological function and pathophysiology of human lung (43, 44). In addition, this offers a clear advantage over other immunostimulatory RNAs in terms of intrinsic toxicity. For example, while the commonly used PRR agonist poly(I:C) activates multiple signaling pathways, including RIG-I, MDA5, and TLR3 (45-47), and induces the production of several pro-inflammatory cytokines and chemokines, such as TNF-α, IL-1, IL-6, and IL-8 (48), the novel immunostimulatory RNAs described herein do not. In addition, the fact that the RNAs described herein specifically induce IFN-I but not IFN-III makes them safer for clinical use against endemic viruses, because IFN-III can disrupt the lung epithelial barrier upon virus recognition (40).

[0258] The finding that these immunostimulatory RNAs can induce IFN-I in highly differentiated primary human lung epithelial and endothelial cells in microfluidic organ chips that replicate human pathophysiology provides further support for their clinical use as preventive or therapeutic agents against COVID-19 or other future viral pandemics. To explore these clinical applications, the concept of intranasal or inhaled RNA formulations (similar to asthma inhalers) that can optimize the efficiency of RNA delivery and locally increase endogenous IFN-β levels in the airways several times over to prevent the spread of infection in the context of viral pandemics such as COVID-19 is particularly noteworthy.

[0259] References TIFF2026123077000011.tif24147TIFF2026123077000012.tif245147TIFF2026123077000013.tif245147TIFF2026123077000014.tif171147

[0260] Example 3: Materials and Methods cell culture

[0261] A549 cells (ATCC CCL-185), A549-Dual® cells (InvivoGen), RIG-I KO A549-Dual® cells (InvivoGen), MDA5 KO A549-Dual® cells (InvivoGen), TLR3 KO A549 cells (Abcam), MDCK cells (ATCC CRL-2936), and LLC-MK2 cells (ATCC CCL-7.1) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and penicillin-streptomycin (Life Technologies). HAP1 cells, IRF3 KO HAP1 cells, and IRF7 KO HAP1 cells were purchased from Horizon Discovery Ltd and cultured in Iskov-modified Dulbecco's medium (IMDM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and penicillin-streptomycin (Life Technologies). All cells were maintained in a humidified incubator at 37°C and 5% CO2. All cell lines used in this study were confirmed to be mycoplasma-free using the LookOut Mycoplasma PCR Detection Kit (Sigma). Cell lines were certified by ATCC, InvivoGen, Abcam, or Horizon Discovery Ltd. Primary human lung airway epithelial basal stem cells (Lonza, USA) were cultured in airway epithelial cell growth medium (Promocell, Germany) for 75 cm³. 2 In tissue culture flasks, cells were grown to 60-70% confluence. Primary human alveolar epithelial cells (Cell Biologics, H-6053) were cultured using alveolar epithelial growth medium (Cell Biologics, H6621). Primary human pulmonary microvascular endothelial cells (Lonza, CC-2527, P5) were cultured using human endothelial cell growth medium (Lonza, CC-3202) for 75 cm³. 2 The tissue was grown in a tissue culture flask until it reached 70-80% confluence.

[0262] virus The viruses used in this study included SARS coronavirus-2 (SARS-CoV-2), human coronavirus HCoV-NL63, influenza A / WSN / 33 (H1N1), and influenza A / Hong Kong / 8 / 68 (H3N2). The SARS-CoV-2 isolate USA-WA1 / 2020 (NR-52281) was deposited by the U.S. Centers for Disease Control and Prevention and obtained from BEI Resources at the NIH and NIAID, and propagated as previously described (Blanco-Melo et al., 2020). HCoV-NL63 was obtained from ATCC and propagated in LLC-MK2 cells. Influenza A / WSN / 33 (H1N1) was generated using reverse genetic techniques, and influenza A / Hong Kong / 8 / 68 (H3N2) was obtained from ATCC. Both influenza virus strains were propagated in MDCK cells. HCoV-NL63 titers were measured in LLC-MK2 cells using the Reed-Muench method. Influenza virus titers were measured by a plaque formation assay (Si et al., 2020).

[0263] Transfection of cell lines RNA and scrambled negative control dsRNA were all synthesized by Integrated DNA Technologies, Inc. (IDT). Cells were divided into 3 × 10⁶ cells. 5 Seed cells / well into a 6-well plate, or 10 4Cells were seeded in 96-well plates at the indicated cell density and cultured for 24 hours prior to transfection. Transfection was performed using the TransIT-X2 Dynamic Delivery System (Mirus), with some modifications to the manufacturer's instructions. Unless otherwise noted, the transfection mixture was prepared by adding 6.8 μL of 10 μM RNA stock solution and 5 μL of transfection reagent to 200 μL of Opti-MEM (Invitrogen). For transfection in 6-well plates, 200 μL of the transfection mixture was added to each well; for transfection in 96-well plates, 10 μL of the transfection mixture was added to each well. After transfection, cell samples were collected at the indicated time and subjected to RNA-seq (Genewiz, Inc.), TMT mass spectrometry, qRT-PCR, Western blotting, or Quanti-Luc assay (InvivoGen).

[0264] RNA-seq and gene ontogene analysis RNA-seq was processed using Genewiz with a standard RNA-seq package that includes polyA selection and sequencing with 150bp paired-end reads in Illumina HiSeq. Sequence reads were trimmed using Trimmomatic v.0.36 to remove possible adapter sequences and low-quality nucleotides. The trimmed reads were mapped to the human (Homo sapiens) GRCh38 reference genome using STAR Aligner v.2.5.2b. Unique gene hit counts were calculated using feature counts in Subread package v.1.5.2, and differential expression analysis was then performed using DESeq2. Gene ontology analysis was performed using DAVID (Huang da et al., 2009). Volcano plots and heatmaps were created using GraphPad Prism. RNA-seq data from A549 cells treated with poly(I:C) were obtained from Gene Expression Omnibus under accession number GSE124144 (Burke et al., 2019).

[0265] Proteomics analysis using tandem mass-tagged mass spectrometry Cells were collected on ice. The cell pellet was syringe-lysed in 8M urea containing a protease inhibitor and 200mM EPPS pH 8.5. The protein concentration of each sample was determined by BCA assay. The samples were reduced in 5mM TCEP, alkylated with 10mM iodoacetamide, and quenched with 15mM DTT. 100 μg of protein was precipitated with chloroform-methanol and resuspended in 100 μL of 200mM EPPS pH 8.5. The proteins were digested overnight at room temperature with Lys-C at a protease-to-peptide ratio of 1:100, with gentle shaking. For further digestion, trypsin was used at the same ratio as with Lys-C, at 37°C for 6 hours. After digestion, 30 μL of acetonitrile (ACN) was added to each sample to a final volume of 30%. 200 μg of TMT reagent (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C) was added to 10 μL of ACN for each sample. After labeling for 1 hour, 2 μL of each sample was combined, desalted, and analyzed by mass spectrometry. The total intensity was determined in each channel, and the normalization factor was calculated. After quenching with 0.3% hydroxylamine, the 11 samples were combined in a 1:1 ratio based on the normalization factor. The mixture was desalted by solid-phase extraction, fractionated by basic pH reversed phase (BPRP) high-performance liquid chromatography (HPLC), collected on a 96 six-well plate, and combined to form a total of 24 fractions. Twelve fractions were desalted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Navarrete-Perea et al., 2018).

[0266] Mass spectrometry data were collected using an Orbitrap Fusion Lumos mass spectrometer connected to a Proxeon NanoLC-1200 UHPLC. A 35 cm Accucore 50 resin (2.6 μm, 150 Å, ThermoFisher Scientific) was packed into a 100 μm capillary column. The scan sequence was started with the MS1 spectrum (Orbitrap analysis, resolution 120,000, 375–1500 Th, auto-gain control (AGC) target 4E5, maximum injection time 50 ms). Ion interference was reduced using SPS-MS3 analysis (Gygi et al., 2019; Paulo et al., 2016). Next, the top 10 precursors were selected for MS2 / MS3 analysis. The MS2 analysis consisted of collision-induced dissociation (CID), quadrupole ion trap analysis, auto-gain control (AGC) 2E4, NCE (normalized collision energy) 35, q-value 0.25, maximum injection time 35 ms, and isolation window of 0.7. After acquiring each MS2 spectrum, the MS3 spectrum was collected. Here, multiple MS2 fragment ions were captured into an MS3 precursor population using an isolation waveform with multiple frequency notches. The MS3 precursors were fragmented by HCD and analyzed using Orbitrap (NCE 65, AGC 1.5E5, maximum injection time 120 ms, resolution 50,000 at 400 Th).

[0267] Mass spectra were processed using a Sequest-based pipeline (Huttlin et al., 2010). Spectra were converted to mzXML using a modified version of ReAdW.exe. Database searches included all entries from the human UniProt database (downloaded 2014-02-04). This database was linked to one consisting of all protein sequences in reverse order. The search was performed using a precursor ion tolerance of 50 ppm for total protein level analysis. The product ion tolerance was set to 0.9 Da. Lysine residues and TMT tags on the peptide N-terminus (+229.163 Da) and carboamidomethylation of cysteine ​​residues (+57.021 Da) were set as static modifications, and oxidation of methionine residues (+15.995 Da) was set as a variable modification.

[0268] Peptide-spectrum matching (PSM) was adjusted to a 1% false discovery rate (FDR) (Elias and Gygi, 2007, 2010). PSM filtering was performed using linear discriminant analysis (LDA) as previously described (Huttlin et al., 2010), taking into account the following parameters: XCorr, ΔCn, missed cleavages, peptide length, charge state, and precursor mass accuracy. For TMT-based reporter ion quantification, the summed signal-to-noise (S:N) ratio was extracted for each TMT channel to find the closest matching centroid for the expected mass of the TMT reporter ion. For protein level comparison, PSMs were identified, quantified, collapsed to a 1% peptide false detection rate (FDR), and then further collapsed to a 1% final protein level FDR, resulting in a final peptide level FDR of <0.1%. Furthermore, protein assembly was performed according to the principles of parsimony to obtain the minimum set of proteins necessary to explain the observed peptides. Proteins were quantified by accumulating the reporter ion number across all matched PSMs, as previously described (Huttlin et al., 2010). Low-quality PSMs, MS3 spectra with a TMT reporter integrated signal-to-noise ratio of less than 100, or no MS3 spectra were excluded from quantification (McAlister et al., 2012). Each reporter ion channel was accumulated for all quantified proteins and normalized assuming that the protein loadings of all samples tested were equal.

[0269] qRT-PCR Total RNA was extracted from cells using the RNeasy Plus mini-kit (QiaGen, catalog number 74134) according to the manufacturer's instructions. Next, cDNA was synthesized using the AMV reverse transcriptase kit (Promega) according to the manufacturer's instructions. To detect gene levels, quantitative real-time PCR was performed using either the GoTaq qPCR master mix kit (Promega) with 20 μL of reaction mixture containing gene-specific primers, or the PrimePCR assay kit (Bio-Rad) according to the manufacturer's instructions. The expression levels of the target gene were normalized against GAPDH.

[0270] Antibodies and Western blotting The antibodies used in this study were anti-IRF3 (Abcam, ab68481), anti-IRF3 (phospho S396) (Abcam, ab138449), anti-GAPDH (Abcam, ab9385), and goat anti-rabbit IgG H&L (HRP) (Abcam, ab205718). Cells were collected and lysed on ice in RIPA buffer (Thermo Scientific, catalog no. 89900) supplemented with Halt® protease and phosphatase inhibitor cocktail (Thermo Scientific, catalog no. 78440). Cell lysates were subjected to Western blotting. GAPDH was used as a loading control.

[0271] Confocal immunofluorescence microscopy Cells were rinsed with PBS, fixed with 4% paraformaldehyde (Alfa Aesar) for 30 minutes, permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) in PBS (PBST) for 10 minutes, blocked with 10% goat serum in PBST (Life Technologies) for 1 hour at room temperature, incubated overnight at 4°C with anti-IRF3 (phospho S396) (Abcam, ab138449) antibody diluted in blocking buffer (1% goat serum in PBST), and then incubated with Alexa Fluor 488 conjugate secondary antibody (Life Technologies) for 1 hour at room temperature. After secondary antibody staining, nuclei were stained with DAPI (Invitrogen). Fluorescence imaging was performed using a confocal laser scanning microscope (SP5 X MP DMI-6000, Germany), and image processing was performed using Imaris software (Bitplane, Switzerland).

[0272] Surface plasmon resonance The interaction between double-stranded RNA-1 and cellular RNA sensor molecules (RIG-I (Abcam, catalog no. ab271486), MDA5 (Creative-Biomart, catalog no. IFIH1-1252H), and TLR3 (Abcam, catalog no. ab73825)) was analyzed by SPR using a Biacore T200 system (GE Healthcare) at 25°C (Creative-Biolabs Inc.). RNA-1 (synthesized by IDT Inc.), conjugated to biotin at its 3' end, was immobilized on the SPR sensor chip at a final level of approximately 60 response units (RUs). RNA sensors at various concentrations, diluted in running buffer (10×HBS-EP+; GE Healthcare, catalog no. BR100669), were injected as analytes at a flow rate of 30 μl / min, a contact time of 180 seconds, and a dissociation time of 300 seconds. The surface was regenerated with 2M NaCl for 60 seconds. Data analysis was performed on a Biacore T200 computer using Biacore T200 evaluation software.

[0273] Organ chip culture Microfluidic 2-channel organ chip devices and automated ZOE® equipment used to culture them were obtained from Emulate Inc. (Boston, Massachusetts, USA). Our methods for culturing human lung airway chips (Si et al., 2020, Si et al., 2019) and alveolar chips have been previously described. In this study, the alveolar chip method was modified by coating the inner channels of the devices overnight at 37°C with 200 ug / ml collagen IV (5022-5MG, Advanced Biomatrix) and 15 μg / ml laminin (L4544-100UL, Sigma). On the following day (day 1), primary human lung microvascular endothelial cells (Lonza, CC-2527, P5) and primary human alveolar epithelial cells (Cell Biologics, H-6053) were placed in the bottom and top channels of the chip, with 8 and 1.6 × 10¹⁶ cells respectively. 6 Slight modifications were made by sequentially seeding cells at a density of cells / ml under static conditions. On day 2, the chip was inserted into Pods® (Emulate Inc.) placed in a ZOE® instrument, and the apical and basal channels were perfused with epithelial growth medium (Cell Biologics, H6621) and endothelial growth medium (Lonza, CC-3202), respectively (60 μL / hour). On day 5, 1 μM dexamethasone was added to the apical medium to enhance barrier function. On day 7, an air-liquid interface (ALI) was introduced into the epithelial channel by removing all the medium from this channel, and all cells continued to be nourished via the medium perfused into the lower vascular channel. On day 9, this medium was replaced with EGM-2MV containing 0.5% FBS. Two days later, to mimic lung respiration on-chip, a periodic (0.25 Hz) 5% mechanical strain was applied to the engineered alveolar-capillary interface using a ZOE® instrument. RNA was transfected on day 15.

[0274] RNA transfection in human lung airways and alveolar chips Human airway or alveolar chips were transfected with double-stranded RNA by adding a mixture of RNA and transfection reagent (Lipofectamine RNAiMAX) to the apical and basal channels of an organ chip, incubating under static conditions at 37°C for 6 hours, and then re-establishing ALI. 48 hours after transfection, on-chip cultured tissues were collected using an RNeasy microkit (QiaGen) by first introducing 100 µl of lysis buffer into the apical channel to lyse epithelial cells, and then introducing 100 µl into the basal channel to lyse endothelial cells. Lysates were subjected to qPCR analysis for IFN-β gene expression.

[0275] Natural SARS-CoV-2 infection and inhibition by RNA treatment ACE2-expressing A549 cells (donated by Brad Rosenberg) were transfected with the indicated RNA. 24 hours after transfection, the transfected ACE2-A549 cells were infected with SARS-CoV-2 (MOI=0.05) for 48 hours. Cells were collected in Trizol (Invitrogen), and total RNA was isolated using the Zymo RNA miniprep kit according to the manufacturer's protocol and treated with DNAse-I. Using the KAPA SYBR FAST ONE-STEP qRT-PCR kit (Roche) and following the manufacturer's instructions, α-tubulin (forward: TIFF2026123077000015.tif3128; Reverse: TIFF2026123077000016.tif3128) and SARS-CoV-2 N mRNA (forward: TIFF2026123077000017.tif3128; Reverse: We performed qRT-PCR on TIFF2026123077000018.tif3128.

[0276] Natural SARS-CoV-1 and MERS-CoV infection and inhibition by RNA treatment Vero E6 cells (ATCC number CRL 1586) were cultured in DMEM (Quality Biological) supplemented with 10% (v / v) fetal bovine serum (Sigma), 1% (v / v) penicillin / streptomycin (Gemini Bio-products), and 1% (v / v) L-glutamine (2 mM final concentration, Gibco). Cells were maintained at 37°C (5% CO2). Two days before transfection, Vero E6 cells were placed in a 6-well plate at a rate of 1.5 × 10⁶ cells per well. 5 Cells were plated individually. RNA-1, RNA-2, and scrambled control RNA were transfected into each well using the Transit X2 delivery system (MIRUS; MIR6003) in OptiMEM (Gibco 31985-070). SARS-CoV (Urbani strain, BEI#NR-18925) and MERS-CoV (Jordan strain, provided by NIH) were added at an MOI of 0.01. At 72 hours post-infection, the culture medium was collected and used for a plaque assay to quantify the viral PFU / mL.

[0277] Hamster efficacy research A method for conducting efficacy studies in golden hamsters using the natural SARS-CoV-2 isolate USA-WA1 / 2020 (NR-52281) has been previously described (13). In the prevention study, RNA-1 diluted in PBS was used with SARS-CoV-2 virus (10 in 100 μl of PBS). 2 In the treatment experiment, RNA-1 diluted in 5% glucose containing in vivo-jetPEI® delivery reagent (Genesee Scientific catalog no. 55-202G; 20 ug in 50 uL) was administered via intranasal injection starting one day prior to the administration of PFU's third-passage virus, and continued daily for two days. 3Intranasal administration of PFU was started one day after the first administration and continued daily for two days. In all experiments, animals were sacrificed one day after the last treatment and their lungs were collected for analysis. Animals were anesthetized by intraperitoneal injection of 100 μl of ketamine and xylazine (3:1), kept warm under unconsciousness, and the entire lungs were collected for analysis by RT-qPCR or plaque assay.

[0278] Lung RNA was extracted by phenol-chloroform extraction and DNase treatment using a DNA-free(trademark) DNA removal kit (Invitrogen). RT-qPCR for subgenome nucleocapsid (N)RNA (sgRNA) and actin was performed using the KAPA SYBR FAST qPCR Master Mix Kit (Kapa Biosystems) on a LightCycler 480 Instrument II (Roche) with the following primers: Actin forward primer: TIFF2026123077000019.tif3128, Actin Reverse Primer: TIFF2026123077000020.tif3128, N sgRNA forward primer: TIFF2026123077000021.tif3128, N sgRNA reverse primer: TIFF2026123077000022.tif3128. Relative sgRNA levels were quantified by normalizing sgRNA levels relative to actin expression.

[0279] Quantitative and statistical analysis All data are expressed as mean ± standard deviation (SD). N represents the number of biological replicas. Statistical significance of differences in in vitro experiments was determined using a paired two-sided Student t-test when comparing differences between two groups, and using a one-way ANOVA with multiple comparisons when comparing samples between groups containing three or more samples. For in vivo experiments, the significance of RNA-1-mediated inhibition of viral load was estimated using an unpaired one-sided Student t-test. For all experiments, differences were considered statistically significant at p<0.05 (*, p<0.05; **, p<0.01; ***, p<0.001; ns, no significant difference).

[0280] References TIFF2026123077000023.tif125147

[0281] Example 4: Novel double-stranded RNA inhibits infection by multiple influenza strains. The double-stranded RNAs described herein were tested for their ability to inhibit infection in human and primate cell cultures. The double-stranded RNAs described herein inhibited more than 95% of influenza infection in human lung epithelial cells (Figure 23). When tested in monkey kidney cells infected with the common cold coronavirus HCoV-NL63, the RNA double-stranded RNAs described herein inhibited more than 95% of coronavirus infection in monkey kidney cells (Figure 24).

[0282] Most notably, the RNA double helix described herein inhibited SARS-CoV-2 virus infection in human lung epithelial cells overexpressing ACE2 (Figure 25).

[0283] Methods - Cell Culture and Virus: Vero E6 cells (ATCC number CRL 1586) were cultured in DMEM (Quality Biological®) supplemented with 10% (v / v) fetal bovine serum (Sigma), 1% (v / v) penicillin / streptomycin (Gemini Bio-products®), and 1% (v / v) L-glutamine (2 mM final concentration, Gibco®). Cells were maintained at 37°C (5% CO2). Two days prior to transfection, Vero E6 cells were plated into 6-well plates at a rate of 1.5E5 cells per well. RNA-A, RNA-B, and scrambled control RNA were transfected into each well of the 6-well plate using a Transit X2® delivery system (MIRUS®; MIR6003) in OptiMEM (Gibco® 31985-070). SARS-CoV (Urbani strain, BEI#NR-18925) and MERS-CoV (Jordan strain, provided by NIH) were added at an MOI of 0.01. The culture medium was collected 72 hours after infection and used for a plaque assay to quantify the viral pfu / ml (e.g., Coleman CM, Frieman MB. 2015. Growth and Quantification of MERS-CoV Infection. Curr Protoc Microbiol 37:15E.2.1-15E.2.9).

[0284] Example 5: Novel double-stranded RNA inhibits SARS-CoV-2 infection in vivo. The double-stranded RNA described herein was tested in vivo by transpulmonary administration to hamsters infected with SARS-CoV-2. Induction of type I interferon by double-stranded RNA administered on day -1, day 0, and day +1 of infection was sufficient to significantly reduce viral load in these animals (Figure 26).

[0285] In summary, the dsRNAs described herein, when delivered to the pulmonary airways, can locally induce a higher IFN response than systemically injected IFN protein preparations. Furthermore, these dsRNAs do not induce the systemic inflammatory response seen with other immunostimulatory RNAs, thus minimizing toxicity. The dsRNAs described herein can be used for both prophylaxis and treatment, particularly in COVID-19 and influenza infections.

Claims

1. An immunostimulatory oligonucleotide double helix containing SEQ ID NO:1 at its 5' end.

2. The immunostimulatory oligonucleotide double helix according to claim 1, wherein the oligonucleotide double helix is ​​RNA.

3. The immunostimulatory oligonucleotide bichain according to claim 1 or 2, wherein the oligonucleotide bichain comprises a 5'-monophosphate group.

4. The immunostimulatory oligonucleotide duplex according to any one of claims 1 to 3, wherein the oligonucleotide duplex has a length of at least 20 nucleic acid bases.

5. The immunostimulatory oligonucleotide duplex according to claim 1, wherein the oligonucleotide duplex is a double-stranded RNA.

6. The immunostimulatory oligonucleotide duplex according to claim 1, wherein the oligonucleotide duplex is sufficient to induce interferon (IFN) production in cells that come into contact with the duplex.

7. The method according to claim 6, wherein the IFN production is type I IFN production.

8. The immunostimulatory oligonucleotide bistrand according to claim 1, wherein the oligonucleotide bistrand activates the RIG-I-IRF3 pathway.

9. The immunostimulatory oligonucleotide duplex according to claim 1, wherein the oligonucleotide duplex reduces the viral titer or viral load in cells or populations of cells that come into contact with the duplex.

10. The immunostimulatory oligonucleotide duplex according to claim 1, wherein the oligonucleotide duplex increases STAT1 and STAT2 in cells in which the duplex is in contact.

11. A method for inducing an antiviral response in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-stranded compound according to any one of claims 1 to 10 to a subject in need of such response.

12. A method for treating a viral infection in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-stranded compound according to any one of claims 1 to 10 to a subject in need thereof.

13. The method according to claim 11 or 12, wherein the subject requiring the method is infected with a virus or is at risk of being infected with a virus.

14. The method according to claim 11 or 12, further comprising the step of diagnosing the subject having a viral infection or being at risk of having a viral infection prior to the administration step.

15. The method according to claim 11 or 12, further comprising the step of receiving the results of an assay that diagnoses the subject having a viral infection or being at risk of having a viral infection, prior to the step of administration.

16. Viral infections include John Cunningham virus, measles virus, lymphocytic choriomeningitis virus, arbovirus, rabies virus, rhinovirus, parainfluenza virus, respiratory syncytial virus, herpes simplex virus, herpes simplex virus type 1, herpes simplex virus type 2, human herpesvirus 6, adenovirus, cytomegalovirus, Epstein-Barr virus, mumps virus, influenza A virus, influenza B virus, coronavirus, SARS coronavirus, SARS-CoV-2 virus, and type A coronavirus. The method according to any one of claims 11 to 15, which is caused by a virus selected from the group consisting of Kusackievirus, Coxsackievirus type B, Poliovirus, HTLV-1, Hepatitis A, B, C, D, and E viruses, Varicella-zoster virus, Smallpox virus, Molluscum contagiosum, Human papillomavirus, Parvovirus B19, Rubella virus, Human immunodeficiency virus, Rotavirus, Norovirus, Astrovirus, Ebola virus, Marburg virus, Dengue virus (DENV), and Zika virus.

17. The method according to any one of claims 11 to 16, wherein the viral infection is an infection of tissue selected from the group consisting of central nervous system tissue, eye tissue, upper respiratory system tissue, lower respiratory system tissue, lung tissue, kidney tissue, bladder tissue, spleen tissue, heart tissue, gastrointestinal tissue, epidermal tissue, reproductive tissue, nasal cavity tissue, laryngeal tissue, tracheal tissue, bronchial tissue, oral tissue, blood tissue, and muscle tissue.

18. The method according to any one of claims 11 to 17, wherein the administration is systemic.

19. The method according to any one of claims 11 to 17, wherein the administration is localized to the site of viral infection.

20. The method according to any one of claims 11 to 19, further comprising the step of administering at least one additional therapeutic agent.

21. The method according to claim 20, wherein at least one additional therapeutic agent is an antiviral therapeutic agent.

22. A method for treating influenza infection in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-stranded compound according to any one of claims 1 to 10 to a subject having an influenza infection.

23. The method according to claim 22, wherein the influenza infection is either an influenza A infection or an influenza B infection.

24. The method according to claim 22 or 23, further comprising the step of administering at least one additional antiviral therapeutic agent.

25. A method for treating coronavirus disease in a subject, comprising the step of administering an immunostimulatory oligonucleotide double-stranded compound according to any one of claims 1 to 10 to a subject having coronavirus disease.

26. The method according to claim 25, wherein the coronavirus disease is COVID-19.

27. The method according to claim 25 or 26, further comprising the step of administering at least one additional antiviral therapeutic agent.

28. The method according to claim 25 or 26, further comprising the step of administering plasma obtained from a subject who has recovered from coronavirus disease.

29. A method for increasing the efficacy of an antiviral therapeutic agent, comprising the step of administering an immunostimulant oligonucleotide double-strand according to any one of claims 1 to 10 and at least one antiviral therapeutic agent.

30. Antiviral drugs include abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir (agenese), amodiaquin, apirimod, arbidol, atazanavir, atripra, atovaquone, paravir, baloxavir marboxil (Xofluza®), biktarvy, boceprevir (Victrelis®), cidofovir, clofazimine, clomiphene, clofazamine, cobicistat (Tybost®), Combivir (fixed-dose drug), daclatasvir (Daklinza®), darunavir, delavirdin, descovy, didanosine, docosanol, dolutegravir, doravirine (Pifeltro®), ecoliebel, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine (Intelence®), famciclovir, favipiravir, fenofibrate, homivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir (Cytovene®), Ibasitabine, Ibalizumab (Trogarzo®), Idoxuridine, Imiquimod, Immunovir, Indinavir, Inosine, Integrase inhibitors, Type I interferon, Type II interferon, Type III interferon, Interferon, Ivermectin, Lamivudine, Lasaroside, Letermovir (Prevymis®), Lopinavir, Lobilide, Mannose-binding lectin, Maraviroc, Methisazone, Moroxidine, Nafamostat, Nelfinavir, Nevirapine, Nexav ir (registered trademark), nilotinib, nitazoxanide, Norvir, nucleoside analog, oseltamivir (Tamiflu (registered trademark)), pazopanib, pegylated interferon alfa-2a, pegylated interferon alfa-2b, penciclovir, peramivir (Rapivab (registered trademark)), preconalil, podophyllotoxin, protease inhibitors (pharmacology), pionarizine, pyramidine, raltegravir, remdesivir, reverse transcriptase inhibitors, ribavirin, rilpivirine (Edurant (registered trademark)), rimantadine, ritonavir, saquinavir,The method according to claim 29, comprising a drug selected from the group consisting of simeprevir (Olysio®), sofosbuvir, stabuzin, synergistic enhancer (antiretroviral drug), tafenoquin, telaprevir, terbivudine (Tyzeka®), tenofovir alafenamide, tenofovir disoproxil, tenofovir, toremifene, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir (Valtrex), valganciclovir, bermurafenib, venetoclax, bicribiloc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza®), and zidovudine.

31. The method according to claim 29 or 30, wherein an immunostimulatory oligonucleotide double-strand and at least one antiviral therapeutic agent are administered substantially simultaneously.

32. The method according to claim 29 or 30, wherein an immunostimulatory oligonucleotide double-strand and at least one antiviral therapeutic agent are administered at different time points.

33. A pharmaceutical composition comprising an immunostimulant oligonucleotide double helix according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

34. A pharmaceutical composition comprising an immunostimulatory oligonucleotide double-strand according to any one of claims 1 to 10 and at least one antiviral therapeutic agent.

35. The composition according to claim 33 or 34, formulated for airway administration.

36. The composition according to claim 35, formulated for aerosol administration, nebulizer administration, or tracheal lavage administration.

37. A method for inducing interferon (IFN) production, comprising the step of administering to a subject in need of interferon production an immunostimulatory oligonucleotide double-strand according to any one of claims 1 to 10, or a pharmaceutical composition according to any one of claims 33 to 36, thereby increasing IFN production after administration.

38. The method according to claim 37, wherein the IFN production is the production of type I IFN, type II IFN, or type III IFN.

39. The method according to claim 37 or 38, wherein the IFN production is the production of type I IFN.

40. The method according to any one of claims 37 to 39, wherein the type I IFN is IFN-α, IFN-β, IFN-ε, IFN-κ, or IFN-ω.

41. The method according to claim 38, wherein the type II IFN is IFN-γ.

42. The method according to claim 37, wherein increased IFN production increases the cell's resistance to viral infection.

43. A method for treating an IFN-related disease, comprising the step of administering an immunostimulatory oligonucleotide double chain according to any one of claims 1 to 10 to a subject in need thereof.

44. The method according to claim 43, wherein the subject requiring such treatment has an IFN-related disease or is at risk of developing an IFN-related disease.

45. The method according to claim 43, further comprising the step of diagnosing a subject who has or is at risk of having an IFN-related disease prior to the administration step.

46. The method according to claim 43, further comprising the step of receiving the results of an assay that diagnoses a subject having or being at risk of having an IFN-related disease, prior to the administration step.

47. The method according to claim 43, wherein the IFN-related disease is a disease characterized by reduced IFN levels compared to a reference level.

48. The method according to claim 43, wherein the IFN-related disease is a disease characterized by reduced type I IFN levels compared to a reference level.

49. The method according to any one of claims 43 to 48, wherein the IFN-related disease is selected from the group consisting of viral infectious diseases, bacterial infectious diseases, fungal infectious diseases, parasitic infectious diseases, cancer, and autoimmune diseases.

50. The method according to any one of claims 43 to 49, further comprising the step of administering at least one additional therapeutic agent.

51. The method according to claim 50, wherein at least one additional therapeutic agent is an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, an anticancer agent, or an anti-autoimmune agent.

52. A composition comprising an immunostimulatory oligonucleotide double chain according to any one of claims 1 to 10 and at least one antibacterial therapeutic agent.

53. A composition comprising an immunostimulatory oligonucleotide double-chain according to any one of claims 1 to 10 and at least one antifungal therapeutic agent.

54. A composition comprising an immunostimulatory oligonucleotide double-chain according to any one of claims 1 to 10 and at least one antiparasitic therapeutic agent.

55. A composition comprising an immunostimulatory oligonucleotide double chain according to any one of claims 1 to 10 and at least one anticancer drug.

56. A composition comprising an immunostimulatory oligonucleotide double chain according to any one of claims 1 to 10 and at least one anti-autoimmune therapeutic agent.

57. The composition according to any one of claims 52 to 56, further comprising a pharmaceutically acceptable carrier.

58. An immunostimulatory oligonucleotide double helix conjugated to an antigen or vaccine, containing SEQ ID NO:1 at its 5' end.

59. A composition comprising an immunostimulatory oligonucleotide double chain according to claim 58.

60. A composition comprising an immunostimulatory oligonucleotide double-chain and a vaccine according to any one of claims 1 to 10.

61. A composition comprising an immunostimulant oligonucleotide bichain and nanoparticles according to any one of claims 1 to 10.

62. Nanoparticles comprising an immunostimulatory oligonucleotide double chain according to any one of claims 1 to 10.

63. A composition comprising an immunostimulant oligonucleotide bichain and nanoparticles as described in claim 58.

64. Nanoparticles comprising the immunostimulant oligonucleotide double chain described in claim 58.

65. The composition according to any one of claims 59 to 64, further comprising a pharmaceutically acceptable carrier.

66. A method of administering vaccines, to those who need them. a. The immunostimulatory oligonucleotide bistrand described in claim 58, b. The composition according to any one of claims 59 to 64, or c. An immunostimulatory oligonucleotide double helix according to any one of claims 1 to 10, and a vaccine A method comprising the step of administering a substance.

67. A method to increase the effectiveness of a vaccine, and to those who need it. a. The immunostimulatory oligonucleotide bistrand described in claim 58, b. The composition according to any one of claims 59 to 64, or c. An immunostimulatory oligonucleotide double helix according to any one of claims 1 to 10, and a vaccine A method comprising the step of administering a substance.

68. The composition according to claim 33 or 34, formulated for intravenous, intramuscular, intraperitoneal, subcutaneous, or intrathecal administration.

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