Liposomal sting agonists for intranasal treatment of respiratory infections

Abstract

The invention relates to a composition that includes a particle, a first modulator associated with the particle, and a second modulator in free form in the composition. The first and second modulators may include a pattern recognition receptor agonist, an activator of the immune system, and a combination thereof. The invention also relates to methods of treating a disease or condition in a subject by administering the aforementioned composition to the subject.

Description

PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011TITLE LIPOSOMAL STING AGONISTS FOR INTRANASAL TREATMENT OF RESPIRATORY INFECTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority from, and incorporates by reference the entire disclosure of, U. S. Provisional Patent Application 63 / 728,211, filed on December 5, 2024.BACKGROUND

[0002] Viral pathogens, including influenza viruses, constantly develop drug-resistant strains and variants, making viral respiratory infections one of the most challenging global health threats.SUMMARY

[0003] The present disclosure provides compositions comprising a particle and one or more modulators useful for treating diseases and conditions described herein.

[0004] In some embodiments, the present disclosure provides a composition comprising: a particle, wherein a first modulator is associated with the particle, and a second modulator that is in free form in the composition, wherein the first and second modulators are each independently selected from the group consisting of a pattern recognition receptor agonist, an activator of the immune system, and a combination thereof.

[0005] In some embodiments, the first and second modulators are the same. In some embodiments, the first and second modulators are each independently selected from the group consisting of an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, CF501, MSA-2, SHR1032, C-178, H-151, divalent cations, and a combination thereof. In some embodiments, the first and second modulators are each independently a compound of formula I, as defined herein. In some embodiments, the first modulator comprises an endogenous agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, the first modulator comprises 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2 ’,3 ’-cGAMP). In somePCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011embodiments, the second modulator comprises an endogenous agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, the second modulator comprises 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP). In some embodiments, the molar ratio of the first modulator to the second modulator is about 1:2 to about 1:20. In some embodiments, the molar ratio of the first modulator to the second modulator is about 1:10.

[0006] In some embodiments, the composition comprises a lipid-based particle. In some embodiments, the lipid-based particle is a liposome. In some embodiments, the lipid-based particle is anionic. In some embodiments, the lipid-based particle comprises one or more anionic lipids, one or more neutral lipids, and one or more polyethylene glycol (PEG)-lipids. In some embodiments, the PEG moiety of the one or more PEG-lipids has an average molecular weight of 500-5000 Da. In some embodiments, the lipid-based particle comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), cholesterol, or a combination thereof. In some embodiments, the lipid-based particle comprises DPPC, DPPG, DPPE-PEG2000, and cholesterol in a molar ratio of about 10:1:1:1.

[0007] In some embodiments, the composition further comprises an antigen. In some embodiments, the antigen is operable to elicit an immune response in a subject against a virus. In some embodiments, the antigen comprises a protein or peptide derived from a virus, or a polynucleotide encoding the protein or peptide. In some embodiments, the antigen comprises a protein or peptide derived from a virus. In some embodiments, the virus is selected from the group consisting of an influenza virus, an influenza A virus, an influenza B virus, an influenza H5N1 virus, a SARS-CoV-2 virus, a respiratory virus, or a combination thereof.

[0008] In some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, the method comprising administering to the subject a composition described herein.

[0009] In some embodiments, the present disclosure provides a method of treating a disease or condition in a subject, the method comprising administering a composition to a subject, wherein the composition comprises a particle; and a modulator, wherein the modulator is selected from the group consisting of a pattern recognition receptor agonist, an activator of the immune system, and a combination thereof.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0010] In some embodiments, the disease or condition is a viral infection. In some embodiments, the viral infection is caused by a virus selected from the group consisting of an influenza virus, an influenza A virus, an influenza B virus, an H5N1 virus, a respiratory virus, a SARS-CoV-2 virus, and a combination thereof. In some embodiments, the subject is a human being. In some embodiments, the subject is immunocompromised. In some embodiments, the subject is 60 years of age or older. In some embodiments, the subject is receiving or has received an antiviral drug. In some embodiments, the composition induces Ifnb and Cxcl10 expression in the subject. In some embodiments, the composition is administered through intranasal administration. In some embodiments, the composition is administered through inhalational administration. In some embodiments, the composition is administered in a single dose.

[0011] In some embodiments of methods provided herein, the modulator is selected from the group consisting of an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3 ', 5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, CF501, MSA-2, SHR1032, C-178, H-151, divalent cations, and a combination thereof. In some embodiments, the modulator is a compound of formula I, as defined herein. In some embodiments, the modulator comprises an endogenous agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, the modulator comprises 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’, 3’ -cGAMP).

[0012] In some embodiments of methods provided herein, the composition comprises a lipid-based particle. In some embodiments, the lipid-based particle is a liposome. In some embodiments, the lipid-based particle is anionic. In some embodiments, the lipid-based particle comprises one or more anionic lipids, one or more neutral lipids, and one or more PEG-lipids. In some embodiments, the PEG moiety of the one or more PEG-lipids has an average molecular weight of 500-5000 Da. In some embodiments, the lipid-based particle comprises 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), cholesterol, or a combination thereof. In some embodiments, the lipid-based particle comprises DPPC, DPPG, DPPE-PEG2000, and cholesterol in a molar ratio of about 10:1:1:1.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0013] In some embodiments of methods provided herein, the composition further comprises an antigen. In some embodiments, the antigen is operable to elicit an immune response in a subject against a virus. In some embodiments, the antigen comprises a protein or peptide derived from a virus, or a polynucleotide encoding the protein or peptide. In some embodiments, the antigen comprises a protein or peptide derived from a virus. In some embodiments, the virus is selected from the group consisting of an influenza virus, an influenza A virus, an influenza B virus, an influenza H5N1 virus, a SARS-CoV-2 virus, a respiratory virus, or a combination thereof.BRIEF DESCRIPTION OF DRAWINGS

[0014] FIG. 1A provides a depiction of a composition that includes a particle, a modulator, and an optional antigen for treating a viral infection, in accordance with various embodiments of the present disclosure.

[0015] FIGS. IB and 1C illustrate methods of treating a viral infection in a subject.

[0016] FIGS. 2A-2I provide results showing the safety and efficacy of NanoSTING formulations.FIG. 2A provides a schematic of NanoSTING (NS) and NanoSTING-2’,3’-cGAMP (NS-CG) formulations. The diagram shows the co-formulation of 2’,3’-cGAMP (CG) with NanoSTING, leading to NS-CG formulation. FIG. 2B provides a THP-1 dual assay showing luminescence (RLU) response after 12 and 24 hours. The assay tested different doses of cGAMP (CG) and NanoSTING (NS). FIG. 2C provides an experimental setup of mouse model treatments. Mice were administered either PBS, CG (2’,3’-cGAMP) alone, NS (NanoSTING), or NS-CG (Nano STING-2 ’,3’ -cGAMP) at different doses. 24 hours later, the mice were euthanized, and lung and nasal tissues were collected for RT-qPCR to assess Ifnb and Cxcl10 gene expression, along with lung histopathology. FIG. 2D shows an RT-qPCR fold change of Ifnb and Cxcl10 in nasal tissues after treatment with NanoSTING (4, 6, and 12 pg) compared to PBS. Blue bars (circles) represent Ifnb, while red bars (squares) represent Cxcl10. FIG. 2E shows RT-qPCR fold change of Ifnb and Cxcl10 in nasal tissues after treatment with 2’,3’-cGAMP (10, 20, and 40 pg) compared to PBS. Blue bars (circles) represent Ifnb, and red bars (squares) represent Cxcl10. FIG. 2F shows RT-qPCR fold change of Ifnb and Cxcl10 in nasal tissues after treatment with NS4-CG10, NS4-CG20, and NS4-CG40. Blue bars (circles) represent Ifnb, and red bars (squares) represent Cxcl10. FIG.2G shows RT-qPCR fold change of Ifnb and Cxcl10 in lung tissues after treatment with NS4-CG10, NS4-CG20, and NS4-CG40. Blue barsPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011(circles) represent Ifnb, and red bars (squares) represent Cxcl10. FIG. 2H shows histopathology images of lung tissue from different treatment groups (CG 10, 20, 40 pg, NS 4, 6, 12 pg, PBS, and NS4-CG40). The images show lung tissue morphology and inflammation across different treatments.FIG. 2I shows a pathology score of lungs for the different treatment groups, showing low scores overall, indicating minimal lung pathology across treatments.

[0017] FIGS. 3A-3D show that single-dose therapeutic administration of NanoSTING-CG offers protection against Influenza A comparable to 10 doses of oseltamivir. FIG.3A shows a schematic of an experimental design. BALB / c mice were challenged with an aerosolized dose of 2 x 104CCID50of Influenza A / California / 04 / 2009 (HINldpm) virus. Following the challenge, groups of mice received the following treatments: a single dose of NS4-CG40 (4 μg of NanoSTING mixed with 40 μg of 2’,3’-cGAMP), Oseltamivir administered at 40 mg / kg / day (with the first dose given 4 hours post-infection, followed by 9 subsequent doses every 12 hours), or a placebo treatment (PBS). Blood samples were taken on day 0, and treatments were administered as indicated. Mice were euthanized at the end of the study (day 20) to assess endpoints, including percent survival and weight loss. FIG. 3B shows body weight change over time for different treatment groups. FIG.3C shows Kaplan-Meier survival curves showing percent survival of mice in the different treatment groups. FIG.3D shows viral titers in lung tissues at day 20, presented as Log10CCID50 / mL. Statistical significance is indicated by *p < 0.05 and **p< 0.01.

[0018] FIGS. 4A-4C show that single-dose therapeutic administration of NanoSTING-CG offers protection against Influenza B superior to oseltamivir. FIG.4A shows a study timeline. BALB / c mice were challenged with an aerosolized dose of 2 x 104CCID50Influenza B (B / Victoria / 2 / 87) virus. The treatment groups included NS4-CG40 (administered once on either day 1 or day 2 post-infection), Oseltamivir administered at 40 mg / kg / day (with the first dose given 4 hours post-infection, followed by 9 additional doses every 12 hours over 5 days), and a placebo group (PBS). Mice were euthanized on day 20 to assess endpoints such as survival and body weight change. FIG. 4B shows a percent body weight change over time in the different treatment groups. FIG. 4C shows Kaplan-Meier survival curves for mice in each treatment group.

[0019] FIGS. 5A-5F show that NanoSTING induces Ifnb and Cxcl10 expression in immunocompromised and aged mice models. FIG. 5A provides a schematic of an experimental design for RT-qPCR analysis of nasal tissue (NT) to assess Ifnb and Cxcl10 expression. Mice werePCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011treated with cyclophosphamide (CFX) via intraperitoneal injection (IP) at 0 hours, followed by intranasal (IN) administration of NS4-CG40 at 48 hours. Mice were euthanized at 72 hours for analysis. FIG. 5B shows RT-qPCR results showing fold change in Ifnb (blue, circles) and Cxcl10 (red, squares) expression in nasal tissues of different treatment groups. FIG. 5C shows a schematic of an experimental design for RT-qPCR analysis of nasal tissue. Mice were treated with dexamethasone (Dexa, IP) at 0 hours, followed by intranasal NS4-CG40 at 24 hours. Mice were euthanized at 48 hours for analysis. FIG. 5D shows RT-qPCR results showing fold change in Ifnb and Cxcl10 expression in nasal tissues of different treatment groups. NS4-CG40 upregulated both genes, with no significant change when combined with dexamethasone treatment. FIG. 5E shows a schematic of experimental design to assess the effect of age on RT-qPCR analysis of Ifnb and Cxcl10 expression. Young BALB / c (2 months) and aged B.129 mice (18-22 months) were administered 20 pg NS intranasally and euthanized at 48 hours for nasal tissue collection and analysis. FIG.5F shows RT-qPCR results showing fold change in Ifnb and Cxcl10 expression in nasal tissues of young and aged mice compared to the control group.

[0020] FIGS. 6A-6B show NS4-CG40 pre-treatment and post-treatment mitigates H5N1 -induced weight loss in mice, comparable to oseltamivir. FIG.6A shows a study timeline. The panel illustrates the experimental design for evaluating the efficacy of NS4-CG40 and Oseltamivir in mice challenged with H5N1 avian influenza virus (Influenza A / Vietnam / 1203 / 2004 H5N1 virus). Mice were divided into four treatment groups: i) Mice pre-treated with NS4-CG40 (Px) 24 hours before the viral challenge; ii) Mice treated with NS4-CG40 (Tx) 24 or 48 hours post-viral challenge; iii) Mice treated with Oseltamivir (70 mg / kg) beginning 4 hours post-challenge, followed by 9 additional doses every 12 hours; and iv) a control group that received no challenge (no virus exposure) or PBS (placebo).FIG. 6B shows body weight change. This panel shows the percentage body weight change of mice over the 20-day period post-infection. Weight loss and recovery trends are compared across different treatment groups. Oseltamivir-Tx: Mice treated with Oseltamivir post-challenge; NanoSTING-Px-24h: Mice pre-treated with NS4-CG40 24 hours prior to challenge; NanoSTING-Tx-24h and 48h: Mice treated with NS4-CG40 24 or 48 hours after challenge; PBS: Placebo-treated mice; No challenge: Mice not exposed to H5N1 AIV.

[0021] FIGS. 7A-7I demonstrate the stability and efficacy of NanoSTING formulations. FIG. 7A provides a schematic of NanoSTING (NS), a liposomal formulation encapsulating cGAMP, a naturalPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011STING agonist. FIG.7B provides particle size distribution ofNanoSTING measured over a 12-month period using dynamic light scattering. FIG.7C provides Zeta potential measurements ofNanoSTING over a 12-month period. FIG. 7D provides quantification of encapsulated cGAMP in NanoSTING over a year using HPLC. FIG. 7E provides a schematic of RT-qPCR experimental design. Mice were intranasally administered PBS, NanoSTING (0.2 mg / kg), or free cGAMP (2 mg / kg), and euthanized after 24 hours for tissue collection (nasal tissue and lungs). FIG. 7F shows an RT-qPCR analysis of Ifnb1 (black bars) and Cxcl10 (gray bars) expression in nasal tissues from mice treated with PBS, NanoSTING, or cGAMP. FIG.7G shows independent replication in a second cohort of mice showing robust Ifnb1 and Cxcl10 induction in nasal tissues following NanoSTING administration. FIG. 7H shows RT-qPCR analysis of Ifnb1 and Cxcl10 expression in lung tissues after NanoSTING treatment.FIG. 71 shows representative H & E-stained lung sections from mice 24 hours after treatment with PBS, NanoSTING, or cGAMP (scale bar = 100 pm), Magnification 20x.

[0022] FIGS. 8A-8D show that single-dose therapeutic administration of NanoSTING offers protection against Influenza A comparable to 10 doses of oseltamivir. FIG. 8A provides a schematic of the experimental design. BALB / c mice were challenged with an aerosolized dose of 1 x 104.3CCID50of Influenza A / California / 04 / 2009 (HINldpm) virus. Following the challenge, groups of mice received the following treatments: a single dose ofNanoSTING (0.2 mg / kg), oseltamivir administered at 30 mg / kg / day (with the first dose given 4 hours post-infection, followed by 9 subsequent doses every 12 hours), or a placebo treatment (PBS). Blood samples were taken on day 0, and treatments were administered as indicated. Mice were euthanized at the end of the study (day 20) to assess endpoints, including percent survival and weight loss. FIG. 8B shows a change in body weight over time for different treatment groups. FIG. 8C shows Kaplan-Meier survival curves showing percent survival of mice in the different treatment groups, p-values were calculated using the log-rank (Mantel-Cox) test. FIG. 8D shows viral titers in lung tissues at day 20, presented as Log10CCID50 / mL. Statistical significance is indicated by *p < 0.05 and **p < 0.01.

[0023] FIGS. 9A-9C show that single-dose therapeutic administration of NanoSTING offers protection against Influenza B superior to oseltamivir. FIG. 9A provides a study timeline. BALB / c mice were challenged with an aerosolized dose of 1 x 104.3CCID50of Influenza B strain B / Brisbane / 60 / 2008 (Victoria lineage) virus. The treatment groups included intranasal NanoSTING (0.2 mg / kg), oral oseltamivir administered at 30 mg / kg / day (with the first dose given 4 hours post-PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011infection, followed by 9 additional doses every 12 hours over 5 days), or placebo (PBS). Mice were euthanized on day 20 to assess endpoints such as survival and body weight change. FIG. 9B shows a change in body weight over time for different treatment groups. FIG. 9C shows Kaplan-Meier survival curves for mice in each treatment group, / ^-values were calculated using the log-rank (Mantel-Cox) test.

[0024] FIGS. 10A-10F show that NanoSTING induces activation of Ifnb1 and Cxcl10 in immunocompromised and aged mice models. FIG. 10A provides a schematic of experimental design for RT-qPCR analysis of nasal tissue (NT) to assess Ifnb1 and Cxcl10 expression. Mice were treated with cyclophosphamide (CFX) via intraperitoneal injection (IP) at 0 hours, followed by intranasal (IN) administration of NanoSTING (0.2 mg / kg) at 48 hours. Mice were euthanized at 72 hours for analysis.FIG. 10B shows RT-qPCR results showing fold change in Ifnb1 and Cxcl10 expression in nasal tissues of different treatment groups. FIG. 10C shows a schematic of experimental design for RT-qPCR analysis of nasal tissue. Mice were treated with dexamethasone (DX, IP) at 0 hours, followed by intranasal NanoSTING (0.2 mg / kg) at 24 hours. Mice were euthanized at 48 hours for analysis. FIG.10D shows RT-qPCR results showing fold change in Ifnb1 and Cxcl10 expression in nasal tissues of different treatment groups. FIG. 10E shows a schematic of experimental design to assess the effect of age on RT-qPCR analysis of Ifnb1 and Cxcl10 expression. Young BALB / c (2 months) and aged B.129 mice (18-22 months) were administered NanoSTING (0.1 mg / kg) intranasally and euthanized at 48 hours for nasal tissue collection and analysis. FIG. 10F shows RT-qPCR results showing fold change in Ifnb1 and Cxcl10 expression in nasal tissues of young and aged mice compared to the control group.

[0025] FIGS. 11A-11G provide data showing that NanoSTING treatment protects against HP Al A(H5N1) infection. FIG. 11A provides a schematic of the experimental timeline showing five treatment groups: mice received either NanoSTING (0.2 mg / kg) prophylactically 24 hours before viral challenge (Px-24h), therapeutically at 24-, 48- or 72 hours post-challenge (Tx-24h, Tx-48h and Tx-72h), oseltamivir (75 mg / kg / day) starting 4 hours post-challenge for 10 doses, PBS (placebo), or no virus challenge (non-challenged controls). FIGS. 11B-11G show body weight changes being monitored daily for 15 days across all treatment groups. FIG. 11B displays weight trajectories for placebo mice, while FIG. 11C shows weight loss in Oseltamivir-treated mice. FIGS. 11D-11G illustrate weight changes in mice treated with NanoSTING pretreatment (FIG. 11D), and NanoSTINGPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011post-treatment at 24 hours (FIG. 11E), 48 hours (FIG. 11F), or 72 hours (FIG. 11G) after challenge.FIGS. 11H-11I show the assessment of lung viral titers on Day 5 post-infection. FIG. 11H shows titers from mice receiving NanoSTING post-treatment (Tx-24h, Tx-48h) or oseltamivir. FIG. 11I shows viral titers in mice pretreated with NanoSTING (Px-24h) or oseltamivir.DETAILED DESCRIPTION

[0026] It is to be understood that both the foregoing general description and the following detailed description are illustrative and explanatory, and are not restrictive of the subject matter, as claimed. In this application, the use of the singular includes the plural, the word “a” or “an” means “at least one”, and the use of “or” means “and / or”, unless specifically stated otherwise. Furthermore, the use of the term “including”, as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements or components comprising one unit and elements or components that include more than one unit unless specifically stated otherwise.

[0027] The section headings used herein are for organizational purposes and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated herein by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines a term in a manner that contradicts the definition of that term in this application, this application controls.

[0028] An urgent demand exists for effective, safe, and affordable therapeutics to combat viral infections. In particular, respiratory viral infections, such as influenza, present a significant challenge in modern medicine. Numerous embodiments of the present disclosure aim to address the aforementioned need.

[0029] Definitions

[0030] The term “about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by 20 percent up or down (higher or lower). In some embodiments, the term “about” modifies a numerical value above andPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011below the stated value by 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% up or down (higher or lower).

[0031] The term "aliphatic" or "aliphatic group", as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "carbocycle," "cycloaliphatic" or "cycloalkyl"), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-6 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. In some embodiments, "cycloaliphatic" (or "carbocycle" or "cycloalkyl") refers to a monocyclic C3-C6 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

[0032] The term "alkyl," as used herein, refers to saturated, straight- or branched-chain hydrocarbon radicals derived from an aliphatic moiety containing between one and six carbon atoms by removal of a single hydrogen atom. Unless otherwise specified, alkyl groups contain 1-12 carbon atoms. In certain embodiments, alkyl groups contain 1-8 carbon atoms. In certain embodiments, alkyl groups contain 1-6 carbon atoms. In some embodiments, alkyl groups contain 1-5 carbon atoms, in some embodiments, alkyl groups contain 1-4 carbon atoms, in some embodiments alkyl groups contain 1-3 carbon atoms, and in some embodiments alkyl groups contain 1-2 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert- butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.

[0033] The term "alkenyl," as used herein, denotes a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Unless otherwise specified, alkenyl groups contain 2-12 carbon atoms. InPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011certain embodiments, alkenyl groups contain 2-8 carbon atoms. In certain embodiments, alkenyl groups contain 2-6 carbon atoms. In some embodiments, alkenyl groups contain 2-5 carbon atoms, in some embodiments, alkenyl groups contain 2-4 carbon atoms, in some embodiments alkenyl groups contain 2-3 carbon atoms, and in some embodiments alkenyl groups contain 2 carbon atoms. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, l-methyl-2-buten-l-yl, and the like.

[0034] The term "alkynyl," as used herein, refers to a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. Unless otherwise specified, alkynyl groups contain 2-12 carbon atoms. In certain embodiments, alkynyl groups contain 2-8 carbon atoms. In certain embodiments, alkynyl groups contain 2-6 carbon atoms. In some embodiments, alkynyl groups contain 2-5 carbon atoms, in some embodiments, alkynyl groups contain 2-4 carbon atoms, in some embodiments alkynyl groups contain 2-3 carbon atoms, and in some embodiments alkynyl groups contain 2 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

[0035] The term "aryl" used alone or as part of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl," refers to monocyclic and bicyclic ring systems having a total of five to 10 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term "aryl" may be used interchangeably with the term "aryl ring". In some embodiments, an 8-10 membered bicyclic aryl group is an optionally substituted naphthyl ring. In certain embodiments of the present invention, "aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term "aryl," as it is used herein, is a group in which an aromatic ring is fused to one or more non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl, phenanthridinyl, or tetrahydronaphthyl, and the like.

[0036] The term "halogen" or "halo" means F, CI, Br, or I.

[0037] The term "heteroatom" means one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quatemized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+(as in N-substituted pyrrolidinyl)).PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0038] The terms "heteroaryl" and "heteroar-," used alone or as part of a larger moiety, e.g., "heteroaralkyl,'1or "heteroaralkoxy," refer to groups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 n electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pteridinyl. The terms "heteroaryl" and "heteroar-", as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono-or bicyclic. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. The term "heteroaralkyl" refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.

[0039] As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-27 / -pyrrolyl), NH (as in pyrrolidinyl), or NR (as in A-substituted pyrrolidinyl).

[0040] A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclic group,” “heterocyclic moiety,” and “heterocyclic radical,” are used interchangeably herein, and also include groups in which a heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted with a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted.

[0041] As used herein, the term "partially unsaturated" refers to a ring moiety that includes at least one double or triple bond. The term "partially unsaturated" is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.

[0042] The term "unsaturated," as used herein, means that a moiety has one or more units of unsaturation.

[0043] As described herein, compounds of the disclosure may contain “substituted” moieties. In general, the term “substituted” means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0044] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; -(CH2)0-4R°; -(CH2)0-1OR°; -0(CH2)o-4R°, -O-(CH2)o 4C(O)OR°; -(CH2)O-4CH(OR°)2; -(CH2)O-4SR°; -(CH2)o-4Ph, which may be substituted with R°; — (CH2)o 0('CH2)O 1 Ph which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o 40(CH2)o 1 -pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)O N(R°)2; -(CH2)O^N(R°)C(0)R°; -N(R°)C(S)R°; -(CH2)O-PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-0114N(R°)C(O)NR°2; -N(R°)C(S)NR°2; -(CH2)O4N(RO)C(O)OR°; - N(R°)N(R°)C(O)R°; -N(RO)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; (CH2)0^C(O)R°; C(S)R°; (CH2)O4C(0)0RO; -(CH2)O4C(O)SR°; -(CH2)O4C(O)OSiR°3; -(CH2)0 4OC(O)R°; -OC(O)(CH2)04S R°; (CH2)O 4SC(O)RO; (CH2)( 4C(O)NR°2; C(S)NRO2; C(S)SR°; SC(S)SR°, -(CH2)O-4OC(O)NRO2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)RO; -C(NOR°)R°; -(CH2)O4SSRO; -(CH2)OS(O)2RC; -(CH2)O 4S(O)2ORO; -(CH2)O 4OS(O)2RO; -S(O)2NRO2; -(CH2)O-4S(O)RO; -N(RO)S(O)2NR°2; -N(RO)S(O)2R°; -N(OR°)R°; -C(NH)NRO2; -(CH2)O4P(O)2RO; -(CH2)O 4P(O)RO2; -(CH2)O 4OP(O)RO2; -(CH2)O 4OP(O)(OR°)2; SiR°3; -(Ci-4straight or branched alkylene)O-N(R°)2; or-(Ci-4straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, Ci-6 aliphatic, -CH2Ph, -0(CH2)o iPh, -CH2-(5-6 membered heteroaryl ring), or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0045] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o 2 *, -(haloR*), -(CH2)O2OH, -(CH2)O 2OR*, -(CH2)O 2CH(OR*)2; -O(haloR’), -CN, -N3, -(CH2)02C(O)R’, -(CH2)O2C(O)OH, -(CH2)O 2C(O)OR*, -(CH2)O2SR*, -(CH2)O -2SH, -(CH2)O2NH2, -(CH2)O-2NHR*, -(CH2)O2NR*2, -NO2, -SiR*3, -OSiR -C(O)SR* -(Ci-4straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from Ci-4 aliphatic, -CH2Ph, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.

[0046] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =0, =S, =NNR*2, =NNHC(O)R*, =NNHC(0)0R*, =NNHS(O)2R*, =NR*, =N0R*, -O(C(R*2))2-3O-, or-S(C(R*2))2-3S-, wherein each independent occurrence of R* isPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(CR* 2)2 30-wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0047] Suitable substituents on the aliphatic group of R* include halogen, -R’, -(haloR*), -OH, -OR’, -O(haloR’), -CN, -C(O)OH, -C(O)OR’, -NH2, -NHR’, -NR’2, or -NO2, wherein each R’ is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C 1 4 aliphatic, -CH2PI1, 0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0048] Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include -R'. -NRT2, -C(O)Rf, -C(O)ORt, -C(O)C(O)RT, -C(O)CH2C(O)R+, -S(O)2Rf, -S(O)2NRT2, -C(S)NR^2, -C(NH)NR^2, or -N(R')S(O)2R'; wherein each R' is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R', taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0049] Suitable substituents on the aliphatic group of Rfare independently halogen, -R’, -(haloR*), -OH, -OR’, -O(haloR’), -CN, -C(O)OH, -C(O)OR’, -NH2, -NHR’, -NR’2, or -NO2, wherein each R* is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, -CH2PI1, -0(CH2)o iPh, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0050] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans andPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19.

[0051] As used herein, the term “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration of a composition to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. In some embodiments, treatment is therapeutic treatment. In some embodiments, treatment is preventative treatment.

[0052] Compositions

[0053] In some embodiments, the present disclosure pertains to a composition. In some embodiments, the composition includes a particle. In some embodiments, the composition also includes one or more modulators. In some embodiments, the composition also includes an antigen. As set forth in more detail herein, the compositions of the present disclosure can include various types of particles, modulators, and / or antigens in various arrangements.

[0054] Modulators

[0055] The compositions of the present disclosure can include various modulators. For instance, in some embodiments, the modulator includes, without limitation, a pattern recognition receptor agonist, an activator of the immune system, or a combination thereof.

[0056] In some embodiments, a modulator is a pattern recognition receptor agonist. Exemplary pattern recognition receptor agonists include, but are not limited to, MF59, AS03, MPLA (AS01), QS-21 (AS01), CpG 1018, poly(ICLC), poly(IC12U), ARNAX, R837, R848, CpG7909, MGN1703, IC31, flagellin 2’,3’-cGAMP, 3’,3’-cGAMP, cAIMP, c-di-AMP, LTA-BS, C12-iE-DAP, FSL-1, TL8-506, scleroglucan, poly(A: U), poly(I: C) HMW, poly(dA:dT)naked, poly(dG:dC)naked, imiquimod, gardiquimod, poly(I: C) LMW, Pam2CSK4, MDP, ODN 4084-F, loxoribine, iE-DAP, FLA-BS, c-di-GMP, adilipoline, Pam3CSK4, and LPS-RS. See, e.g., Garcia, G., Jr. et al., Cell Reports Medicine, May 16, 2023; 4(5), 101024; and Ong, G. H. et al., Front. Cell. Infect. Microbiol. 2021 Oct 6;11:745016.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0057] In some embodiments, a modulator is an activator of the immune system. Activators of the immune system include, but are not limited to, cyclic dinucleotides (CDNs), non-cyclic dinucleotides, CDN-infused exosomes, engineered bacterial vectors, and hybrid small molecule-nucleic acids.

[0058] In some embodiments, a modulator is a modulator of the stimulator of interferon genes (STING) pathway. In some embodiments, a modulator is an agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, a modulator is an endogenous agonist of the stimulator of interferon genes (STING) pathway. In some embodiments, a modulator includes, without limitation, an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphateadenosine monophosphate (cGAMP), 2’, 3 ’-cyclic guanosine monophosphate-adenosine monophosphate (2’, 3 ’-cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, ADU-S100, MK-1454, CF501 (CAS No. 2408723-12-4), MSA-2 (CAS No. 129425-81-6), SHR1032 (structure disclosed in Chunying Song, et al. Sci Rep. 2022 May 20;12(l):8579), C-178 (CAS No. 329198-87-0), H-151 (CAS No. 941987-60-6), divalent cations, or a combination thereof.

[0059] In some embodiments, a modulator includes 2’, 3 ’-cyclic guanosine monophosphate-adenosine monophosphate (2’, 3 ’-cGAMP). In some embodiments, a modulator is 2’, 3 ’-cyclic guanosine monophosphate-adenosine monophosphate (2’, 3 ’-cGAMP).

[0060] In some embodiments, a modulator is a compound of formula I:or a pharmaceutically acceptable salt thereof, wherein:Ring A and Ring B are each independently selected from the group consisting of:PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011each X1and X2is independently -CR- or -N-;each X3is independently -C(R)2-, -O-, or -NR-;Xcand Xdare each independently -OR, -SR, -N(R)2, BH3, or optionally substituted C1-12 aliphatic; Xeand Xfare each independently -O-, -S-, or -N(R)-;each R1and R2is independently selected from the group consisting of hydrogen, halogen, -NO2, - CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, - S(O)2R, - C(0)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, - N(R)C(=NR)N(R)2, - C(=NR)N(R)2, -C=NOR, -N(R)C(0)N(R)2, -N(R)SO2N(R)2, - N(R)SO2R, -OC(O)N(R)2, and optionally substituted C1-12 aliphatic or C1-4 alkoxy-Ci-4 alkyl;each R3, R4, R5, R6, and R7is independently selected from the group consisting of hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, - C(O)CH2C(O)R, - S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, - N(R)N(R)2, - N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, - N(R)SO2N(R)2, -N(R)SO2R, - 0C(0)N(R)2, or an optionally substituted group selected from C1-12 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each R is independently selected from the group consisting of hydrogen or an optionally substituted group selected from C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:two R groups on the same nitrogen are taken together with their intervening atoms to form an optionally substituted 3-7 membered saturated, partially unsaturated, or heteroaryl ring having 1- 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.R4

[0061] In some embodiments of formula I, Ring A is. In some embodiments,Ring A is. In some embodiments, Ring A is. In some embodiments,Ring A isPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0062] In some embodiments of formula I, Ring B is. In some embodiments,Ring B is. In some embodiments, Ring B is. In some embodiments,R4Sd NDRing B is

[0063] In some embodiments of formula I, X1is -CR-. In some embodiments, X1is -N-.

[0064] In some embodiments of formula I, X2is -CR-. In some embodiments, X2is -N-.

[0065] In some embodiments of formula I, X3is - C(R)2-. Tn some embodiments, X3is -O-. In some embodiments, X3is -NR-.

[0066] In some embodiments of formula I, Xcis -OR (e.g., -OH). In some embodiments, Xcis -SR (e.g., -SH). In some embodiments, Xcis -N(R)2. In some embodiments, Xcis BH3. In some embodiments, Xcis optionally substituted C1-12 aliphatic.

[0067] In some embodiments of formula I, Xdis -OR (e g., -OH). In some embodiments, Xdis -SR (e.g., -SH). In some embodiments, Xdis -N(R)2. In some embodiments, Xdis BH3. In some embodiments, Xdis optionally substituted Ci-12 aliphatic.

[0068] In some embodiments of formula I, Xeis -O-. In some embodiments, Xeis -S-. In some embodiments, Xeis -N(R)-.

[0069] In some embodiments of formula I, Xfis -O-. In some embodiments, Xfis -S-. In some embodiments, Xfis -N(R)-.

[0070] In some embodiments of formula I, R1is hydrogen, halogen, -OR, -SR, -N(R)2, and optionally substituted Ci-12 aliphatic or C1-4 alkoxy-Ci-4 alkyl. In some embodiments, R1is hydrogen. In somePCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011embodiments, R1is halogen. In some embodiments, R1is -OR. In some embodiments, R1is -OH. In some embodiments, R1is fluoro. In some embodiments, R1is Ci-12 aliphatic. In some embodiments, R1is C1-6 aliphatic. In some embodiments, R1is C1-3 aliphatic. In some embodiments, R1is methyl. In some embodiments, R1is C1-4 alkoxy-Ci-4 alkyl. In some embodiments, R1is methoxy-ethyl.

[0071] In some embodiments of formula I, R2is hydrogen, halogen, -OR, -SR, -N(R)2, and optionally substituted C1-12 aliphatic or C1-4 alkoxy-Ci-4 alkyl. In some embodiments, R2is hydrogen. In some embodiments, R2is halogen. In some embodiments, R2is -OR. In some embodiments, R2is -OH. In some embodiments, R2is fluoro. In some embodiments, R2is C1-12 aliphatic. In some embodiments, R2is Ci-6 aliphatic. In some embodiments, R2is C1-3 aliphatic. In some embodiments, R2is methyl. In some embodiments, R2is C1-4 alkoxy-Ci-4 alkyl. In some embodiments, R2is methoxy-ethyl.

[0072] In some embodiments of formula I, R3is hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -S(O)2R, - C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, -N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, -N(R)SO2N(R)2, -N(R)SO2R, -OC(O)N(R)2, or optionally substituted C1-12 aliphatic. In certain embodiments, R3is hydrogen. In some embodiments, R3is halogen. In certain embodiments, R3is -NO2. In some embodiments, R3is -CN. In certain embodiments, R3is -OR. In some embodiments, R3is Ci -12 aliphatic. In some embodiments, R3is Ci-6 aliphatic.

[0073] In some embodiments of formula I, R4is hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -S(O)2R, - C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, -N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, -N(R)SO2N(R)2, -N(R)SO2R, -OC(O)N(R)2, or optionally substituted C1-12 aliphatic. In certain embodiments, R4is hydrogen. In some embodiments, R4is halogen. In certain embodiments, R4is -NO2. In some embodiments, R4is -CN. In certain embodiments, R4is -OR. In some embodiments, R4is C1-12 aliphatic. In some embodiments, R4is C1-6 aliphatic.

[0074] In some embodiments of formula I, R5is hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -S(O)2R, - C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, -N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, -N(R)SO2N(R)2, -N(R)SO2R, -OC(O)N(R)2, or optionally substituted Ci-12aliphatic. In certain embodiments, R5is hydrogen. In some embodiments, R5is halogen. In certain embodiments, R5is -PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011NO2. In some embodiments, R5is -CN. In certain embodiments, R5is -OR. In some embodiments, R5is C1-12 aliphatic. In some embodiments, R5is C1-6 aliphatic.

[0075] In some embodiments of formula I, R6is hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -S(O)2R, - C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, -N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, -N(R)SO2N(R)2, -N(R)SO2R, -OC(O)N(R)2, or optionally substituted Ci-12aliphatic. In certain embodiments, R6is hydrogen. In some embodiments, R6is halogen. In certain embodiments, R6is -NO2. In some embodiments, R6is -CN. In certain embodiments, R6is -OR. In some embodiments, R6is Ci-i2aliphatic. In some embodiments, R6is Ci-6 aliphatic.

[0076] In some embodiments of formula I, R7is hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, -S(O)2R, - C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, -N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, -N(R)SO2N(R)2, -N(R)SO2R, -OC(O)N(R)2, or optionally substituted Ci-12 aliphatic. In certain embodiments, R7is hydrogen. In some embodiments, R7is halogen. In certain embodiments, R7is -NO2. In some embodiments, R7is -CN. In certain embodiments, R7is -OR. In some embodiments, R7is Ci-i2 aliphatic. In some embodiments, R7is C1-6 aliphatic.

[0077] In some embodiments of formula I, each R is independently hydrogen or an optionally substituted C1-6 aliphatic. In some embodiments, each R is hydrogen. In some embodiments, a R is hydrogen. In some embodiments, each R is optionally substituted C1-6 aliphatic (e.g., optionally substituted C1-6 alkyl). In some embodiments, a R is optionally substituted C1-6 aliphatic (e.g., optionally substituted C1-6 alkyl).

[0078] The compositions of the present disclosure can include various amounts of modulators. For instance, in some embodiments, a composition of the present disclosure can include 0.1 micrograms to 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).

[0079] In some embodiments, modulators of the present disclosure are associated with the particles of the present disclosure. As used herein in reference to a modulator and a particle in a composition, “associated with” means that the modulator is encapsulated within the particle or is bound to, adsorbed to, or otherwise complexed with the particle, e g., the surface of the particle, in the composition. In some embodiments, a modulator that is associated with a particle is not substantially separated from the particle by filtration. For instance, in some embodiments, modulators of the present disclosure arePCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011encapsulated within the particle. In some embodiments, modulators of the present disclosure are on the surface of a particle. In some embodiments, a modulator includes 2’,3’-cGAMP that is associated with a particle of the composition (e.g., encapsulated within the particle).

[0080] In some embodiments, modulators of the present disclosure are in free form in the composition. As used herein in reference to a modulator in relation to a particle in a composition, “in free form” means that the modulator is not associated with the particle in the composition. In some embodiments, a modulator includes 2’,3’-cGAMP that is in free form in the composition.

[0081] In some embodiments, the present disclosure provides compositions comprising a first modulator and a second modulator. In some embodiments, a first modulator is associated with a particle (e g., encapsulated within a particle) of a composition disclosed herein. In some embodiments, a second modulator is in free form in the composition.

[0082] In some embodiments, the present disclosure provides compositions comprising (1) a first modulator associated with a particle (e.g., encapsulated within a particle) of the composition; and (2) a second modulator in free form in the composition. In some such embodiments, the first and second modulators are the same modulator (e.g., 2’,3’-cGAMP). In some such embodiments, the first and second modulators are different modulators (e.g., wherein one of the modulators is 2’,3’-cGAMP).

[0083] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:20. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:12. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2.5 to about 1:10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:5. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:20. InPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1: 12. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:10 to about 1:20. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:10 to about 1:12.

[0084] In some embodiments, provided compositions comprise a first modulator that is associated with a particle and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:20. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:12. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2.5 to 1:10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:5. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1:20. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1:12. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1:10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:10 to 1:20. In some embodiments, provided compositionsPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1: 10 to 1:12.

[0085] In some embodiments, provided compositions comprise a first modulator that is associated with a particle and a second modulator in free form in the composition in a molar ratio of about 1:2. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:2.5. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:5. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1: 12. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:20.

[0086] In some embodiments, provided compositions comprise a first modulator that is associated with a particle and a second modulator in free form in the composition in a molar ratio of 1:2. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:2.5. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:5. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1: 10. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1: 12. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:20.

[0087] In some embodiments, provided compositions comprise a first modulator that is associated with a particle and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:20, wherein the first modulator and the second modulator are each a compound ofPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1: 12, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2.5 to about 1:10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:5, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:20, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:12, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:10 to about 1:20, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:10 to about 1: 12, wherein the first modulator and the second modulator are each a compound of formula I.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0088] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:20, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:12, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2.5 to 1:10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:5, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1:20, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1: 12, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1: 10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:10 to 1:20, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulatorPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011in free form in the composition in a molar ratio of from 1: 10 to 1:12, wherein the first modulator and the second modulator are each a compound of formula I.

[0089] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:2, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:2.5, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:5, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:12, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:20, wherein the first modulator and the second modulator are each a compound of formula I.

[0090] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:2, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:2.5, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:5, wherein the first modulator and the second modulator are each a compound of formula I. In somePCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:10, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:12, wherein the first modulator and the second modulator are each a compound of formula I. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:20, wherein the first modulator and the second modulator are each a compound of formula I.

[0091] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1: 12, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2.5 to about 1:10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:5, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:2 to about 1:10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:12,PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:5 to about 1:10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:10 to about 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from about 1:10 to about 1: 12, wherein the first modulator and the second modulator are each 2’,3’-cGAMP.

[0092] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:12, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2.5 to 1:10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:5, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:2 to 1:10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011to 1:12, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:5 to 1:10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:10 to 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of from 1:10 to 1:12, wherein the first modulator and the second modulator are each 2’,3’-cGAMP.

[0093] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:2, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:2.5, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:5, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:12, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of about 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP.

[0094] In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molarPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011ratio of 1:2, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:2.5, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:5, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1: 10, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:12, wherein the first modulator and the second modulator are each 2’,3’-cGAMP. In some embodiments, provided compositions comprise a first modulator that is associated with a particle of the composition and a second modulator in free form in the composition in a molar ratio of 1:20, wherein the first modulator and the second modulator are each 2’,3’-cGAMP.

[0095] In some embodiments, compositions that include one or more modulators in free form (e.g., one or more modulators outside of and unassociated with a particle of the composition) can elicit a more immediate response from a target tissue or cell and / or decrease the cost and / or complexity of manufacture of the composition. In some embodiments, encapsulating one or more modulators within a particle as described herein can increase uptake efficiency, tissue targeting, stability, and / or efficacy of the composition or a method of its use, such as by controlling (e.g., maintaining) spatial concentration of the one or more modulators and / or by presenting a recognition signal (e.g., an antigen) to a target cell.

[0096] The present disclosure encompasses the recognition that compositions including (1) one or more modulators associated with a particle; and (2) one or more modulators in free form provide advantages and benefits in comparison to compositions not comprising modulators in both free form and associated with a particle. For example, in some embodiments, provided compositions (i.e., those comprising one or more modulators in both free form and within a particle) demonstrated enhanced activation of interferon-stimulated genes, such aslfribl and Cxcl10, promoted robust antiviral activityPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011(e.g., against Influenza A & B viruses); improved therapeutic efficacy; demonstrated faster viral clearance; and provided better overall outcomes (i.e., in comparison to compositions not comprising modulators in both free form and associated with a particle).

[0097] Antigens

[0098] The compositions of the present disclosure can optionally include various antigens. For instance, in some embodiments, the antigen is operable to elicit an immune response in a subject against a virus. In some embodiments, the antigen is or comprises an attenuated or killed version of a pathogen, or portion thereof, that causes a disease (e.g., one or more of the viruses described herein). In some embodiments, the antigen includes a protein or peptide derived from a virus (e.g., recombinant proteins or peptides), or a polynucleotide encoding the protein or peptide (e.g., a plasmid DNA molecule and / or minigenes expressing the protein or peptide). In some embodiments, the antigen includes a protein or peptide derived from a virus. In some embodiments, the antigen includes a polynucleotide encoding a protein or peptide derived from a virus.

[0099] In some embodiments, the antigen includes a protein derived from a virus, or a polynucleotide encoding the protein. In some embodiments, the protein is in the form of a fusion protein, a protein fragment, a full protein, or a combination thereof.

[0100] In some embodiments, the virus includes, without limitation, an influenza virus, an influenza A virus, an influenza B virus, an H5N1 virus, a respiratory virus, or a combination thereof. In some embodiments, the virus is a coronavirus, a parainfluenza virus, an adenovirus, an enterovirus, a respiratory syncytial virus, a rhinovirus, a DNA virus, an RNA virus, or a combination thereof.

[0101] In some embodiments, the antigen includes, without limitation, an antigen of an influenza virus, an influenza A virus, an influenza B virus, an H5N1 virus, a respiratory virus, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, a fusion protein thereof, or a combination thereof. In some embodiments, the antigen comprises an antigen of a coronavirus, a parainfluenza virus, an adenovirus, an enterovirus, a respiratory syncytial virus, a rhinovirus, a DNA virus, an RNA virus, a full protein thereof, a protein fragment thereof, a peptide fragment thereof, a fusion protein thereof, or a combination thereof. In some embodiments, the antigen comprises an antigen of a severe acute respiratory syndrome coronavirus (SARS-CoV), a severe acute respiratory syndrome-related coronavirus (SARSr-CoV), a human coronavirus 229E (HCoV-229E), a human coronavirus NL63 (HCoV-NL63), a human coronavirus OC43 (HCoV-OC43), a human coronavirusPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011HKU1 (HCoV-HKU1), a Middle East respiratory syndrome-related coronavirus (MERS-CoV), a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a variant of SARS-CoV-2 (e.g., 20A. EU1, or spike variant D614G), or a combination thereof.

[0102] In some embodiments, an antigen comprises a spike protein or a portion thereof. In some embodiments, an antigen comprises at least one component of a coronavirus spike protein (S-protein). In some embodiments, an antigen comprises a monomeric form of a coronavirus spike protein (S-protein). In some embodiments, an antigen comprises a multimeric form of a coronavirus spike protein (S-protein). In some embodiments, an antigen comprises a monomeric form of the SARS-CoV2 spike protein (S), a monomeric form of the receptor binding domain (RBD) of the SARS-CoV2 spike protein (S), a multimeric form of the SARS-CoV2 spike protein (S), a multimeric form of the receptor binding domain (RBD) of the SARS-CoV2 spike protein (S), a dimeric form of the SARS-CoV2 spike protein (S), a dimeric form of the receptor binding domain (RBD) of the SARS-CoV2 spike protein (S), a trimeric form of the SARS-CoV2 spike protein (S), a trimeric form of the receptor binding domain (RBD) of the SARS-CoV2 spike protein (S), or a combination thereof. In some embodiments, an antigen of a composition can comprise a chimeric protein (e.g., a chimeric spike protein). In some embodiments, an antigen can comprise a monomeric or multimeric form of the SARS-CoV2 spike protein (S) containing the D614G mutation, A222V mutation, S477N mutation, D80Y mutation, S98F mutation, or a combination thereof. In some embodiments, the antigen can be a mixture of SARS-CoV2 spike proteins harboring different mutations. In some embodiments, an antigen comprises an (e g., coronavirus) alpha variant spike protein monomer, an (e.g., coronavirus) alpha variant spike protein trimer, a (e.g., coronavirus) beta variant spike protein monomer, a (e.g., coronavirus) beta variant spike protein trimer, a (e.g., coronavirus) gamma variant spike protein monomer, a (e.g., coronavirus) gamma variant spike protein trimer, a (e.g., coronavirus) delta variant spike protein monomer, a (e.g., coronavirus) delta variant spike protein trimer, a receptor binding domain (RBD) portion of an (e.g., coronavirus) alpha variant spike protein, an RBD portion of a (e.g., coronavirus) beta variant spike protein, an RBD portion of a (e.g., coronavirus) gamma variant spike protein, and / or an RBD portion of a (e.g., coronavirus) delta variant spike protein.

[0103] In some embodiments, an antigen comprises a nucleocapsid protein or a portion thereof. In some embodiments, an antigen comprises a monomeric or multimeric form of the SARS-PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011CoV2 nucleocapsid (N) protein. In some embodiments, an antigen comprises the monomeric or multimeric form of the SARS-CoV2 nucleocapsid (N) protein containing the A220V mutation.

[0104] In some embodiments, an antigen is suitable for developing immunity against a cancer. In some embodiments, an antigen comprises an attenuated or killed version of a tumor cell or portion thereof associated with a cancer. In some embodiments, an antigen comprises a peptide or protein associated with a cancer. In some embodiments, an antigen comprises a surface protein (e.g., a receptor protein) of a cancer cell. In some embodiments, an antigen comprises a mutated protein of a cancer cell. In some embodiments, an antigen comprises a synthetic long peptide targeting cancer mutations.

[0105] In some embodiments, the antigen includes a fusion protein of a virus, or a nucleotide encoding the fusion protein.

[0106] The antigens of the present disclosure may be associated with the particles of the present disclosure in various manners. For instance, in some embodiments, the antigen is associated with an outer surface of the particle. In some embodiments, the antigen is encapsulated within the particle.

[0107] In some embodiments, the association of an antigen with a particle of the present disclosure may increase the efficacy of the composition in treating a viral infection in a subject, such as by spatially concentrating an antigen and a modulator of the composition (e.g., at a target tissue). In some embodiments, association of an antigen with a particle (e.g., on an outer surface of a particle) that is associated with a modulator (e.g., an encapsulated modulator) can significantly increase the targeting and / or delivery of the modulator to a target tissue of interest (e g., an intranasal compartment and / or a lung compartment of a subject).

[0108] The compositions of the present disclosure can include various amounts of antigens. For instance, in some embodiments, a composition of the present disclosure can include 0.1 micrograms to 200 micrograms of an antigen.

[0109] Particles

[0110] The compositions of the present disclosure can include various particles. For instance, in some embodiments, the particles include, without limitation, a lipid-based particle, a carbon-based particle, a metal -based particle, or a combination thereof.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0111] In some embodiments, the particles comprise one or more pulmonary surfactantbiomimetic molecules. For example, in some embodiments, the particles are those described in WO 2021 / 071823, the contents of which are incorporated by reference herein.

[0112] In some embodiments, the particles of the present disclosure include a lipid-based particle. In some embodiments, the lipid-based particle is in the form of a liposome. In some embodiments, the lipid-based particle includes one or more lipids.

[0113] In some embodiments, the one or more lipids include, without limitation, one or more anionic lipids, one or more neutral lipids, and one or more polyethylene glycol (PEG)-lipids. In some embodiments, the PEG moiety of a PEG-lipid has an average molecular weight of 500-5000 Da.

[0114] In some embodiments, the one or more lipids include, without limitation, 1,2-dipalmitoyl-sn-gly cero-3 -phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3 -phospho-(l'-rac-glycerol) (DPPG), cholesterol, a PEG-lipid (e g., DPPE-PEG), or a combination thereof. In some embodiments, the lipid-based particle includes DPPC, DPPG, cholesterol, and PEG-lipid (e.g., DPPE-PEG) at a molar ratio of about 10: 1: 1: 1. In some embodiments, the lipid-based particle includes DPPC, DPPG, cholesterol, and PEG-lipid (e.g., DPPE-PEG) at a molar ratio of 10:1:1:1.

[0115] In some embodiments, the one or more lipids include, without limitation, 1,2-dipalmitoyl-sn-gly cero-3 -phosphocholine (DPPC), 1,2-dipalmitoyl-sn-gly cero-3 -phospho-( 1 '-rac-glycerol) (DPPG), cholesterol, 1,2-dipalmitoyl-sn-gly cero-3 -phosphoethanol amine-N-[methoxy(polyethylene glycol)-2000] DPPE-PEG2000, or a combination thereof. In some embodiments, the lipid-based particle includes DPPC, DPPG, cholesterol, and DPPE-PEG2000 at a molar ratio of 10:1:1:1. In some embodiments, the lipid-based particle includes DPPC, DPPG, cholesterol, and DPPE-PEG2000 at a molar ratio of about 10:1:1:1.

[0116] In some embodiments, a particle can include a membrane or wall. In some embodiments, a membrane or wall of a particle can define an interior space. In some embodiments, an interior space of a particle can include one or more modulators. In some embodiments, one or more antigens can be associated with a membrane or wall of the particle (e.g., an outer surface of a membrane or wall of the particle).

[0117] In some embodiments, the particles of the present disclosure include a plurality of molecules. In some embodiments, the particles of the present disclosure or a portion thereof can be anionic (e.g., can include an anionic membrane or one or more anionic lipids). For example, in somePCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011embodiments, a lipid-based particle of the present disclosure can include an anionic lipid (e.g., DPPE-PEG2000 or DPPG) or a neutral lipid (e.g., DPPE, DPPC, or cholesterol). In some embodiments, a particle of the present disclosure includes no cationic lipids.

[0118] In some embodiments, a particle of the present disclosure can be cationic. In some embodiments, a particle of the present disclosure can include a cationic (e.g., positively charged) lipid. For example, in some embodiments, a particle of the present disclosure can include, without limitation, l,2-dipalmitoyl-3-trimethylammonium-propane chloride (DPTAP) or l,2-dioleoyl-3-trimethylammonium propane (DOTAP).

[0119] In some embodiments, a particle of the present disclosure can be zwitterionic. In some embodiments, a particle of the present disclosure or a portion thereof can have a net zero charge. In some embodiments, a particle of the present disclosure or a portion thereof can be uncharged. In some embodiments, a particle of the present disclosure can include dipalmitoylphosphatidylcholine, dipalymitoylphosphatidylglycerol, l,2-bis(diphenylphosphino)ethane (DPPE), cholesterol, or a combination thereof.

[0120] In some embodiments, a particle of the present disclosure can include a poly(ethyleneglycol)-lipid (e.g., a PEG-lipid). In some embodiments, a PEG-lipid is DPPE-PEG. In some embodiments, a PEG-lipid is DPPE-PEG2000.

[0121] In some embodiments, a particle of the present disclosure can include DPPC (1,2-dipalmitoyl-sn-glycero-3 -phosphocholine), DPPG (l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol)), DPPE-PEG2000 (l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), cholesterol, l,2-dipalmitoyl-3-trimethylammonium-propane chloride (DPTAP), l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), or a combination thereof.

[0122] In some embodiments, a particle of the present disclosure can include a combination of DPPC and DPPG, for example, at a molar ratio of about 10:1. In some embodiments, a particle of the present disclosure can include a combination of DPPC and cholesterol, for example, at a molar ratio of about 10: 1. In some embodiments, a particle of the present disclosure can include a combination of DPPC and DPPE-2000, for example, at a molar ratio of about 10:1. In some embodiments, a particle of the present disclosure can include a combination of DPPG and cholesterol, for example, at a molar ratio of about 1: 1. In some embodiments, a particle of the present disclosure can include a combination of cholesterol and DPPE-PEG2000, for example, at a molar ratio of about 1: 1. In some embodiments,PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011a particle of the present disclosure can include a combination of DPPG and DPPE-PEG2000, for example, at a molar ratio of about 1:1.

[0123] In some embodiments, a particle of the present disclosure can include a combination of DPPC and DPPG, for example, at a molar ratio of 10: 1. In some embodiments, a particle of the present disclosure can include a combination of DPPC and cholesterol, for example, at a molar ratio of 10:1. In some embodiments, a particle of the present disclosure can include a combination of DPPC and DPPE-2000, for example, at a molar ratio of 10:1. In some embodiments, a particle of the present disclosure can include a combination of DPPG and cholesterol, for example, at a molar ratio of 1:1. In some embodiments, a particle of the present disclosure can include a combination of cholesterol and DPPE-PEG2000, for example, at a molar ratio of 1:1. In some embodiments, a particle of the present disclosure can include a combination of DPPG and DPPE-PEG2000, for example, at a molar ratio of 1: 1.

[0124] In some embodiments, a particle of the present disclosure can include DPPC, DPPG, cholesterol, and DPPE-PEG2000. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 10:1:1:1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 20:1:1:1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 5: 1: 1: 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 10:2: 1: 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 10:1:2:1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively.

[0125] In some embodiments, a particle of the present disclosure can include DPPC, DPPG, cholesterol, and DPPE-PEG2000. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:1:1:1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 20:1:1:1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 5: 1: 1: 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the presentPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011disclosure can be composed of a molar ratio of 10:2:1:1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10: 1:2: 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively.

[0126] In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 10:1:1:2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 10:2:2: 1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 10:1:2:2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of about 10:2:1:2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively.

[0127] In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:1:1:2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:2:2:1 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:1:2:2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively. In some embodiments, a particle of the present disclosure can be composed of a molar ratio of 10:2:1:2 of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively.

[0128] In some embodiments, the particles of the present disclosure can include a nanoparticle (e.g., a liposomal nanoparticle). In some embodiments, the present disclosure provides a composition comprising nanoparticles having an average particle diameter (e.g., mean hydrodynamic particle diameter) of less than 400 nanometers (nm), less than 300 nm, less than 200 nm, less than 150 nm, less than 120 nm, less than 115 nm, less than 111 nm, less than 110 nm, less than 105 nm, less than 100 nm, less than 95 nm, less than 90 nm, less than 85 nm, or less than 80 nm.

[0129] In some embodiments, the present disclosure provides a composition comprising particles having an average outer diameter of 1 nanometer to 500 nanometers, 1 nanometer to 750 nanometers, or 1 nanometer to 1,000 nanometers. In some embodiments, the particles of the present disclosure can have an outer diameter of 1 nanometer to 10 nanometers, 1 nanometer to 15 nanometers, 1 nanometer to 20 nanometers, 1 nanometer to 30 nanometers, 1 nanometer to 50 nanometers, 1PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011nanometer to 75 nanometers, 1 nanometer to 100 nanometers, 1 nanometer to 150 nanometers, 1 nanometer to 200 nanometers, 1 nanometer to 250 nanometers, 1 nanometer to 300 nanometers, 1 nanometer to 400 nanometers, 1 nanometer to 500 nanometers, 10 nanometers to 15 nanometers, 10 nanometers to 20 nanometers, 10 nanometers to 30 nanometers, 10 nanometers to 50 nanometers, 10 nanometers to 75 nanometers, 10 nanometers to 100 nanometers, 10 nanometers to 150 nanometers, 10 nanometers to 200 nanometers, 10 nanometers to 250 nanometers, 10 nanometers to 300 nanometers, 15 nanometers to 20 nanometers, 15 nanometers to 30 nanometers, 15 nanometers to 50 nanometers, 15 nanometers to 75 nanometers, 15 nanometers to 100 nanometers, 15 nanometers to 150 nanometers, 15 nanometers to 200 nanometers, 15 nanometers to 250 nanometers, 15 nanometers to 300 nanometers, 20 nanometers to 30 nanometers, 20 nanometers to 50 nanometers, 20 nanometers to 75 nanometers, 20 nanometers to 100 nanometers, 20 nanometers to 150 nanometers, 20 nanometers to 200 nanometers, 20 nanometers to 250 nanometers, 20 nanometers to 300 nanometers, 30 nanometers to 50 nanometers, 30 nanometers to 75 nanometers, 30 nanometers to 100 nanometers, 30 nanometers to 150 nanometers, 30 nanometers to 200 nanometers, 30 nanometers to 250 nanometers, 30 nanometers to 300 nanometers, 50 nanometers to 75 nanometers, 50 nanometers to 100 nanometers, 50 nanometers to 150 nanometers, 50 nanometers to 200 nanometers, 50 nanometers to 250 nanometers, 50 nanometers to 300 nanometers, 75 nanometers to 100 nanometers, 75 nanometers to 150 nanometers, 75 nanometers to 200 nanometers, 75 nanometers to 250 nanometers, 75 nanometers to 300 nanometers, 100 nanometers to 150 nanometers, 100 nanometers to 200 nanometers, 100 nanometers to 250 nanometers, 100 nanometers to 300 nanometers, 150 nanometers to 200 nanometers, 150 nanometers to 250 nanometers, 150 nanometers to 300 nanometers, 200 nanometers to 250 nanometers, 200 nanometers to 300 nanometers, or 250 nanometers to 300 nanometers. In some embodiments, a particle can have an outer diameter of 1 nanometer, 10 nanometers, 15 nanometers, 20 nanometers, 30 nanometers, 50 nanometers, 75 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 400 nanometers, or 500 nanometers.

[0130] In some embodiments, the particles of the present disclosure have an outer diameter of at least 1 nanometer, 10 nanometers, 15 nanometers, 20 nanometers, 30 nanometers, 50 nanometers, 75 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 400 nanometers, or 500 nanometers. In some embodiments, the particles of the present disclosure can have an outer diameter of at most 1 nanometer, 10 nanometers, 15 nanometers, 20 nanometers, 30PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011nanometers, 50 nanometers, 75 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 400 nanometers, 500 nanometers, 750 nanometers, or 1,000 nanometers.

[0131] In some embodiments, an outer diameter of a particle is measured as an average outer diameter of a population of particles, e.g., measured for 1 day, 2 days, 3 days, up to 96 hours, from 4 to 7 days, more than 7 days, from 7 to 30 days, or more than 30 days at 25 °C. In some embodiments, an outer diameter of a particle is measured as an average outer diameter of a population of particles, e.g., measured for 1 day, 2 days, 3 days, up to 96 hours, from 4 to 7 days, more than 7 days, from 7 to 30 days, or more than 30 days at 37 °C. In some embodiments, an outer diameter of a particle is measured using dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA).

[0132] One or more modulators and / or antigens of the present disclosure may be associated with the particles of the present disclosure in various manners. For instance, in some embodiments, the antigens and modulators of the present disclosure can be positioned on different regions of the particles of the present disclosure.

[0133] In some embodiments, one or more modulators of the present disclosure are associated with the particle and are present in free form in the composition. In some such embodiments, the composition further comprises an antigen. In some embodiments, one or more antigens of the present disclosure are associated with an outer surface of a particle of the present disclosure, while one or more modulators of the present disclosure are associated with the particle and are present in free form in the composition. In some embodiments, one or more antigens of the present disclosure are associated with a particle of the present disclosure, while one or more modulators of the present disclosure are associated with the particle and are present in free form in the composition.

[0134] In some embodiments, a modulator of the present disclosure can be encapsulated in a particle. In some embodiments, all or a portion of an antigen of the present disclosure can be associated with (e.g., attached to, adhered to, adsorbed onto, electrostatically interacted with, covalently bound to, noncovalently bound to, integrated into, or formulated onto) a surface of the particle. In some embodiments, all or a portion of an antigen is encapsulated within a particle. In some embodiments, the association (e.g., adsorption) of an antigen with a surface of a particle can increase stability of the particle and / or increase delivery efficiency (e.g., to a target tissue) after administration.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0135] In some embodiments, one or more antigens of the present disclosure can be encapsulated within a particle of the present disclosure. In some embodiments, one or more modulators of the present disclosure can be associated with an outer surface of the particle. In some embodiments, one or more modulators of the present disclosure can be in free form within the composition.

[0136] In some embodiments, one or more antigens of the present disclosure can be encapsulated in a particle of the present disclosure while one or more modulators of the present disclosure are associated with an outer surface of the particle. In some embodiments, one or more antigens of the present disclosure can be associated with an outer surface of a particle of the present disclosure while one or more modulators of the present disclosure may be in free form within the composition. In some embodiments, one or more antigens of the present disclosure can be associated with an outer surface of a particle of the present disclosure. In some embodiments, one or more modulators of the present disclosure can be encapsulated within the particles of the present disclosure.

[0137] In some embodiments, one or more antigens of the present disclosure can be associated with an outer surface of a particle of the present disclosure while one or more modulators of the present disclosure are encapsulated within the particle. In some embodiments, one or more antigens and one or more modulators of the present disclosure can both be encapsulated within a particle of the present disclosure. In some embodiments, one or more antigens and one or more modulators of the present disclosure may both be associated with a surface of a particle of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be integrated into a membrane of a particle of the present disclosure. In some embodiments, one or more modulators of the present disclosure can be integrated into a membrane of a particle of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be encapsulated within a particle of the present disclosure while one or more modulators are integrated into a membrane of a particle of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be associated with an outer surface of a particle of the present disclosure while one or more modulators are integrated into a membrane of a particle of the present disclosure.

[0138] In some embodiments, one or more modulators of the present disclosure can be encapsulated within a particle of the present disclosure while one or more antigens of the present disclosure are integrated into a membrane of the particle. In some embodiments, one or more modulators can be associated with an outer surface of a particle of the present disclosure while one orPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011more antigens of the present disclosure are integrated into a membrane of the particle. In some embodiments, the incorporation of both an antigen and a modulator of the present disclosure within a particle can facilitate coordinated cytosolic delivery.

[0139] In some embodiments, the modulators of the present disclosure are associated with (e.g., encapsulated within) the particle and in free form in the composition. In some embodiments, the antigens of the present disclosure are associated with an outer surface of a particle.

[0140] The compositions of the present disclosure can include various amounts of particles. For instance, in some embodiments, a composition of the present disclosure can include 0.1 micrograms to 200 micrograms of a particle of the present disclosure.

[0141] Composition forms

[0142] The compositions of the present disclosure may be in various forms. For instance, in some embodiments, the compositions of the present disclosure may be formulated for intranasal or inhalational delivery. In some embodiments, the compositions are formulated for intranasal delivery. In some embodiments, the compositions are formulated for inhalational delivery.

[0143] In some embodiments, the composition is lyophilized. In some embodiments, the composition is in liquid form. In some embodiments, the composition is suitable for use in treating a viral infection.

[0144] An example of a composition of the present disclosure is illustrated in FIG. 1 as composition 10, which includes a lipid-based particle 12 (e.g., a liposome), a modulator 16 (e.g., a STING agonist, such as 2’,3’-cGAMP), and an optional antigen 14 (e.g., a fusion protein of a virus). In this example, modulator 16 is in free form in the composition. Modulator 16 in this embodiment may also be encapsulated within lipid-based particle 12. Additionally, in this embodiment, an optional antigen 14 is associated with the outer surface of lipid-based particle 12. In some embodiments, the composition of FIG. 1 does not comprise an antigen 14.

[0145] In some embodiments, it can be advantageous to formulate the compositions of the present disclosure within a desired pH range. For example, formulation of a composition described herein (e.g., for intranasal delivery) between pH 4.0 and pH 7.5 (e.g., pH 4.5 to pH 6.5 or pH 5.5 to pH 6.5) can avoid irritation and / or histological damage to the intranasal tissue, which may occur if extremely acidic or basic formulations are used for intranasal delivery (e.g., because nasal cavity pHPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011can be from about 5.5 to 6.5). In some embodiments, a composition of the present disclosure can have a pH of4.0 to 7.5.

[0146] In some embodiments, it can be advantageous to formulate a composition of the present disclosure within a desired osmolarity range. For example, formulation of a composition of the present disclosure (e.g., for intranasal delivery) having an osmolarity from 50 mOsm / kg to 900 mOsm / kg can improve absorption while avoiding potential epithelial damage (eg., which may occur from compositions with very low osmolarities) and avoiding increases to mucosal secretions (e.g., which may occur from compositions with very high osmolarities) (e.g., because nasal cavity osmolarity can be about 280 mOsm / kg). In some embodiments, a composition of the present disclosure can have an osmolarity of 50 mOsm / kg to 900 mOsm / kg.

[0147] In some embodiments, the compositions of the present disclosure can be advantageous to formulate compositions described herein within a desired viscosity range. For example, formulation of a composition of the present disclosure (e.g., for intranasal delivery) having a viscosity, for example, from 1.1 cP (centipoise) to 50 cP (e.g., 1.5 cP to 50 cP) can increase residence time in the nasal cavity while adversely affecting droplet size (e.g., which can affect spray pattern and / or distribution within the nasal cavity). In some embodiments, a composition of the present disclosure can have a viscosity of 1 cP (centipoise) to 100 cP.

[0148] The compositions of the present disclosure can be in various forms. For instance, in some embodiments, the compositions of the present disclosure can be in the form of a solubilized liquid. In some embodiments, the compositions of the present disclosure are suitable for intranasal and / or inhalational administration to a subject. In some embodiments, intranasal delivery of the compositions of the present disclosure can be used to target intranasal compartment tissues. In some embodiments, inhalational administration of the compositions of the present disclosure can be used to target lung compartment tissues.

[0149] In some embodiments, the compositions of the present disclosure also include one or more stabilizers. In some embodiments, the stabilizers include, without limitation, anti-oxidants, sequestrants, ultraviolet stabilizers, or a combination thereof.

[0150] In some embodiments, the compositions of the present disclosure also include one or more surfactants. In some embodiments, the surfactants include, without limitation, anionicPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011surfactants, sugars, cationic surfactants, zwitterionic surfactants, non-ionic surfactants, or a combination thereof.

[0151] In some embodiments, the compositions of the present disclosure also include one or more excipients. In some embodiments, the excipients include, without limitation, lactose, sucrose, starch powder, cellulose esters of alkanoic acids, trehalose, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, trehalose, sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, or a combination thereof.

[0152] In some embodiments, a composition of the present disclosure is in lyophilized form (e.g., freeze-dried). Lyophilization of a composition of the present disclosure (or a component thereof) can increase the storage stability (e.g., shelf stability) of a composition. In some embodiments, one or more components of a composition of the present disclosure can be lyophilized.

[0153] In some embodiments, lyophilization of a composition of the present disclosure (or a component thereof) can allow for easy preparation of the composition. In some embodiments, such lyophilization allows for use of the compositions of the present disclosure (e.g., administration to a subject) in regions without easy access to material preparation facilities. For instance, a modulator, a lipid-based particle, and / or an antigen described herein can be lyophilized and then rehydrated and mixed (e.g., as described herein) to formulate a composition described herein at a site of administration to a subject that is remote from a permanent medical or pharmaceutical facility.

[0154] In some embodiments, a lyophilized composition of the present disclosure (or a portion thereof, such as a lyophilized particle or a lyophilized antigen or a lyophilized modulator) can be stored at 4 °C. In some embodiments, a lyophilized composition of the present disclosure (or a portion thereof) can be stored at 4 °C and used for up to 1 week, up to 2 weeks, up to 3 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, or up to 12 months.

[0155] In some embodiments, the compositions of the present disclosure may be in liquid form. In some embodiments, the compositions of the present disclosure may be in solid form.

[0156] In some embodiments, a composition of the present disclosure can include a divalent ion, such as a divalent cation. In some embodiments, a composition of the present disclosure includes a divalent cation encapsulated within, adsorbed onto, covalently coupled to, electrostatically interactedPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011with, or formulated onto a membrane of a particle of the present disclosure. In some embodiments, a composition can include a divalent cation that includes, without limitation, Mn2+, Mg2+, Ca2+, and Zn2. For example, a composition of the present disclosure can include a modulator (e.g., a STING agonist), a particle (e.g., a lipid-based nanoparticle), and a divalent cation, such as Mn2+, Mg2+, Ca2+, or Zn2+.

[0157] Methods of treating a viral infection

[0158] Additional embodiments of the present disclosure pertain to methods of treating a viral infection in a subject. As illustrated in FIG. IB, such methods generally include administering a composition of the present disclosure to the subject (step 20) to result in the treatment of a viral infection in the subject (step 22). As set forth in more detail herein, the methods of the present disclosure can have numerous uses and embodiments.

[0159] Subjects

[0160] The methods of the present disclosure may be utilized to treat a viral infection in various subjects. For instance, in some embodiments, the subject does not exhibit symptoms of a viral infection. In some embodiments, the subject exhibits symptoms of a viral infection.

[0161] The compositions of the present disclosure may be administered to various subjects. For instance, in some embodiments, the subject is a mammal (e.g., a human). In some embodiments, the subject is a human being. In some embodiments, the subject is a domesticated animal. For example, in some embodiments, the subject can be a dog or a cat. In some embodiments, a subject can be a cow, a horse, a non-human primate, a mouse, a rat, a rabbit, a guinea pig, a goat, a sheep, a giraffe, a zebra, a lion, a tiger, or a bear.

[0162] In some embodiments, the subject can be vulnerable to or suffering from a viral infection. For example, in some embodiments, the compositions of the present disclosure may be administered to a subject who has been exposed to or who has been infected with a virus. In some embodiments, a subject can be selected for treatment as a result of exhibiting one or more symptoms of a viral infection. For example, a subject may be selected for treatment based on having one or more symptoms, including persistent coughing, elevated body temperature (e.g., greater than 100.40C by forehead skin measurement), body chills, achy joints, difficulty breathing or catching one’s breath, fluid in the lungs, fatigue, headache, loss of taste or smell, or a positive viral infection test, such as a PCR test.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0163] In some embodiments, a subject may be a subject vulnerable to a viral infection. For instance, in some embodiments, the subject may be selected for treatment with a composition of the present disclosure based on a demographic risk factor, such as obesity, advanced age (e.g., 65 years old or older), immune impairment, or pregnancy. In some embodiments, a subject may be selected for treatment based on a risk of infection by a virus, for instance, if the subject has an occupation involving close interaction with customers, frequent interaction with at-risk populations, handling of biological samples, or close contact with potentially infected individuals.

[0164] In some embodiments, the subject is immunocompromised. In some embodiments, the subject is moderately to severely immunocompromised. In some embodiments, subjects with the following medical conditions may be immunocompromised: active treatment for solid tumor or hematologic malignancy; hematologic malignancy (e.g., chronic lymphocytic leukemia, non-Hodgkin lymphoma, multiple myeloma, or acute leukemia) regardless of treatment status; receipt of solid-organ transplant or an islet transplant and taking immunosuppressive therapy; receipt of chimeric antigen receptor (CAR)-T-cell therapy or hematopoietic stem cell transplant (within 2 years of transplantation or taking immunosuppressive therapy); moderate or severe primary immunodeficiency (e.g., common variable immunodeficiency disease, severe combined immunodeficiency, DiGeorge syndrome, or Wiskott-Aldrich syndrome); advanced or untreated HIV infection (e.g., subjects with HIV and CD4 cell counts less than 200 / mm3, a history of an AIDS-defining illness without immune reconstitution, or clinical manifestations of symptomatic HIV); or active treatment with high-dose corticosteroids (e.g., >20 mg prednisone or equivalent per day administered for >2 weeks), alkylating agents, antimetabolites, transplant-related immunosuppressive drugs, cancer chemotherapeutic agents classified as severely immunosuppressive, tumor necrosis factor blockers, or other biologic agents that are immunosuppressive or immunomodulatory. In some embodiments, subject with the following medical conditions may be immunocompromised: AIDS or CD4+count <200; complement deficiency; graft-versus-host disease; HIV infection; immunoglobulin deficiency / immunodeficiency; immunosuppressive therapy; leukemia; Hodgkin or non-Hodgkin lymphoma; metastatic cancer; multiple myeloma; solid organ malignancy; steroid therapy; or transplant history involving hematopoietic stem cells or solid organs. See, e.g., Singson, J. R. C. et al., MMWRMorb. Mortal. Wkly. Rep. 2022 Jul 8;71(27): 878-884; and <archive.cdc.gov / www_cdc_gov / coronavirus / 2019-ncov / need-PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011extra-precautions / people-who-are-immunocompromised.html>, accessed December 3, 2024, the contents of each of which are hereby incorporated by reference.

[0165] In some embodiments, the subject is elderly. For instance, in some embodiments, the subject is 60 years of age or older. In some embodiments, the subject is 65 years of age or older. In some embodiments, the subject is 70 years of age or older. In some embodiments, the subject is 75 years of age or older. In some embodiments, the subject is 80 years of age or older. In some embodiments, the subject is 85 years of age or older.

[0166] Administration

[0167] The compositions of the present disclosure may be administered to subjects in various manners. For instance, in some embodiments, the composition is administered in at least one dose. In some embodiments, the composition is administered in one dose. In some embodiments, the composition is administered in two doses. In some embodiments, the composition is administered in multiple doses.

[0168] The compositions of the present disclosure may be administered to subjects by various methods. For instance, in some embodiments, the administration occurs by methods that include, without limitation, intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, subcutaneous administration, spray-based administration, aerosol-based administration, in ovo administration, oral administration, intraocular administration, intratracheal administration, intranasal administration, inhalational administration, or a combination thereof. In some embodiments, the compositions of the present disclosure are administered through intranasal administration. In some embodiments, the compositions of the present disclosure are administered through inhalational administration.

[0169] In some embodiments, a composition of the present disclosure may be administered to a subject before exposure to a virus, for example, to prevent the subject from acquiring a viral infection (e.g., as illustrated in FIG. IB). In some embodiments, a composition of the present disclosure may be administered in the form of a vaccine.

[0170] In some embodiments, a method of the present disclosure can include administering a dose of a composition of the present disclosure (e.g., via intranasal administration) to a subject (e.g., a human subject) after exposure or suspected exposure to a virus. In some embodiments, a composition of the present disclosure may be administered to a subject after exposure to a virus, forPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011example, to treat (e.g., ameliorate or, in some embodiments, cure) a viral infection, for example, after the subject has acquired a viral infection (e.g., as illustrated in FIG. 1C). In some embodiments, the compositions of the present disclosure may be administered to a subject at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, or at least 28 days after exposure or suspected exposure to a virus.

[0171] In some embodiments, compositions described herein are administered as monotherapy. In some embodiments, the compositions of the present disclosure can be administered in combination with other prophylactic treatments (e.g., vaccine). In some embodiments, provided compositions are administered to a subject who has previously been vaccinated against a disease or infection described herein.

[0172] In some embodiments, the compositions of the present disclosure can be administered in combination with other therapeutic treatments. In some embodiments, the other therapeutic treatments include, without limitation, antiviral drugs, antibiotic treatment, targeted inhibition, or a combination thereof. In some embodiments, a composition of the present disclosure can include and / or can be administered in a treatment regimen (e.g., administered concurrently or non-concurrently) with an adjuvant (e.g., one or more antibodies, one or more vaccines, one or more small molecules, one or more nucleic acids, and / or one or more peptides or proteins, such as an interleukin (e.g, IL-21)).

[0173] The administration of the compositions of the present disclosure can have various effects on a subject. For instance, in some embodiments, the administration of the compositions of the present disclosure to the subject can induce Ifnb and Cxcl10 expression in the subject.

[0174] Treatment of viral infections

[0175] The methods of the present disclosure may have various uses. For instance, in some embodiments, the methods of the present disclosure may be used to treat a viral infection in a subject. In some embodiments, the methods of the present disclosure may be used to prevent a viral infection in a subject. For instance, in some embodiments, the methods of the present disclosure may be used to prevent the establishment of a viral infection in the subject, to prevent progression of a viralPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011infection in the subject, to prevent the transmission of a viral infection to a second subject, or a combination thereof. In some embodiments, the methods of the present disclosure may initiate an innate immune response that leads to associated adaptive immunity for a viral infection.

[0176] In some embodiments, the administered compositions of the present disclosure can rapidly activate a subject’s innate immune system to facilitate a broad-spectrum response against a virus. For instance, in some embodiments, a single intranasal dose of a composition of the present disclosure can: (1) treat infections throughout the respiratory tree and minimize symptoms, (2) reduce transmission by decreasing infections in the nasal passage, (3) protect against a virus, and / or (4) provide durable protection against reinfection by stimulating adaptive immunity.

[0177] In some embodiments, administered compositions of the present disclosure can enable sustained release of modulators (e.g., 2’,3’-cGAMP) to both the nasal compartment and the lung over a period of 48 hours or more following administration to a subject. In some embodiments, the administered compositions of the present disclosure can activate multiple pathways and facilitate a type-I interferon (IFN-I) mediated response in a subject.

[0178] In some embodiments, the compositions of the present disclosure can elicit an immune response in the subject against a viral infection. In some embodiments, the compositions of the present disclosure can elicit such immunity through at least one of innate immunity, mucosal immunity, systemic immunity, cellular immunity, humoral immunity, T-cell immunity, production of systemic neutralizing antibodies, induction of IgG responses, induction of IgA responses, induction of IgM responses, induction of T-cell responses, induction of mucosal IgA responses in lung and nasal compartments, induction of Thl T-cell responses, induction of CD8+ T-cell responses, induction of CD4+ T cell responses, induction of NK cell responses, activation or inhibition of the stimulator of interferon genes (STING) pathway, or a combination thereof.

[0179] The methods of the present disclosure may be utilized to treat viral infections caused by various viruses. For instance, the virus may include, without limitation, an influenza virus, an influenza A virus, an influenza B virus, an H5N1 virus, a respiratory virus, or a combination thereof.

[0180] In some embodiments, provided methods are useful for treating a viral infection caused by influenza. In some embodiments, provided methods are useful for treating an influenza A viral infection (e.g., Califomia / 04 / 2009 (H1N1), Hong Kong 2369 / 2009 (H1N1), Texas / 37 / 2024 (H5N1), or clade 2.3.4.4b A(H5N1) infection). In some embodiments, provided methods are useful for treatingPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011an influenza B infection (e.g., B / Victoria / 2 / 87 viral infection). In some embodiments, the influenza virus is an oseltamivir-sensitive strain. In some embodiments, the influenza virus is a treatmentresistant strain.

[0181] In some embodiments, provided methods are useful for treating a viral infection caused by a parainfluenza virus, an adenovirus, an enterovirus, a coronavirus, a respiratory syncytial virus, a rhinovirus, a DNA virus, an RNA virus, variants thereof, or a combination thereof.

[0182] In some embodiments, provided methods are useful for treating a viral infection caused by a coronavirus. In some embodiments, the coronavirus includes, without limitation, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome-related coronavirus (SARSr-CoV), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a variant of SARS-CoV-2 (e.g., 20A. EU1, or spike variant D614G), or a combination thereof. In some embodiments, the coronavirus is a SARS-CoV-2 virus, an alpha variant thereof (e.g., B.l.1.7), a delta variant thereof (e.g., B.1.617.2), an omicron variant thereof (e.g., B.1.1.529), or a combination thereof.

[0183] In some embodiments, provided methods are useful for treating a cancer. In some embodiments, the cancer is tracheal cancer, lung cancer, bronchial cancer, epithelial cancer, blood cancer, breast cancer, melanoma, ovarian cancer, gynecological cancer, leukemia, lymphoma, prostate cancer, bladder cancer, colon cancer, glioma, sarcoma, glioblastoma, or a combination thereof. In some embodiments, the cancer is lung cancer.

[0184] Advantages and Applications

[0185] The methods and compositions of the present disclosure can have numerous advantages and applications. For instance, in some embodiments, the methods and compositions of the present disclosure can be utilized to treat viral infections independent of viral mutations. In some embodiments, the compositions of the present disclosure can serve as an agonist of a host immune response and hence bypass development of resistance to treatment.

[0186] In some embodiments, the methods and compositions of the present disclosure can have ease of administration in a safe and cost-effective manner. For instance, in some embodiments, the methods and compositions of the present disclosure can provide lasting immunity at a single dose. InPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011some embodiments, the methods and compositions of the present disclosure are stable at room temperature.

[0187] In some embodiments, the methods and compositions of the present disclosure provide protection in aged and / or immunocompromised subjects. In some embodiments, the methods and compositions of the present disclosure may be utilized to provide protection against highly pathogenic viruses, such as the H5N1 virus.

[0188] Additional embodiments

[0189] Reference will now be made to more specific embodiments of the present disclosure and experimental results that provide support for such embodiments. However, Applicant notes that the disclosure below is for illustrative purposes only and is not intended to limit the scope of the claimed subject matter in any way. The results described are reflective of the particular conditions outlined in each example.

[0190] Example 1. NanoSTING-CG induces dose-dependent Ifnb1 and Cxcl10 expression in the nasal tissue and lungs

[0191] Viral pathogens, including influenza viruses, constantly develop drug -resistant strains and variants, making viral respiratory infections one of the most important global health threats. A liposomally incorporated STING agonist, hereby referred to as NanoSTING (NS), has been developed that evokes a potent antiviral immune response. Applicant’s studies showed that intranasal NanoSTING, when combined with free 2’,3’-cGAMP (CG), referred to as “NanoSTING-CG,” synergistically enhanced activation of interferon-stimulated genes, such as Ifnb1 and Cxcl10, promoting robust antiviral activity. In particular, NanoSTING-CG significantly enhances the antiviral response, providing a robust, broad-spectrum protection against Influenza A & B viruses, including the H5N1 virus (pathogenic avian influenza virus). This synergy not only improved therapeutic efficacy but also contributed to faster viral clearance and better overall outcomes.

[0192] A single dose of NanoSTING-CG demonstrated broad-spectrum efficacy in murine models of influenza A and B, providing superior protection compared to 10 doses of oseltamivir, with significant reductions in viral titers and improved survival rates. Importantly, NanoSTING was effective even in immunocompromised and aged mice, highlighting its potential use in for high-risk populations. Mechanistically, NanoSTING acts through the STING pathway, rapidly upregulating antiviral cytokines in both the nasal turbinates and lungs.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0193] Safety evaluations confirmed minimal lung toxicity at therapeutic doses, supporting its use as safe and potent intranasal antivirals. Also, NanoSTING-CG protected weight loss against highly pathogenic H5N 1 challenge. These results suggest that NanoSTING-CG can serve as an off-the-shelf, low-cost prophylactic or therapeutic for respiratory viral infections.

[0194] Example 1.1. Formulation of NanoSTING-GC

[0195] NanoSTING is a liposomal formulation that incorporates the endogenous STING agonist, 2’,3’-cGAMP. 2’,3’-cGAMP can naturally be transported across cell membranes using several transporters, including SLC19A1 and LRRC8A transporters, which play crucial roles in its uptake into immune cells and activation of STING pathway. Applicant’s prior work demonstrated that, upon liposomal incorporation to yield NanoSTING, the 2’,3’-cGAMP is delivered primarily to epithelial cells and myeloid cells within the nasal compartment.

[0196] NanoSTING is a formulation comprising liposomes composed of a molar ratio of 10:1:1:1 DPPC, DPPG, cholesterol, and DPPE-PEG2000 with 2’,3’-cGAMP incorporated therein. NanoSTING was prepared according to methods described in WO 2023 / 141328, the contents of which are hereby incorporated by reference. NanoSTING compositions and variations thereof are also described in WO 2022 / 020542 and WO 2022 / 098701, the contents of each of which are hereby incorporated by reference.

[0197] Applicant aimed to investigate if using a mixture of free 2’,3’-cGAMP (CG) and NanoSTING (NS) (FIG. 2A) altered the cellular targets of 2’,3’-cGAMP mediated activation. Accordingly, Applicant investigated the ability of NanoSTING and naked 2’,3’-cGAMP to induce interferon response THP-1 dual reporter cells that conditionally secrete luciferase downstream of an IRF promoter. Applicant stimulated THPl-dual cells with NS and CG at doses ranging from 1.25-5 pg and checked the luciferase activity in the supernatant at 12 and 24 h. Throughout the disclosure, a dose of NanoSTING (NS) refers to the dose of cGAMP incorporated with the liposome of NanoSTING, e.g., a 5 pg NS dose refers to an amount of NanoSTING composition that incorporates 5 pg of cGAMP. NS treatment resulted in dose dependent increase in luciferase activity at 12 hours, increasing by 24 hours, with the highest response at 5 pg, indicating sustained activation. Controls showed minimal activity (FIG.2B).

[0198] Applicant evaluated the ability of NanoSTING to activate the STING pathway in BALB / c mice by measuring the expression of key effector cytokines, C-X-C motif chemokine ligandPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-01110 (Cxcl10) and interferon beta (Ifnb1) following intranasal administration. Mice were divided into groups receiving increasing doses of NanoSTING (4, 6, and 12 pg) intranasally. Histopathological analysis and RT-qPCR were conducted to identify the optimal NanoSTING dose that effectively activated the STING pathway while minimizing toxicity for intranasal administration (FIG. 2C). At 4 pg NS dose, Ifnb1 induction increased by 922-fold and Cxcl10 by 50-fold compared to PB S controls. The 6 pg NS dose further elevated Ifnb1 to 4473 -fold and Cxcl10 to 107-fold. The highest dose of 12 pg resulted in 10,650-fold Ifnb1 and 256-fold Cxcl10 induction. These results highlighted NanoSTING’ s potent ability to stimulate antiviral immune responses, with maximal activation at 12 pg (FIG.2D).

[0199] Histopathological analysis of lung tissue 24 hours after intranasal administration revealed no significant tissue damage or inflammation compared to PBS-treated controls (FIG. 2H).However, at the higher dose of 12 pg NanoSTING, there was neutrophil infiltration and necrotic debris in the lungs, as indicated by an elevated pathology score (FIG. 21). Given these results, Applicant decided to proceed with 4 pg NanoSTING dose for all future studies, as it demonstrated the most potent immune response with minimal toxicity.

[0200] Next, Applicant intranasally administered increasing doses of naked 2’,3’-cGAMP (10, 20, and 40 pg) to three groups of mice to determine the optimal concentration for inducing maximal Ifnb expression. The Applicant’s goal was to identify the most effective dose of 2’,3’-cGAMP to enhance Ifnb induction when spiked outside of the NanoSTING formulation. To validate the activation of Ifnb, Applicant performed RT-qPCR on the nasal tissue samples. For Ifnb expression, the 10 pg and 20 pg doses of 2’,3’-cGAMP induced 1073-fold and 1071-fold upregulation compared to the PBS-treated control. However, at the highest dose of 40 pg, Ifnb expression decreased to a 568-fold induction, suggesting a saturation effect at higher concentrations where the response plateaued. Similarly, Cxcl10 expression exhibited a dose-dependent pattern. The 10 pg and 20 pg doses resulted in 42 -fold and 50-fold induction, respectively. At 40 pg, Cxcl10 expression increased to 55-fold, indicating that both Ifnb and Cxcl10 show saturation at higher concentrations of 2’,3’-cGAMP (FIG.2E). The histopathology data revealed no toxicity at all doses tested (FIGS. 2H-I).

[0201] Based on these observations, Applicant chose the 40 pg dose of 2’,3’-cGAMP for spiking NanoSTING to maximize immune stimulation while accounting for saturation effect. To further explore immune enhancement, Applicant added varying amounts of 2’,3’-cGAMP (10, 20, andPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-01140 pg) to a fixed concentration of 4 pg of NanoSTING and dosed the animals intranasally and collected lungs and nasal tissue for RT-qPCR post 24 of dosing the mice. In nasal tissue, Ifnb expression showed a marked increase with NanoSTING 4pg-naked 2’,3’-cGAMP 40pg (NS4-CG40), reaching a fold induction of 1441-fold compared to the PBS control. Similarly, Cxcl10 expression also demonstrated a strong response, with a fold induction of 192-fold at the same dosage. Lower doses of 2’,3’-cGAMP, specifically 10 pg and 20 pg, also yielded substantial increases, indicating a dose-dependent relationship in the induction of both cytokines (FIG. 2F).

[0202] In lung tissue, the results were consistent with those observed in nasal tissue. The highest dose of NS4-CG40 again resulted in a significant fold induction for Ifnb at 68-fold and for Cxcl10 at 71 -fold compared to the PBS control (FIG. 2G). Based on these findings, Applicant conducted the histopathological analysis ofNS4-CG40 to assess potential toxicity. The results showed no signs of inflammation in the lung tissue, confirming its safety at this dose.

[0203] These findings, combined with robust immune stimulation observed with both nasal and lung tissue when NanoSTING was supplemented with 2’,3’-cGAMP spiked outside the liposomes, demonstrated that this formulation synergistically stimulated innate immune responses in both the nasal and lung tissues without inducing toxicity (FIGS.2H-2I).

[0204] Example 1.2, NanoSTING-CG provides protection against Influenza A superior to Oseltamivir

[0205] Applicant assessed the protective effects of NanoSTING against aerosolized Influenza A virus in a murine model. BALB / c mice were challenged with 2 x 104CCID50 of Influenza A / California / 04 / 2009 (H1N1dpm) strain and 24 h later treated with either NS4-CG40 (administered intranasally), oseltamivir (40 mg / kg / day for five days), or a placebo (PBS). The animals were monitored for changes in body weight until day 20. Percent survival and viral titers (lungs) were the primary end points of the study (FIG. 3A). The results demonstrated that the placebo group experienced a severe decline in body weight, losing approximately 30% within 10 days (mean peak weight loss: -29.6%), indicative of significant disease progression. In contrast, both NS4-CG40 and oseltamivir-treated groups exhibited reduced weight loss (mean peak weight loss: -26.9%), with NS4-CG40 treated mice showing a more pronounced recovery after day 10 (mean peak weight loss: -18.7%) (FIG. 3B)PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0206] By the end of the study (day 20), the percent survival was 0% for the placebo group, while both the NS4-CG40 and oseltamivir (40 mg / kg / d) groups achieved 60% survival, with six out of ten animals surviving in each group. Notably, the NS4-CG40 group showed less severe weight loss compared to oseltamivir, indicating its potential as a more effective therapeutic option in mitigating the adverse effects associated with viral infections (FIG. 3C). These findings suggested that NanoSTING enhanced resilience better than oseltamivir in this experimental model. Furthermore, viral titers in the lungs were significantly lower in both NanoSTING and oseltamivir-treated groups compared to the placebo group, indicating more rapid viral clearance (FIG. 3D). These findings suggested that NanoSTING provided superior protection and recovery compared to Oseltamivir, with a single dose proving as effective as ten doses of Oseltamivir. These findings reinforced NanoSTING’ s potential as a therapeutic alternative for influenza treatment.

[0207] Example 1.3, NanoSTING-CG provides protection against Influenza B superior to Oseltamivir

[0208] BALB / c mice were challenged with 2 x 104CCID50 of Influenza B (B / Victoria / 2 / 87) strain on Day 0 and NS4-CG40 was administered intranasally 24 h post-infection, while oseltamivir was given daily for five days. The animals were monitored for changes in body weight until day 20. Percent survival was the primary end point of the study (FIG. 4A). In the placebo group, mean peak body weight loss of -28.8% was observed by day 7. In contrast, both the NS4-CG40 group (with a mean peak weight loss of -11.4%) and the oseltamivir-treated group (with a mean peak weight loss of -25.8%) experienced less severe weight loss. Remarkably, the NanoSTING treatment not only resulted in the least weight loss but also facilitated a more rapid recovery in body weight compared to both oseltamivir and placebo groups (FIG. 4B).

[0209] Consistent with these observations, the PBS control group exhibited a survival rate of 0% by day 7. In contrast, the NS4-CG40 group demonstrated a remarkable survival rate of 87.5%, with 7 out of 8 animals remaining alive by the end of the study. Meanwhile, the oseltamivir group showed a survival rate of 50%, with 4 out of 8 animals surviving, highlighting the effectiveness of NS4-CG40 in improving survival outcomes compared to both oseltamivir and placebo (FIG. 4C).Collectively, these findings suggest that NS4-CG40 is highly effective in mitigating body weight loss and promoting recovery, and significantly enhancing survival rates compared to oseltamivir, thereby indicating its capacity to improve outcomes in this context.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0210] Example 1.4, NanoSTING-CG induces potent immune response in immunocompromised and aged mouse models

[0211] Given the increased susceptibility of immuno-compromised and aged populations to severe influenza outcomes, Applicant aimed to evaluate the immunostimulatory effects of NS4-CG40 in these vulnerable models. The expression of Ifnb and Cxcl10 in the nasal tissue was examined in immunocompromised mouse models upon intranasal administration with NS4-CG40. Applicant induced immunosuppression with Cyclophosphamide (CFX) or Dexamethasone (Dexa). Groups of mice were pretreated with CFX, IP, 48 h before or Dexa, IP, 24 h before, followed by intranasal dosing with NS4-CG40, and animals were euthanized post 24 h of the dosing (FIGS. 5A and 5C).

[0212] RT-qPCR analysis of nasal tissue collected at 24h post-treatment showed significant upregulation of both Ifnb and Cxcl10 in the NS4-CG40 treated groups compared to the controls, indicating NanoSTING's potent immunostimulatory effects. However, no significant differences were observed between the CFX + NS4-CG40 or Dexa + NS4-CG40 groups and the NS4-CG40 alone group, suggesting that immunosuppression by CFX or Dexa did not diminish NanoSTING’ s ability to activate these immune pathways (FIGS. 5B and 5D).

[0213] Applicant also evaluated the influence of age on NanoSTING-induced immune responses by comparing young (2-month-old BALB / c mice) and aged (18-22-month-old B.129 mice) mice. Both groups were administered 20 pg of NanoSTING intranasally, and nasal tissues were harvested at 48h for gene expression analysis (FIG. 5E). As shown in FIG. 5F, both Ifnb and Cxcl10 were strongly induced in young and aged mice compared to the untreated control (FIG. 5F). No significant differences in gene induction were observed between the two age groups, indicating that age did not impair NanoSTING’s ability to activate these key antiviral pathways in nasal tissue.

[0214] Example 1.5, NS4-CG40 attenuate H5N1-Induced weight loss in mice

[0215] Applicant then investigated whether NS4-CG40 was able to mitigate these severe symptoms from highly pathogenic H5N1, which induces severe respiratory illness and significant weight loss in infected animals, in a mouse challenge model. In this Example, mice were challenged with H5N1 AIV (Influenza A / Vietnam / 1203 / 2004) strain and treated with NS4-CG40 as pre-treatment (24 hours before challenge) or post-treatment (24 or 48 hours after challenge). Applicant recorded changes in body weight of mice for 20 days. Oseltamivir at 75 mg / kg post-challenge served as positive control. PBS-treated and non-challenged groups were used as negative controls (FIG. 6A).PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011

[0216] As depicted in FIG. 6B, weight loss among the PBS-treated mice began directly after H5N1 challenge and continued through Day 8 with a peak loss of greater than 20% of initial body weights. In contrast, the Oseltamivir-treated group had initial weight loss comparable to the PBS-treated mice but recovered at around Day 8, and by Day 15, it attained near baseline body weight. The pre-treatment with NS4-CG40, therefore had completely prevented severe weight loss; only a slight decrease in body weight was observed within the first few days post-challenge.

[0217] The mice in this group recovered quicker than their Oseltamivir-treated group, eventually reaching their baseline or slightly above baseline body weight by Day 8. The post-treatment in NS4-CG40, either NanoSTING-Tx-24h or NanoSTING-Tx-48h post-challenge, resulted in an intermediate effect. Animals treated 24 hr post-challenge exhibited mild weight loss and were back to normal by Day 10. Animals treated with 48 hr post-challenge were delayed in recovering to normal weight, similar to the Oseltamivir group. The mice that were not challenged did not show significant weight loss; instead, they maintained the same body weights throughout the duration of the study (FIG. 6B)

[0218] Data demonstrated that NS4-CG40 is highly protective against H5Nl-induced weight loss in mice and effective under both pre- and post-treatment regimens. Of note, pretreatment with NS4-CG40 gave a quick recovery with better weight retention compared to post-treatment and the standard antiviral, Oseltamivir, although NS4-CG40 was given as a single dose against the multiple doses of Oseltamivir. These results support the potential of NS4-CG40 for use as an effective therapeutic agent against severe influenza infections, including highly pathogenic strains such as H5N1.

[0219] Example 2. Therapeutic intranasal delivery of NanoSTING provides broad protection against seasonal and highly pathogenic influenza strains

[0220] In this Example, Applicant reports the development of NanoSTING, a liposomally encapsulated STING agonist (cGAMP), as a single-dose intranasal treatment of influenza. Applicant demonstrates that NanoSTING is stable under simple refrigeration conditions, with no loss of encapsulated cGAMP, for up to a year. A single dose of NanoSTING administered intranasally induced robust type I interferon responses in the nasal and lung tissue of mice without observable toxicity. In mouse challenge models with influenza A (H1N1 or highly pathogenic H5N1) and influenza B, NanoSTING exhibited therapeutic protection comparable to or superior to ten doses ofPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011oseltamivir. NanoSTING demonstrated therapeutic efficacy even when administered 48-72 hours post-infection. Furthermore, NanoSTING maintained its activity in aged and immunocompromised mice, as evidenced by the robust induction of interferon responses in nasal tissues, highlighting its potential for use in vulnerable individuals. These attributes of NanoSTING support its potential use as a promising host-directed anti-viral with a large therapeutic window and broad-spectrum efficacy

[0221] Example 2, 1, NanoSTING activates type I interferon responses in nasal and lung tissue without inducing toxicity

[0222] NanoSTING in this Example is a liposomal formulation that encapsulates the endogenous STING agonist, cGAMP (FIG. 7A). cGAMP can naturally be transported across cell membranes using several transporters, including SLC19A1 and LRRC8A transporters, which play a crucial role in its uptake primarily into immune cells and activation of the STING pathway.

[0223] To assess the long-term stability of NanoSTING at 4°C, Applicant measured the particle diameter and zeta potential at various time points over 12 months. NanoSTING maintained a consistent particle size (120 ± 30 nm) with a low poly dispersity index (PDI <0.1) throughout all time points tested (FIG. 7B). The zeta potential remained unchanged during this same period of observation (FIG. 7C). Applicant confirmed these DLS results independently using NanoSight nanoparticle tracking analysis (NTA). Applicant was also able to visually confirm the shape and size of the NanoSTING liposomes using NTA. These findings confirm that NanoSTING maintains its physicochemical characteristics with simple refrigeration. Applicant quantified the amount of encapsulated cGAMP using HPLC, and these results showed no change in the amount of cGAMP over time, supporting NanoSTING's structural integrity and indicating minimal drug leakage for up to a year (FIG. 7D).

[0224] One of the advantages of liposomal encapsulation is that it reduces the effective dose of cGAMP necessary for efficacy. Applicant compared the ability of NanoSTING and cGAMP to activate the STING pathway in BALB / c mice by measuring the expression of key effector cytokines, C-X-C motif chemokine ligand 10 Cxcl10) and interferon beta (IfnbB) following intranasal administration. Mice were divided into groups and administered a single intranasal dose of either PBS (Control), NanoSTING (0.2 mg / kg encapsulated cGAMP), or cGAMP (2 mg / kg), and euthanized 24 hours after administration for collection of nasal tissue and lungs (FIG. 7E). In the nasal tissue, NanoSTING-mediated activation of Ifnb1 (1,200 ± 500 fold compared to PBS 1.1 ± 0.3) wasPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011comparable to cGAMP-mediated activation of Ifnb1 (600 ± 100 fold compared to PBS) (FIG. 7F).Upregulation of Cxcl10 followed a similar pattern with both treatments showing no significant differences (FIG. 7F). These findings demonstrate that NanoSTING achieves comparable activation of the STING pathway at one-tenth the dose of free cGAMP.

[0225] To validate Applicant’s initial observations in the nasal compartment and to explore whether STING activation extends to lungs, Applicant repeated the experiment, isolated nasal tissue for confirmation, and evaluated lung tissue in parallel. Applicant assessed the upregulation of Ifnb1 and Cxcl10 gene expression in both nasal tissue and lungs. Consistent with intranasal delivery, the magnitude of activation in the nasal compartment was again higher and more uniform across the animals (Ifnb1, 2800 ± 800 fold compared to PBS, 1.9 ± 0.9) whereas the activation in the lung was 10-fold lower and more heterogenous across the animals (Ifnb1, 100 ± 100 fold compared to PBS, 1.6 ± 0.7) (FIGS. 7G-7H). To evaluate safety in the lung, Applicant conducted hematoxylin and eosin (H& E) staining to assess the safety of NanoSTING following intranasal administration in lung tissues. Mice treated with NanoSTING (0.2 mg / kg) displayed normal lung architecture, with no evidence of inflammation, confirming its safety (FIG.71). Similarly, mice treated with cGAMP (2 mg / kg) showed no histological abnormalities. Collectively, these findings confirm that NanoSTING activates innate immune responses without inducing toxicity (FIG. 71).

[0226] Example 2,2, NanoSTING provides protection against Influenza A, superior to oseltamivir

[0227] Applicant assessed the protective effects of NanoSTING against aerosolized Influenza A virus in a murine model. BALB / c mice were challenged with 1 x 104CCID50 of Influenza A / California / 04 / 2009 (H1N1dpm) strain and treated with either NanoSTING (single intranasal dose at 24 hours post infection (hpi), oseltamivir (30 mg / kg / day for five days, starting at 12 hpi), or a placebo (PBS) (FIG. 8A). The results demonstrated that the placebo group experienced a severe decline in body weight (mean peak weight loss: -29.6 %), indicative of significant disease progression. In contrast, the oseltamivir-treated groups exhibited reduced weight loss (mean peak weight loss: -26.9 ± 0.8%), with NanoSTING-treated mice showing a significantly lower weight loss (mean peak weight loss: -19 ± 2%) (FIG. 8B). Notably, the NanoSTING-treated animals showed faster recovery from weight loss compared to oseltamivir. Treatment with either NanoSTING or oseltamivir significantly improved survival of mice compared to the placebo treated mice (FIG. 8C). ApplicantPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011evaluated viral titers in the lung homogenates; the viral titers were significantly lower in both NanoSTING and oseltamivir-treated groups compared to the placebo group, indicating more rapid viral clearance (FIG. 8D). Collectively, these findings suggest that NanoSTING provides superior recovery compared to oseltamivir with a single dose proving as effective as ten doses of oseltamivir in enabling survival from lethal influenza challenge.

[0228] Example 2.3. NanoSTING provides protection against Influenza B superior to oseltamivir

[0229] To evaluate therapeutic efficacy against influenza B, mice were challenged with 1 x 104.3CCID50of Influenza B strain B / Brisbane / 60 / 2008 (Victoria lineage) on Day 0 and NanoSTING was administered intranasally 24 hpi, while oseltamivir (30 mg / kg / day) was given twice daily for five days starting at 12 hpi (FIG. 9A). In the placebo group, mean peak body weight loss of -28.8 % was observed by day 7. In contrast, the oseltamivir-treated group (with a mean peak weight loss of -26 ± 2 %) experienced less severe weight loss and increased survival (FIGS. 9B and 9C). Similar to the results with influenza A infected animals, the NanoSTING treated animals had less severe weight loss (mean peak weight loss of -11 ± 3 %) and a more rapid recovery in body weight compared to both oseltamivir and placebo groups (FIG. 9B). While the untreated animals did not survive, treatment with either oseltamivir or NanoSTING significantly improved survival (FIG. 9C). Collectively, these findings suggest that NanoSTING is effective in mitigating loss in body weight and promoting recovery, better than oseltamivir, further strengthening its translational potential.

[0230] Example 2.4. NanoSTING induces potent immune response in immunocompromised and aged mouse models

[0231] Given the increased susceptibility of immunocompromised and aged populations to severe influenza outcomes, Applicant aimed to evaluate the immunostimulatory effects of NanoSTING in these models. Lymphopenia, either due to natural reasons like autoimmune diseases or induced due to medications (e.g., chemotherapies for cancer), increases the likelihood of infections and is associated with high risk of mortality among adults. Accordingly, Applicant evaluated the ability of NanoSTING to activate the innate immune system in lymphodepleted animals by using three groups of animals: (1) mice receiving cyclophosphamide (CFX) alone, (2) mice receiving only a single intranasal dose of NanoSTING (0.2 mg / kg), and (3) mice that were pre-treated with CFX and, 48 hours later, treated with a single intranasal dose of NanoSTING (FIG. 10A). Quantification of Ifnb1 andPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011Cxcl10 in the nasal tissue using RT-qPCR confirmed that pre-treatment with CFX had no significant impact on the ability of NanoSTING to upregulate either Ifnb1 (3,000 ± 2,000 fold for NanoSTING treated mice vs 4,500 ± 1,000 fold for CFX + NanoSTING treated mice) or Cxcl10 (110 ± 30 fold for NanoSTING treated mice vs 120 ± 20 fold CFX + NanoSTING treated mice) (FIG. 10B). Hence, NanoSTING retains innate immunostimulatory activity under cyclophosphamide-induced lymphodepletion, supporting its use in vulnerable hosts.

[0232] Corticosteroids have become indispensable tools for controlling inflammation in modern medicine and are used to treat a wide range of inflammatory disorders, including chronic diseases such as rheumatoid arthritis, asthma, and inflammatory bowel disease. Since corticosteroids are also immunosuppressive, Applicant tested whether the treatment with a commonly used corticosteroid, dexamethasone, interferes with NanoSTING. Applicant treated a groups of mice with either: (1) dexamethasone (DX) alone, (2) single-dose intranasal NanoSTING (0.2 mg / kg), or (3) treatment with DX and 24 hours later treated mice with a single intranasal dose of NanoSTING (FIG.10C). Quantification oi Ifnb1 and Cxcl10 in the nasal tissue using qPCR confirmed that pre-treatment with DX had no significant impact on the ability of NanoSTING to upregulate either Ifnb1 (1,300 ± 300 fold for NanoSTING treated mice vs 1,600 ± 300 fold for DX + NanoSTING treated mice) or Cxcl10 (370 ± 90 fold for NanoSTING treated mice vs 400 ± 200 fold for DX + NanoSTING treated mice) (FIG. 10D).

[0233] Older adults (>65 years old) are at higher risk of developing serious complications from influenza infections compared to younger adults. One of the primary reasons for this risk is that the adaptive immune system weakens with advanced age. Applicant sought to determine whether NanoSTING-mediated activation of the innate immune system is preserved in aged mice, serving as a surrogate for elderly humans. Applicant treated groups of old mice (18-22 months) intranasally with low dose of NanoSTING (0.1 mg / kg) to determine their efficacy and compared their responses with those of the young mice (2 months) (FIG. 10E). RT-qPCR confirmed that the upregulation of Ifnb1 (100 ± 20 fold for aged mice vs 90 ± 40 fold for young mice) or Cxcl10 (70 ± 20 fold for aged mice vs 50 ± 20 fold for young mice) in the nasal compartment was no different when comparing aged to young mice, indicating that age did not impair NanoSTING's ability to activate these key antiviral pathways in nasal tissue (FIG. 10F). Taken together, these results demonstrate that NanoSTING-PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011mediated activation of the innate immune system is largely preserved in animal models of the vulnerable populations.

[0234] Example 2.5. NanoSTING attenuates H5Nl-induced weight loss in mice

[0235] Since 2001, there has been a resurgence of highly pathogenic avian influenza HPAI A(H5N1) virus transmission among birds, and in 2024, human infections were reported. Applicant investigated whether NanoSTING could offer protection against HP Al H5N1, which induces significant respiratory illness and weight loss in infected mice. Groups of mice were challenged with H5N1 and treated with a single intranasal dose of NanoSTING as either a pretreatment (24 hours before challenge) or post-treatment (24, 48, or 72 hpi). In this animal model, to afford any protection, we had to use oseltamivir at a high dose (75mg / kg / day) and initiate treatment with oseltamivir 4 hpi, consistent with other published reports (FIG. 11A).

[0236] In this HP Al A(H5N1) model, single-dose NanoSTING pretreatment outperformed oseltamivir treatment in preventing weight loss (mean peak weight loss: NanoSTING -3.2 ± 0.8 % vs oseltamivir -5 ± 1 %) and faster recovery, whereas placebo-treated mice exhibited sustained weight loss (FIGS. 11B-11D). To evaluate the therapeutic window, Applicant initiated NanoSTING treatment 24 / 48 / 72 hpi. Surprisingly, treatment at 24 hpi was the least effective at preventing weight loss (mean peak weight loss: -13 ± 2 %), whereas 48 hpi (mean peak weight loss: -12 ± 3 %) and 72 hpi (mean peak weight loss: -11 ± 3 %) showed better protection (FIGS. 11E-11F). Comparisons of all these groups illustrated that even a single-dose treatment with NanoSTING, given 72 hpi, showed significant improvement in preventing weight loss compared to placebo (FIG. 11G).

[0237] The quantification of viral titers in the lungs of NanoSTING-pretreated mice and oseltamivir-treated mice was significantly lower (2 ± 0.4) compared to that of untreated animals (4.9 ± 0.2), confirming that protection was conferred at least partly through reduced viral replication (FIG.11H) NanoSTING treatment administered at 48 hpi had significantly lower viral titers (2.7 ± 0.3) compared to the placebo 24 hpi group (4.2 ± 0.4) (FIG. 11I). Notably, the reduction in viral titers with a single dose of NanoSTING initiated 48hpi was comparable to the reduction in viral titers observed with 10 doses of oseltamivir initiated 4 hpi (FIG. 11I).

[0238] These results demonstrate that NanoSTING is highly effective in reducing both HPAI (A)H5N1-induced weight loss and viral replication. A single intranasal dose of NanoSTING, whether used as a prophylactic or therapeutic, provided protection comparable to or better than multiple dosesPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011of oseltamivir. These findings support the potential of NanoSTING as a promising antiviral for both prophylactic and therapeutic use against severe influenza infections, including HPAI (A)H5N1, with a broad window of treatment.

[0239] Without further elaboration, it is believed that one skilled in the art can, using the description herein, utilize the present disclosure to its fullest extent. The embodiments described herein are to be construed as illustrative and not as constraining the remainder of the disclosure in any way whatsoever. While the embodiments have been shown and described, many variations and modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. Accordingly, the scope of protection is not limited by the description set out above, but is only limited by the claims, including all equivalents of the subject matter of the claims. The disclosures of all patents, patent applications and publications cited herein are hereby incorporated herein by reference, to the extent that they provide procedural or other details consistent with and supplementary to those set forth herein.

Claims

PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011CLAIMS1. A composition comprising:a particle, wherein a first modulator is encapsulated within the particle, anda second modulator is in free form in the composition,wherein the first and second modulators are each independently selected from the group consisting of a pattern recognition receptor agonist, an activator of the immune system, and a combination thereof.

2. The composition of claim 1, wherein the first and second modulators are the same.

3. The composition of claim 1 or 2, wherein the first and second modulators are each independently selected from the group consisting of an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’, 3 ’-cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, CF501, MSA-2, SHR1032, C-178, H-151, divalent cations, and a combination thereof.

4. The composition of claim 1 or 2, wherein the first and second modulators are each independently a compound of formula I:or a pharmaceutically acceptable salt thereof, wherein:PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011Ring A and Ring B are each independently selected from the group consisting of:each X1and X2is independently -CR- or -N-;each X3is independently -C(R)2-, -O-, or -NR-;Xcand Xdare each independently -OR, -SR, -N(R)2, BH3, or optionally substituted Ci-12 aliphatic; Xeand Xfare each independently -O-, -S-, or -N(R)-;each R1and R2is independently selected from the group consisting of hydrogen, halogen, -NO2, - CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, - S(O)2R, - C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, - N(R)C(=NR)N(R)2, - C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, -N(R)SO2N(R)2, - N(R)SO2R, -OC(O)N(R)2, and optionally substituted Ci-i2aliphatic or C1-4 alkoxy-Ci-4 alkyl;each R3, R4, R5, R6, and R7is independently selected from the group consisting of hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, - C(O)CH2C(O)R, - S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, - N(R)N(R)2, - N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -ONOR, -N(R)C(O)N(R)2, - N(R)SO2N(R)2, -N(R)SO2R, - OC(O)N(R)2, or an optionally substituted group selected from Ci-12 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each R is independently selected from the group consisting of hydrogen or an optionally substituted group selected from C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered saturated or partially unsaturated monocyclic heterocyclic ringPCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:two R groups on the same nitrogen are taken together with their intervening atoms to form an optionally substituted 3-7 membered saturated, partially unsaturated, or heteroaryl ring having 1- 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

5. The composition of any one of claims 1-4, wherein the first modulator comprises an endogenous agonist of the stimulator of interferon genes (STING) pathway.

6. The composition of any one of claims 1-4, wherein the first modulator comprises 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP).

7. The composition of any one of claims 1-6, wherein the second modulator comprises an endogenous agonist of the stimulator of interferon genes (STING) pathway.

8. The composition of any one of claims 1-6, wherein the first and the second modulator each comprises 2’, 3 ’-cyclic guanosine monophosphate-adenosine monophosphate (2’,3’-cGAMP).

9. The composition of any one of claims 1-8, wherein the molar ratio of the first modulator to the second modulator is about 1:2 to about 1:20.

10. The composition of claim 9, wherein the molar ratio of the first modulator to the second modulator is about 1:10.

11. The composition of any one of claims 1-10, wherein the composition comprises a lipid-based particle.

12. The composition of claim 11, wherein the lipid-based particle is a liposome.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-01113. The composition of claim 11 or 12, wherein the lipid-based particle is anionic.

14. The composition of any one of claims 11-13, wherein the lipid-based particle comprises one or more anionic lipids, one or more neutral lipids, and one or more polyethylene glycol (PEG)-lipids.

15. The composition of claim 14, wherein the PEG moiety of the one or more PEG-lipids has an average molecular weight of 500-5000 Da.

16. The composition of any one of claims 11-15, wherein the lipid-based particle comprises 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), cholesterol, or a combination thereof.

17. The composition of claim 16, wherein the lipid-based particle comprises DPPC, DPPG, DPPE-PEG2000, and cholesterol in a molar ratio of about 10:1:1:1.

18. The composition of any one of claims 1-17, wherein the composition further comprises an antigen.

19. The composition of claim 18, wherein the antigen is operable to elicit an immune response in a subject against a virus.

20. The composition of claim 18 or 19, wherein the antigen comprises a protein or peptide derived from a virus, or a polynucleotide encoding the protein or peptide.

21. The composition of claim 20, wherein the antigen comprises a protein or peptide derived from a virus.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-01122. The composition of claim 21, wherein the virus is selected from the group consisting of an influenza virus, an influenza A virus, an influenza B virus, an influenza H5N1 virus, a SARS-CoV-2 virus, a respiratory virus, or a combination thereof.

23. A method of treating a disease or condition in a subject, the method comprising:administering a composition to a subject, wherein the composition comprises:a particle, wherein a first modulator is encapsulated within the particle, anda second modulator is in free form in the composition,wherein the first and second modulators are each independently selected from the group consisting of a pattern recognition receptor agonist, an activator of the immune system, and a combination thereof.

24. The method of claim 23, wherein the disease or condition is a viral infection.

25. The method of claim 24, wherein the viral infection is caused by a virus selected from the group consisting of an influenza virus, an influenza A virus, an influenza B virus, an H5N1 virus, a respiratory virus, a SARS-CoV-2 virus, and a combination thereof.

26. The method of any one of claims 23-25, wherein the subject is a human being.

27. The method of any one of claims 23-26, wherein the subject is immunocompromised.

28. The method of any one of claims 23-27, wherein the subject is 60 years of age or older.

29. The method of any one of claims 23-28, wherein the subject is receiving or has received an antiviral drug.

30. The method of any one of claims 23-29, wherein the composition induces Ifnb and Cxcl10 expression in the subject.PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-01131. The method of any one of claims 23-29, wherein the composition is administered through intranasal administration.

32. The method of any one of claims 23-31, wherein the composition is administered through inhalational administration.

33. The method of any one of claims 23-32, wherein the composition is administered in a single dose.

34. The method of any one of claims 23-33, wherein the first and second modulators are each independently selected from the group consisting of an endogenous agonist of the stimulator of interferon genes (STING) pathway, bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), 2’, 3 ’-cyclic guanosine monophosphate-adenosine monophosphate (2’, 3 ’-cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, CF501, MSA-2, SHR1032, C-178, H-151, divalent cations, and a combination thereof.

35. The method of any one of claims 23-33, wherein the first and second modulators are each independently a compound of formula I:or a pharmaceutically acceptable salt thereof, wherein:Ring A and Ring B are each independently selected from the group consisting of:PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011each X1and X2is independently -CR- or -N-;each X3is independently -C(R)2-, -O-, or -NR-;Xcand Xdare each independently -OR, -SR, -N(R)2, BH3, or optionally substituted C1-12 aliphatic; Xeand Xfare each independently -O-, -S-, or -N(R)-;each R1and R2is independently selected from the group consisting of hydrogen, halogen, -NO2, - CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, -C(O)CH2C(O)R, -S(O)R, - S(O)2R, - C(0)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)N(R)2, - N(R)C(=NR)N(R)2, - C(=NR)N(R)2, -C=NOR, -N(R)C(0)N(R)2, -N(R)SO2N(R)2, - N(R)SO2R, -OC(O)N(R)2, and optionally substituted C1-12 aliphatic or C1-4 alkoxy-Ci-4 alkyl;each R3, R4, R5, R6, and R7is independently selected from the group consisting of hydrogen, halogen, -NO2, -CN, -OR, -SR, -N(R)2, -C(O)R, -CO2R, -C(O)C(O)R, - C(O)CH2C(O)R, - S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, - N(R)N(R)2, - N(R)C(=NR)N(R)2, -C(=NR)N(R)2, -C=NOR, -N(R)C(O)N(R)2, - N(R)SO2N(R)2, -N(R)SO2R, - 0C(0)N(R)2, or an optionally substituted group selected from C1-12 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated monocyclic carbocyclic ring, a 7-10 membered saturated or partially unsaturated bicyclic carbocyclic ring, a 3-7 membered saturated or partially unsaturated heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 7-10 membered saturated or partially unsaturated bicyclic heterocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6 membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each R is independently selected from the group consisting of hydrogen or an optionally substituted group selected from C1-6 aliphatic, phenyl, a 3-7 membered saturated or partially unsaturated carbocyclic ring, a 3-7 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and a 5-6PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011membered heteroaryl ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or:two R groups on the same nitrogen are taken together with their intervening atoms to form an optionally substituted 3-7 membered saturated, partially unsaturated, or heteroaryl ring having 1- 4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

36. The method of any one of claims 23-33, wherein the first and second modulators each independently comprise an endogenous agonist of the stimulator of interferon genes (STING) pathway.

37. The method of any one of claims 23-33, wherein the first and second modulators each independently comprise 2’,3’-cyclic guanosine monophosphate-adenosine monophosphate (2’, 3 ’-cGAMP).

38. The method of any one of claims 23-37, wherein the composition comprises a lipid-based particle.

39. The method of claim 38, wherein the lipid-based particle is a liposome.

40. The method of claim 38 or 39, wherein the lipid-based particle is anionic.

41. The method of claim 39 or 40, wherein the lipid-based particle comprises one or more anionic lipids, one or more neutral lipids, and one or more PEG-lipids.

42. The method of claim 41, wherein the PEG moiety of the one or more PEG-lipids has an average molecular weight of 500-5000 Da.

43. The method of any one of claims 38-42, wherein the lipid-based particle comprises 1,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-PCT Application Attorney Docket No. AF23853. P210WO UH ID No. 2025-011glycerol) (DPPG), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethylene glycol)-2000] (DPPE-PEG2000), cholesterol, or a combination thereof.

44. The method of claim 43, wherein the lipid-based particle comprises DPPC, DPPG, DPPE-PEG2000, and cholesterol in a molar ratio of about 10:1:1:1.

45. The method of any one of claims 23-44, wherein the composition further comprises an antigen.

46. The method of claim 45, wherein the antigen is operable to elicit an immune response in a subject against a virus.

47. The method of claim 45 or 46, wherein the antigen comprises a protein or peptide derived from a virus, or a polynucleotide encoding the protein or peptide.

48. The method of claim 47, wherein the antigen comprises a protein or peptide derived from a virus.

49. The method of claim 48, wherein the virus is selected from the group consisting of an influenza virus, an influenza A virus, an influenza B virus, an influenza H5N1 virus, a SARS-CoV-2 virus, a respiratory virus, or a combination thereof.

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