Nasal vaccines and treatments for respiratory diseases

Abstract

Described herein are compositions and methods for reducing the risk of respiratory disease in a subject or reducing the risk of respiratory disease being transmitted from a first subject to a second subject.In some cases, the compositions or methods described herein may include a modulator (e.g., a pattern recognition receptor, such as a STING agonist, e.g., cGAMP).In some cases, the compositions or methods described herein may include a liposome that can be used to encapsulate one or more STING agonists.In some cases, the liposome of the compositions or methods described herein may include one or more antigens attached to, integrated into, or associated with the liposomal membrane of the liposome.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 301,918, filed January 21, 2022, and U.S. Provisional Patent Application No. 63 / 329,261, filed April 8, 2022. The entirety of each of the foregoing applications is incorporated herein by reference for all purposes.

[0002] Sequence Listing

[0002] This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. Said XML copy was created on January 23, 2023, is named AF23853.P179WO.xml, and is 52,000 bytes in size. Applicant hereby incorporates the data in this Sequence Listing by reference. [Background technology]

[0003]

[0003] There is an urgent need for safe and durable prophylactic and therapeutic compositions to combat a variety of diseases, such as infections caused by respiratory pathogens. Numerous embodiments of the present disclosure address the aforementioned needs. [Brief description of the drawings]

[0004] [Figure 1] FIG. 1 provides a depiction of a composition comprising a liposome, a payload, and an antigen for treating or preventing a disease according to various embodiments of the present disclosure. [Diagram 2]

[0005] 2A and 2B illustrate a method of treating or preventing a disease in a subject. [Figure 3-1]

[0006] FIG. 3A shows an overall schematic of the formulation and nasal delivery of compositions (e.g., NanoSTING compositions) according to embodiments described herein to animals. FIG. 3B illustrates signaling pathways that may be involved in (e.g., activated by) embodiments of methods and compositions (e.g., including NanoSTING delivery). In some cases, THP1-Lucia™ cells can be used to stably express a secreted form of luciferase under the control of a synthetic interferon-responsive promoter, for example, to illustrate induction of type I interferon (IFN) (e.g., via IRF pathway activation) according to embodiments of compositions and methods described herein in an in vitro setting. [Figure 3-2]

[0007] Figures 3C and 3D illustrate the size distribution of NanoSTING liposome particles at 25 Celsius and 37 Celsius, respectively, as measured by dynamic light scattering according to an embodiment. [Figure 3-3]

[0008] Figure 3E illustrates the size distribution of NanoSTING liposome particles as measured by dynamic light scattering according to an embodiment, and Figure 3F illustrates the zeta potential of a NanoSTING composition according to an embodiment described herein as measured by electrophoretic light scattering (ELS). [Diagram 3-4]

[0009] FIG. 3G shows a table of particle size (DH), polydispersity index (PDI), and zeta potential of compositions described herein (e.g., NanoSTING particles) at 25 and 37 Celsius. [Figure 4]

[0010] Figure 4 illustrates the kinetics of luciferase induction in THP1-Dual cells by varying concentrations of NanoSTING according to an embodiment. RLU: Relative Light Units. [Figure 5-1]

[0011] Figure 5A illustrates the experimental design of an experiment in which three to four Balb / c mice were treated with a single dose of NanoSTING (10 µg, 20 µg, or 40 µg). Blood, nasal turbinates, and lungs from euthanized animals were analyzed at 6, 12, 24, 36, and 48 hours, and cGAMP from nasal turbinates was analyzed by ELISA (Figure 5B), cGAMP from lungs was analyzed by ELISA (Figure 5C), cGAMP from serum was analyzed by ELISA (Figure 5D), CXCL10, IFNB1, ISG15, IRF7, Mx1, Mx2, IL-6, TNF, IL-10, and Ifit1 were analyzed by qRT-PCR (RNA extracted from nasal turbinates) (Figures 5E, 5F, 5G, 5H, 5-I, 5J, 5K, 5L, 5M, and 5N, showing fold changes in gene expression, respectively), IFNβ by ELISA, and CXCL10 by ELISA. The results illustrate a method of treating or preventing disease in a subject. Analysis was performed using the Mann-Whitney test. Bars and columns indicate median values. Each dot represents an individual mouse. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 5-2]

[0011] Figure 5A illustrates the experimental design of an experiment in which three to four Balb / c mice were treated with a single dose of NanoSTING (10 μg, 20 μg, or 40 μg). Blood, nasal turbinates, and lungs from euthanized animals were analyzed at 6, 12, 24, 36, and 48 hours, and cGAMP from nasal turbinates was analyzed by ELISA (Figure 5B), cGAMP from lungs was analyzed by ELISA (Figure 5C), cGAMP from serum was analyzed by ELISA (Figure 5D), CXCL10, IFNB1, ISG15, IRF7, Mx1, Mx2, IL-6, TNF, IL-10, and Ifit1 were analyzed by qRT-PCR (RNA extracted from nasal turbinates) (Figures 5E, 5F, 5G, 5H, 5-I, 5J, 5K, 5L, 5M, and 5N, showing fold changes in gene expression, respectively), IFNβ by ELISA, and CXCL10 by ELISA. The results illustrate a method of treating or preventing disease in a subject. Analysis was performed using the Mann-Whitney test. Bars and columns indicate median values. Each dot represents an individual mouse. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 5-3]

[0011] Figure 5A illustrates the experimental design of an experiment in which three to four Balb / c mice were treated with a single dose of NanoSTING (10 μg, 20 μg, or 40 μg). Blood, nasal turbinates, and lungs from euthanized animals were analyzed at 6, 12, 24, 36, and 48 hours, and cGAMP from nasal turbinates was analyzed by ELISA (Figure 5B), cGAMP from lungs was analyzed by ELISA (Figure 5C), cGAMP from serum was analyzed by ELISA (Figure 5D), CXCL10, IFNB1, ISG15, IRF7, Mx1, Mx2, IL-6, TNF, IL-10, and Ifit1 were analyzed by qRT-PCR (RNA extracted from nasal turbinates) (Figures 5E, 5F, 5G, 5H, 5-I, 5J, 5K, 5L, 5M, and 5N, showing fold changes in gene expression, respectively), IFNβ by ELISA, and CXCL10 by ELISA. The results illustrate a method of treating or preventing disease in a subject. Analysis was performed using the Mann-Whitney test. Bars and columns indicate median values. Each dot represents an individual mouse. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 5-4]

[0011] Figure 5A illustrates the experimental design of an experiment in which three to four Balb / c mice were treated with a single dose of NanoSTING (10 μg, 20 μg, or 40 μg). Blood, nasal turbinates, and lungs from euthanized animals were analyzed at 6, 12, 24, 36, and 48 hours, and cGAMP from nasal turbinates was analyzed by ELISA (Figure 5B), cGAMP from lungs was analyzed by ELISA (Figure 5C), cGAMP from serum was analyzed by ELISA (Figure 5D), CXCL10, IFNB1, ISG15, IRF7, Mx1, Mx2, IL-6, TNF, IL-10, and Ifit1 were analyzed by qRT-PCR (RNA extracted from nasal turbinates) (Figures 5E, 5F, 5G, 5H, 5-I, 5J, 5K, 5L, 5M, and 5N, showing fold changes in gene expression, respectively), IFNβ by ELISA, and CXCL10 by ELISA. The results illustrate a method of treating or preventing disease in a subject. Analysis was performed using the Mann-Whitney test. Bars and columns indicate median values. Each dot represents an individual mouse. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 5-5]

[0011] Figure 5A illustrates the experimental design of an experiment in which three to four Balb / c mice were treated with a single dose of NanoSTING (10 μg, 20 μg, or 40 μg). Blood, nasal turbinates, and lungs from euthanized animals were analyzed at 6, 12, 24, 36, and 48 hours, and cGAMP from nasal turbinates was analyzed by ELISA (Figure 5B), cGAMP from lungs was analyzed by ELISA (Figure 5C), cGAMP from serum was analyzed by ELISA (Figure 5D), CXCL10, IFNB1, ISG15, IRF7, Mx1, Mx2, IL-6, TNF, IL-10, and Ifit1 were analyzed by qRT-PCR (RNA extracted from nasal turbinates) (Figures 5E, 5F, 5G, 5H, 5-I, 5J, 5K, 5L, 5M, and 5N, showing fold changes in gene expression, respectively), IFNβ by ELISA, and CXCL10 by ELISA. The results illustrate a method of treating or preventing disease in a subject. Analysis was performed using the Mann-Whitney test. Bars and columns indicate median values. Each dot represents an individual mouse. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 5-6]

[0011] Figure 5A illustrates the experimental design of an experiment in which three to four Balb / c mice were treated with a single dose of NanoSTING (10 μg, 20 μg, or 40 μg). Blood, nasal turbinates, and lungs from euthanized animals were analyzed at 6, 12, 24, 36, and 48 hours, and cGAMP from nasal turbinates was analyzed by ELISA (Figure 5B), cGAMP from lungs was analyzed by ELISA (Figure 5C), cGAMP from serum was analyzed by ELISA (Figure 5D), CXCL10, IFNB1, ISG15, IRF7, Mx1, Mx2, IL-6, TNF, IL-10, and Ifit1 were analyzed by qRT-PCR (RNA extracted from nasal turbinates) (Figures 5E, 5F, 5G, 5H, 5-I, 5J, 5K, 5L, 5M, and 5N, showing fold changes in gene expression, respectively), IFNβ by ELISA, and CXCL10 by ELISA. The results illustrate a method of treating or preventing disease in a subject. Analysis was performed using the Mann-Whitney test. Bars and columns indicate median values. Each dot represents an individual mouse. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 6]

[0012] FIG. 6 shows the DNA sequences of the primers used to obtain the qRT-PCR experimental data shown in FIGS. 5E-5N. [Figure 7-1]

[0013] Figures 7A, 7B and 7C illustrate data from the experiment described in Figure 5A for IFNb from nasal turbinates by quantitative ELISA (Figure 7A), CXCL10 from nasal turbinates by quantitative ELISA (Figure 7B), and lungs (Figure 7C), according to an embodiment. [Figure 7-2]

[0013] Figures 7A, 7B and 7C illustrate data from the experiment described in Figure 5A for IFNb from nasal turbinates by quantitative ELISA (Figure 7A), CXCL10 from nasal turbinates by quantitative ELISA (Figure 7B), and lungs (Figure 7C), according to an embodiment. [Figure 7-3]FIG. 7D shows quantification of cGAMP in mouse serum after treatment with NanoSTING according to an embodiment. FIG. 7E shows detection of IFNb concentration in mouse serum using a quantitative ELISA according to an embodiment. FIG. 7F shows detection of CXCL10 levels in mouse serum using a quantitative ELISA according to an embodiment. For fold changes in gene expression, analysis was performed using the Mann-Whitney test. Bars and columns indicate median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 8-1]

[0014] 8A and 8B show temperature and weight changes, respectively, in a NanoSTING versus control (PBS) study in hamster subjects, in which groups of animals were administered a daily dose of 60 μg NanoSTING (n=4 / group) or PBS (n=4 / group) intranasally for 4 consecutive days. Hamsters were euthanized on day 5 after the final dose was administered on day 4, and lungs were subsequently harvested. [Figure 8-2] Figure 8C shows representative images of dissected hamster nasal turbinates, lungs, and stomachs analyzed with the Evans Blue dye assay. Hamsters (n=4 / group) were administered 0.125% Evans Blue dye intranasally in PBS (40 μL and 120 μL). Figures 8D and 8E show the quantified distribution of Evans Blue dye after intranasal administration. In these experiments, supernatants from homogenized lungs and stomachs were treated with trichloroacetic acid and analyzed for absorbance at 620 nm. The concentration of the dye was interpolated from a standard curve. [Figure 9-1]

[0015] FIG. 9A shows real-time qRT-PCR of folding induction of Cxcl11, Ccl5, Ifnb1, Isg15, Irf7, Mx1, Mx2, Il6, and Il10 mRNA from hamster lungs treated with 60 μg NanoSTING compared to control hamster lungs (n=4). [Figure 9-2] FIG. 9B shows the DNA sequences of the primers used to collect the data in FIG. 9A. [Figure 10-1]

[0016] Figures 10A, 10B, and 10C show activation of IFN-dependent and IFN-independent pathways in the lungs of NanoSTING-treated hamsters using RNA-sequencing. Figure 10A shows a heatmap of the top 50 differentially expressed genes (DEGs) between NanoSTING-treated lungs (marked as green) and control lungs (marked as black). [Figure 10-2] FIG. 10B shows a volcano plot of DEGs comparing NanoSTING-treated with PBS-treated control animals. [Figure 10-3] Figure 10C shows gene set enrichment analyses (GSEA) of C2 and C7 curated pathways visualized using Cytoscape. Nodes (red and blue circles) represent pathways and edges (green lines) represent overlapping genes between pathways. The size of the node represents the number of enriched genes in the pathway and the thickness of the edge represents the number of overlapping genes. Node colors were adjusted for FDR q-values ​​ranging from 0 to 0.25. Clusters of pathways were labeled as groups with similar themes. [Figure 11-1]

[0017] Figure 11A shows GSEA of IFN-independent activity of the activated STING pathway in the lungs of animals treated with NanoSTING. The schematic represents the comparison made between samples collected from the GSE149744 dataset to generate pathway gene sets. [Figure 11-2] FIG. 11B shows the expression of genes in the lungs related to IFN-dependent and IFN-independent antiviral pathways between the NanoSTING and control groups. [Figure 11-3] Figures 11C and 11D show schematics depicting the rate constants and equations governing viral dynamics during natural infection (Figure 11C) and in the presence of NanoSTING treatment (Figure 11D). [Figure 11-4]Figure 11E shows the reduction in viral area under the curve (AUC) at different NanoSTING potencies (RIR) compared to natural infection. Assuming that the action of NanoSTING treatment lasts only 24 hours, treatment begins on day 0. Figure 11F shows a heat map of viral AUC with various NanoSTING potencies and treatment start times. Red boxes represent combinations where viral AUC was reduced by nearly 100%. [Figure 11-5] Figure 11G shows that the peak spontaneous response is independent of the initial viral load. Figure 11H shows that viral kinetics may be independent of the initial viral titer when treated with NanoSTING. E0 is the initial number of infected cells upon viral infection, which is a proxy for viral titer. [Figure 11-6] Figures 11-I, 11J, and 11K show the evolution of viral dynamics at different treatment initiation times and NanoSTING efficacies. [Figure 11-7] Figure 11L shows a heat map of viral AUC with various NanoSTING potencies and treatment initiation times, where the effect of NanoSTING lasted 48 hours after treatment initiation. [Figure 12-1]

[0018] Figures 12A, 12B, 12C, 12D, 12E, and 12F show that NanoSTING protects hamsters during primary challenge and prevents reinfection with pathogenic SARS-CoV-2 delta (B.1.617.2) after treatment with NanoSTING. For viral titers, analysis was performed using the Mann-Whitney test. Figure 12A shows the experimental design in which a group of 12 hamsters was treated with a single dose of 120 μg NanoSTING each and challenged with 104 TCID50 of the SARS-CoV-2 delta mutant strain via the intranasal route on day 0. Viral titers in lung and nasal tissues were determined in half of the hamsters (n=6) on day 6. The remaining 6 hamsters were rechallenged on day 28 and weight changes were followed until day 35. [Figure 12-2]Figures 12B and 12C show the percentage of body weight change compared to baseline at the indicated time intervals. Figure 12F shows the percent of body weight change monitored after rechallenge starting on day 28 through day 35. Figures 12D and 12E show virus titers measured by plaque assay in nasal tissue and lungs on day 6 post-infection. Body weight data were compared via a mixed effects model for repeated measures analysis. Each dot is an individual hamster. Bars and columns show median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 13-1]

[0019] 13A, 13B, and 13C show that prophylactic administration of a single dose of a NanoSTING composition according to an embodiment protects against challenge with SARS-CoV-2 delta variant (B.1.617.2). Hamsters were treated with NanoSTING (120 μg) 24 hours (e.g., "day -1") or 72 hours (e.g., "day -3") prior to challenge with SARS-CoV-2 delta variant. This treatment reduced viral replication and prevented weight loss. [Figure 13-2] Figures 13A, 13B, and 13C show viral titers quantified in lung and nasal tissues by plaque assay on day 2 post-challenge. For viral titers, analysis was performed using the Mann-Whitney test. Body weight data were compared via a mixed effects model for repeated measures analysis. Each dot is an individual hamster and the bar represents the median. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 14]

[0020] Figures 14A, 14B, and 14C show that post-exposure administration of a single dose of a NanoSTING composition according to an embodiment protects against challenge with SARS-CoV-2 delta variant (B.1.617.2). Groups of six hamsters were each challenged with SARS-CoV2 virus (delta variant-B.1.617) and treated 6 hours later with a single dose of NanoSTING (120 μg) (Figure 14A). Animals were euthanized 2 days after infection and analyzed to determine viral load in lung and nasal tissues. Figures 14B and 14C show viral titers from lung and nasal tissues quantified by plaque assay 22 days after challenge. For viral titers, analysis was performed using the Mann-Whitney test. Body weight data was compared via a mixed effects model for repeated measures analysis. Each dot is an individual hamster and the bar indicates the median value. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 15]

[0021] Figures 15A, 15B, 15C, and 15D illustrate that prophylactic administration of a single dose of a NanoSTING composition according to embodiments disclosed herein can protect against SARS-CoV-2 delta variant. Hamsters were treated with NanoSTING (120 μg) 24 hours prior to challenge with SARS-CoV-2 delta variant. This treatment reduced viral replication and prevented weight loss in treated animals. [Figure 16]

[0022] Figures 16A, 16B, 17A, 17B, 17C, and 17D illustrate the protective efficacy of NanoSTING against the interferon (IFN)-evading SARS-CoV-2 alpha mutant (B.1.1.7). Groups of 12 hamsters were each treated with two different doses of NanoSTING (30 μg and 120 μg) and challenged with SARS-CoV-2 alpha mutant (B.1.1.7) 24 hours later (Figure 16A). Body weight changes were monitored daily (Figure 16B). [Figure 17-1]Animals were euthanized on day 5 for histopathology (Figures 17A and 17B), and viral titers in lung and nasal tissues were measured on day 2 (n=6) (Figures 17C and 17D). Lung pathology scores (Figure 17B) and representative hematoxylin and eosin (H&E) images (Figure 17A) showed histopathological changes in the lungs of Syrian hamsters treated with NanoSTING; all images were acquired at 20x; scale bar, 100 μm. [Figure 17-2] Viral titers were quantified in lung and nasal tissues by plaque assay on day 2 post-challenge (FIGS. 17C and 17D). For viral titers, analysis was performed using the Mann-Whitney test. Percent body weights were compared via a mixed effects model for repeated measures analysis. Each dot is an individual hamster. Bars and columns indicate median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 18]

[0023] Figures 18A and 18B show prophylactic administration of a composition disclosed herein (e.g., a NanoSTING composition) prior to challenge with influenza A virus. Figure 18A shows the experimental design for a comparison of pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) treatment followed by challenge with a 4-fold lethal dose 50 (LD50) susceptible strain (Influenza A / California / 04 / 2009 (H1N1) virus) and 14 days of monitoring (n=10 / group) compared to placebo-treated mice. Figure 18B shows weight change in subjects from the experiment shown in Figure 18A. For viral titers, analysis was performed using the Mann-Whitney test. Weight data was compared via a mixed effects model for repeated measures analysis. We compared survival percentages using the log-rank test (Mantel-Cox). Each dot is an individual mouse. Bars and columns indicate median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 19-1]

[0024] Figures 19A, 19B, and 19C show prophylactic administration of a composition disclosed herein (e.g., a NanoSTING composition) prior to challenge with influenza A virus. Figure 19A shows the experimental design for a comparison of pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) followed by challenge with a 4-fold lethal dose 50 (LD50) of resistant strain influenza A / Hong Kong / 2369 / 2009 (H1N1) and 14-day monitoring (n=10 / group) compared to unchallenged mice (n=10 / group). Figure 19B shows the weight change in subjects from the experiment shown in Figure 19A. [Figure 19-2] FIG. 19C shows a Meyer-Kaplan graph of subject survival during the experiment shown in FIG. 19A. For viral titers, analysis was performed using the Mann-Whitney test. Body weight data were compared via a mixed effects model for repeated measures analysis. Survival percentages were compared using the log-rank test (Mantel-Cox). Each dot is an individual mouse. Bars and columns indicate median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 20-1]

[0025] Figures 20A, 20B, and 20C show prophylactic administration of a composition disclosed herein (e.g., a NanoSTING composition) prior to challenge with influenza A virus. Figure 20A shows the experimental design for a comparison of pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) followed by challenge with a 4-fold lethal dose 50 (LD50) of resistant strain influenza A / Hong Kong / 2369 / 2009 (H1N1) and 7 days of monitoring (n=10 / group) compared to unchallenged mice (n=10 / group). Figure 20B shows the weight change in subjects from the experiment shown in Figure 20A. [Figure 20-2]Figure 20C shows infectious virus titers measured by plaque assay in the lungs 7 days after challenge in the experiment shown in Figure 20A. For virus titers, analysis was performed using the Mann-Whitney test. Body weight data were compared via a mixed effects model for repeated measures analysis. Survival percentages were compared using the log-rank test (Mantel-Cox). Each dot is an individual mouse. Bars and columns indicate median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 21]

[0026] Figures 21A and 21B show prophylactic administration of a composition disclosed herein (e.g., a NanoSTING composition) prior to challenge with influenza A virus. Figure 21A shows the experimental design for a comparison of pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) followed by challenge with a 4-fold lethal dose 50 (LD50) of resistant strain influenza A / California / 04 / 2009 (H1N1) and 14 days of monitoring (n=10 / group) compared to unchallenged mice (placebo) (n=10 / group). Figure 21B shows subject weight change from the experiment shown in Figure 21A. Weight data was compared via a mixed effects model for repeated measures analysis. Each dot is an individual hamster and the bar indicates the median value. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 22-1]

[0027] Figures 22A, 22B, 22C, and 22D show that intranasal administration of NanoSTING limits viral transmission and replication in the nasal passages of contact hamsters exposed to the SARS-CoV-2 Omicron (B.1.1.529) variant. Figure 22A shows the experimental design in which groups of eight hamsters were each given 104 TCID50 of SARS-CoV-2 Omicron variant (B.1.1.529) on day 0, and 24 hours later, the indicator hamsters were pair-housed with the contact hamsters (n=8) in clean cages for 4 days. In group II, hamsters were prophylactically pretreated with 120 μg NanoSTING 24 hours prior to infection, and in group III, the contact hamsters were treated with NanoSTING 12 hours after the co-housing period began. The contact and indicator hamsters were euthanized on day 4 of co-housing. Infectious viral particles in nasal tissues on days 2 and 5 after virus challenge following infection were measured by plaque assay (FIGS. 21B and 22C). [Figure 22-2]

[0027] Figures 22A, 22B, 22C, and 22D show that intranasal administration of NanoSTING limits viral transmission and replication in the nasal passages of contact hamsters exposed to the SARS-CoV-2 Omicron (B.1.1.529) variant. Figure 22A shows the experimental design in which groups of eight hamsters were each given 104 TCID50 of SARS-CoV-2 Omicron (B.1.1.529) variant on day 0, and 24 hours later, the indicator hamsters were paired together with the contact hamsters (n=8) in clean cages for 4 days. In group II, hamsters were prophylactically pretreated with 120 μg NanoSTING 24 hours prior to infection, and in group III, the contact hamsters were treated with NanoSTING 12 hours after the start of the cohabitation period. The contact and indicator hamsters were euthanized on day 4 of cohabitation. Infectious viral particles in nasal tissues on days 2 and 5 after virus challenge following infection were measured by plaque assay (FIGS. 21B and 22C). [Figure 22-3]Figure 22D shows the measurement of body weight over time of indicator hamsters intranasally infected with the SARS-CoV-2 Omicron variant. For viral titers, analysis was performed using the Mann-Whitney test. Bars indicate median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. Body weight data were compared via a mixed effects model for repeated measures analysis. Each dot is an individual hamster and bars indicate median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. [Figure 23]

[0028] Figures 23A and 23B show schematic diagrams of a NanoSTING-S composition according to embodiments disclosed herein and an immunogenicity data timeline according to embodiments disclosed herein, respectively. [Figure 24-1]

[0029] Figures 24A, 24B, 24C, 24D, 24E, 24F, 24G, and 24H show the results of a NanoSTING-S composition used in mice according to an embodiment. Lipid NanoSTING-S was used with 10 μg of S protein and 20 μg of NanoSTING, which provides a cross-reactive response to multiple strains of SARS-CoV-2, including the Omicron variant. [Figure 24-2] 24A, 24B, 24C, 24D, 24E, 24F, 24G, and 24H show the results of a NanoSTING-S composition used in mice according to an embodiment. Lipid NanoSTING-S was used with 10 μg of S protein and 20 μg of NanoSTING, which provides a cross-reactive response against multiple strains of SARS-CoV-2, including the Omicron variant. [Figure 25-1]

[0030] Figures 25A, 25B, 25C, and 25D show the impact of the NanoSTING-S vaccine in a hamster challenge model. NanoSTING-S: 20 μg S protein and 40 μg NanoSTING. Weight loss and infectious viral titers in lung and nasal tissues are shown. As shown, the weight loss and infectious viral titer data suggest that the use of a composition comprising NanoSTING-S to achieve immunity in humans will produce favorable results. In some cases, the dose in humans is expected to be 50-200 μg S protein and 200-500 μg NanoSTING. [Figure 25-2]

[0030] Figures 25A, 25B, 25C, and 25D show the effect of NanoSTING-S vaccine in a hamster challenge model. NanoSTING-S: 20 μg S protein and 40 μg NanoSTING. Weight loss and infectious viral titers in lung and nasal tissues are shown. As shown, the weight loss and infectious viral titer data suggest that the use of a composition comprising NanoSTING-S to achieve immunity in humans will produce favorable results. In some cases, the dose in humans is expected to be 50-200 μg S protein and 200-500 μg NanoSTING. [Figure 26-1]

[0031] Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 26-I, 26J, 26K, 26L, 26M, 26N and 26-O show the impact of NanoSTING-N vaccine in mice. This supports the use of NanoSTING-N to achieve immunity in humans that relies on both antibody and T cell responses. The expected dose in humans is expected to be 50-200 μg of N protein and 200-500 μg of NanoSTING. [Figure 26-2]

[0031] Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 26-I, 26J, 26K, 26L, 26M, 26N and 26-O show the impact of NanoSTING-N vaccine in mice. This supports the use of NanoSTING-N to achieve immunity in humans that relies on both antibody and T cell responses. The expected dose in humans is expected to be 50-200 μg N protein and 200-500 μg NanoSTING. [Figure 26-3]

[0031] Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 26-I, 26J, 26K, 26L, 26M, 26N and 26-O show the impact of NanoSTING-N vaccine in mice. This supports the use of NanoSTING-N to achieve immunity in humans that relies on both antibody and T cell responses. The expected dose in humans is expected to be 50-200 μg N protein and 200-500 μg NanoSTING. [Figure 26-4]

[0031] Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 26-I, 26J, 26K, 26L, 26M, 26N and 26-O show the impact of NanoSTING-N vaccine in mice. This supports the use of NanoSTING-N to achieve immunity in humans that relies on both antibody and T cell responses. The expected dose in humans is expected to be 50-200 μg N protein and 200-500 μg NanoSTING. [Figure 26-5]

[0031] Figures 26A, 26B, 26C, 26D, 26E, 26F, 26G, 26H, 26-I, 26J, 26K, 26L, 26M, 26N and 26-O show the impact of NanoSTING-N vaccine in mice. This supports the use of NanoSTING-N to achieve immunity in humans that relies on both antibody and T cell responses. The expected dose in humans is expected to be 50-200 μg N protein and 200-500 μg NanoSTING. [Figure 26-6]

[0032] Figures 26P and 26Q show the IRF response of THP-1 dual cells to poly(dA:dT) / LV and the MTT assay in THP1-dual cells, respectively. According to an embodiment, the dose response of NanoSTING with triton X-100 as a cytotoxicity control. [Figure 27-1]

[0033] Figures 27A, 27B, 27C, 27D, 27E, 27F, and 27G show the NanoSTING-NS vaccine in mice (liposomal particles containing 20 μg N protein, 10 μg S protein, and 20 μg NanoSTING and cGAMP modulator). The ratio of N protein to S protein was optimized at a mass ratio of 2:1. An N:S ratio of at least 1:1 (mass:mass) or greater provides a balance of immune responses to both proteins. This results in a cross-reactive response to multiple strains of SARS-CoV-2. [Figure 27-2] 27A, 27B, 27C, 27D, 27E, 27F, and 27G show the NanoSTING-NS vaccine in mice (liposomal particles containing 20 μg N protein, 10 μg S protein, and 20 μg NanoSTING and cGAMP modulator). The ratio of N protein to S protein was optimized at a mass ratio of 2:1. An N:S ratio of at least 1:1 (mass:mass) or greater provides a balance of immune response to both proteins. This results in a cross-reactive response to multiple strains of SARS-CoV-2. [Figure 27-3]27A, 27B, 27C, 27D, 27E, 27F, and 27G show the NanoSTING-NS vaccine in mice (liposomal particles containing 20 μg N protein, 10 μg S protein, and 20 μg NanoSTING and cGAMP modulator). The ratio of N protein to S protein was optimized at a mass ratio of 2:1. An N:S ratio of at least 1:1 (mass:mass) or greater provides a balance of immune response to both proteins. This results in a cross-reactive response to multiple strains of SARS-CoV-2. [Figure 28-1]

[0034] Figures 28A, 28B, 28C, and 28D show the impact of NanoSTING-NS (NanoSTING-NS: liposomal particles containing 30 μg N protein, 20 μg S protein, and 40 μg NanoSTING and cGAMP modulator) vaccine in a hamster challenge model. Weight loss and infectious viral titers in lung and nasal tissues are shown. This supports the use of NanoSTING-NS to achieve immune neutralization in humans. The expected human dose is expected to be 50-200 μg N protein, 50-200 μg S protein, and 200-500 μg NanoSTING. [Figure 28-2]

[0034] Figures 28A, 28B, 28C, and 28D show the impact of NanoSTING-NS (NanoSTING-NS: liposomal particles containing 30 μg N protein, 20 μg S protein, and 40 μg NanoSTING and cGAMP modulator) vaccine in a hamster challenge model. Weight loss and infectious viral titers in lung and nasal tissues are shown. This supports the use of NanoSTING-NS to achieve immune neutralization in humans. The expected human dose is expected to be 50-200 μg N protein, 50-200 μg S protein, and 200-500 μg NanoSTING. [Figure 29-1]

[0035] Figures 29A and 29B show the effect of the NanoSTING-N vaccine (liposomal particles containing 30 μg of N protein and 40 μg of NanoSTING and cGAMP modulator) in a hamster challenge model. [Figure 29-2] Figures 29C and 29D show weight loss and infectious virus titers in lung and nasal tissues. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005]

[0036] The SARS-CoV-2 pandemic continues to spread with alarming speed, highlighting that existing therapeutic reserves against RNA viruses are woefully inadequate. Vaccines are the preferred defense against SARS-CoV-2, but they have three drawbacks. First, while the current generation of vaccines has been developed with remarkable speed (approximately 10 months), even this speed of development is slow, and vaccines need to be custom manufactured for each emerging virus. Second, the mutational plasticity of RNA viruses such as SARS-CoV-2 facilitates their evolution, and newer variants with immune evasive capabilities are emerging. This in turn requires booster shots for full protection from disease, even if the entire human population is not yet fully vaccinated. As exemplified by the human experience with influenza, requiring additional booster shots reduces human compliance, which in turn facilitates the spread of disease. Third, despite the efficacy of current vaccines in preventing disease, they do not prevent transmission. The evolution of SARS-CoV-2 Omicron (B.1.1.529) variants indicates that the virus can rapidly adapt to facilitate rapid transmission. Thus, while vaccines are necessary, they are insufficient to combat RNA viruses, and the availability of pre- or post-exposure prophylaxis that can both prevent disease and reduce transmission is an urgent and unmet clinical need. As disclosed herein, a single dose of nasal NanoSTING can act as a prophylaxis against multiple respiratory viruses (and treatment-resistant variants).

[0006]

[0037] Monoclonal antibodies, like vaccines, may have high efficacy in preventing disease, but suffer from all of the same disadvantages of vaccines listed above. The SARS-CoV-2 Omicron variant has near-total resistance to neutralization by antibodies. In addition, monoclonal antibodies are expensive therapeutics and are administered in clinical settings, further limiting their widespread use. When efficacy alone is compared, prophylactic administration of antibodies in hamsters (12 hours prior to challenge) provided protection from clinical disease (approximately 2-5% reduction in body weight and approximately 300-fold reduction in viral titers in the lungs) with no effect on transmission. The compositions disclosed herein (e.g., NanoSTING compositions) are simple to administer (e.g., intranasally) and offer a broader administration period (24-72 hours) with comparable efficacy in reducing clinical disease while also reducing transmission.

[0007]

[0038] Oral antivirals that directly inhibit one or more viral proteins have been developed and tested in humans against SARS-CoV-2 (e.g., molnupiravir and paquilovid), but they allow for viral evolution and resistance. Furthermore, oral antivirals are designed as post-exposure prophylaxis to prevent clinical disease and have no impact on viral transmission. In contrast to these pathogen-specific antivirals, the compositions disclosed herein (e.g., NanoSTING compositions) function against multiple respiratory viruses. In addition, the favorable efficacy profile comparing the compositions disclosed herein to these antivirals in small animal models, and the fact that these antivirals were efficacious in humans (30-89% in reducing clinical disease due to SARS-CoV-2), fully validate the clinical translational potential of the compositions disclosed herein (e.g., NanoSTING compositions).

[0008]

[0039] Immunomodulators including defective viral genome particles, cytokines, and small molecule agonists have been tested as antiviral agents. Defective interfering particles (DIPs) have an incomplete genome and inhibit wild-type virus replication when administered therapeutically. These particles have demonstrated efficacy in mitigating disease against both SARS-CoV-2 and influenza in small animal models, but DIPs would be generated against each virus individually. Poliovirus-based defective viral genomes (DVGs) induced broad IFN-I responses and were protective against multiple viruses. Limited replication is essential for DVG efficacy, but their broad applicability is limited due to both safety and the presence of pre-existing antibodies in vaccinated individuals concerns. Lipid nanoparticles complexed with defective genomes can mitigate these concerns and have shown efficacy against SARS-CoV-2 mutant strains in K18-hACE2 mice. However, the generalizability of this approach in the absence of viral replication against other viruses has yet to be demonstrated.

[0009]

[0040] Direct administration of aerosolized interferon to activate antiviral innate immunity has been tested in both animals and humans. In hamsters challenged with SARS-CoV-2, prophylactic or early administration of universal interferon reduced lung damage, provided modest protection against weight loss (10% versus 20% in untreated animals), and reduced infectious viral particles (100-fold). In humans, postexposure prophylaxis with nebulized IFN-α2b was associated with lower in-hospital mortality compared with no administration of IFN-α2b. In contrast, administration of IFN-α2b longer than 5 days after admission delayed recovery and increased mortality, suggesting that the timing of IFN-α2b is important to obtain benefit. The limited impact of IFN-α against COVID19 reflects its marginal efficacy as a prophylactic against influenza in humans.

[0010]

[0041] Other synthetic small molecule agonists of pattern recognition receptors (PRRs), such as stem-loop RNA14 (SLR14), a minimal RIG-I agonist; and diAbzl, a STING agonist, have been tested against SARS-CoV-2 in K18-hACE2 mice. As with all small molecule drugs, their safety, off-target activity, and pharmacokinetics must be thoroughly evaluated before interpretation. In some cases, the compositions disclosed herein (e.g., some NanoSTING compositions disclosed herein) are composed of naturally occurring lipids that have already been tested in humans and cGAMP, an immunotransmitter of danger signals that is conserved across mammals, including humans. These compositions can provide safe and sustained delivery and, as a result, can function as broad-spectrum antiviral agents.

[0011]

[0042] This disclosure shows that the compositions disclosed herein (e.g., NanoSTING compositions) are safe, stable, low-cost, and effective antiviral agents that activate the STING pathway to initiate a broad-spectrum antiviral response effective against multiple respiratory pathogens. The advantage of using cGAMP, a natural immunotransmitter, is that STING activation can result in both interferon-dependent and interferon-independent activity to control viral replication. The broad-spectrum compositions of the present disclosure (e.g., NanoSTING compositions) ensure that activity works against multiple mutant strains, including those that are resistant to other antiviral agents. The ability to activate adaptive immunity ensures that treatment with the compositions of the present disclosure (e.g., NanoSTING compositions) also protects against reinfection and provides durable immunity. The availability of broad-spectrum drugs that can be easily stored for long periods of time ensures preparation to deal with future respiratory viruses as they emerge.

[0012]

[0043] Disclosed herein are compositions, methods, kits, and systems for treating subjects in need thereof, such as subjects with pathogens that can cause respiratory disease.In some cases, the compositions, methods, kits, and / or systems disclosed herein can be useful in preventing subjects (e.g., non-infected subjects) from becoming infected with pathogens and / or from suffering from diseases that pathogens can cause, for example, by preventing the infection of subjects with pathogens or by preventing the progression (e.g., the spread or increase in severity) of conditions (e.g., respiratory disease) caused by pathogens in subjects.In some cases, the compositions, methods, kits, and / or systems disclosed herein can be useful in preventing reinfection of subjects with pathogens (e.g., where the pathogen can cause respiratory disease). In some cases, the compositions, methods, kits, and / or systems disclosed herein may be useful in preventing the transmission of a pathogen (e.g., a pathogen capable of causing a respiratory disease) from a first (e.g., infected) subject to a second (e.g., non-infected) subject. In some cases, the pathogen is a virus. In some cases, the pathogen is a coronavirus. In some cases, the pathogen is an influenza virus.

[0013]

[0044] In various embodiments, the composition comprises lipid-based particles and a modulator, and the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof.In various embodiments, the composition for use in reducing the risk of respiratory disease in a subject after the subject is exposed to a pathogen that can cause respiratory disease comprises lipid-based particles and a modulator, and the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof.In various embodiments, the composition for use in reducing the risk of respiratory disease in a subject before the subject is exposed to a pathogen that can cause respiratory disease comprises lipid-based particles and a modulator, and the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof.In various embodiments, the composition for use in treating respiratory disease in a subject comprises lipid-based particles and a modulator, and the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof. In various embodiments, the composition for use in reducing the rate of progression of respiratory disease in a subject with respiratory disease comprises lipid-based particles and a modulator, the modulator being a pattern recognition receptor agonist, an immune system activator, or a combination thereof.In various embodiments, the composition for use in reducing the risk of transmitting respiratory disease from a subject with respiratory disease to a subject without respiratory disease comprises lipid-based particles and a modulator, the modulator being a pattern recognition receptor agonist, an immune system activator, or a combination thereof.In some cases, the modulator is encapsulated in the lipid-based particles.In some cases, the subject has previously suffered from respiratory disease.In some cases, the lipid-based particles comprise an antigen.In some cases, the lipid-based particles comprise a first antigen and a second antigen.In some cases, the first antigen is a spike protein molecule or a portion thereof.In some cases, the second antigen is a nucleocapsid protein molecule or a portion thereof.In some cases, the lipid-based particle comprises a greater amount of nucleocapsid protein molecules than spike protein molecules. In some cases, the ratio of nucleocapsid protein molecules to spike protein molecules is at least 1:1. In some cases, the composition is formulated for nasal delivery. In some cases, the modulator is an agonist of the STING pathway. In some cases, the lipid-based particle comprises DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a ratio of 10:1:1:1. In some cases, the antigen is associated with the outer surface of the lipid-based particle. In some cases, the disease comprises an infection caused by a pathogen. In some cases, the pathogen is a respiratory pathogen. In some cases, the respiratory pathogen is a virus. In some cases, the virus is selected from influenza virus, parainfluenza virus, adenovirus, enterovirus, coronavirus, respiratory syncytial virus, rhinovirus, DNA virus, RNA virus, variants thereof, or combinations thereof. In some cases, the virus is an influenza virus. In some cases, the influenza virus includes an oseltamivir-susceptible strain, a treatment-resistant strain, or a combination thereof. In some cases, the virus is a coronavirus. In some cases, the coronavirus is a SARS-CoV-2 virus, an alpha variant thereof, a delta variant thereof, an omicron variant thereof, or a combination thereof. In some cases, the composition is lyophilized. In some cases, the composition is in liquid form. In some cases, the modulator is selected from the group consisting of bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), amidobenzimidazole, a derivative of amidobenzimidazole, a nucleotide modulator, a plasmid DNA modulator, a divalent cation, CF501, SHR1032, or a combination thereof. In some cases, the modulator includes cyclic guanosine monophosphate-adenosine monophosphate (cGAMP).In various forms, the kit includes a composition according to any one of the embodiments or forms disclosed herein and includes instructions for use. In various forms, the composition according to any of the embodiments or forms disclosed herein is used in the manufacture of a medicament for treating a respiratory disease.

[0014]

[0045] In various forms, the method includes administering a composition according to any of the embodiments or forms disclosed herein to a subject, wherein the subject does not exhibit symptoms of respiratory disease. In some cases, a sample obtained from the subject has a detectable level of a pathogen associated with respiratory disease. In some cases, a sample obtained from the subject does not have a detectable level of a pathogen associated with respiratory disease. In various forms, the method includes administering a compound according to any of the embodiments or forms disclosed herein to a subject, wherein the subject exhibits symptoms of respiratory disease. In some cases, a sample obtained from the subject has a detectable level of a pathogen associated with respiratory disease. In some cases, the composition is administered in at least one dose. In some cases, the composition is administered in one dose. In some cases, the composition is administered in two doses. In some cases, the composition is administered to the subject before the subject is exposed to a pathogen associated with respiratory disease. In some cases, the composition is administered to the subject at least one day before the subject is exposed to a pathogen associated with respiratory disease. In some cases, the composition is administered to the subject at least 3 days before the subject is exposed to a pathogen associated with respiratory disease. In some cases, the composition is administered via intranasal administration. In some cases, the composition is administered via inhalation administration. In some cases, the method is used to prevent the establishment of disease in a subject. In some cases, the method is used to prevent the progression of disease in a subject. In some cases, the method is used to prevent the transmission of disease to a second subject. In some cases, the method initiates an innate immune response that results in associated adaptive immunity.

[0015] Detailed Description

[0046] Therapeutic agents and vaccines for respiratory diseases have numerous limitations. For example, vaccines provide relief from SARS-CoV2 for a short period of time. However, the rapid evolution of resistant viral variants necessitates additional strategies, such as broad-spectrum antivirals with prophylactic and therapeutic properties.

[0016]

[0047] SARS-CoV2 and influenza are among the major infectious diseases causing death and morbidity worldwide. Drug resistance and viral mutations are major challenges in preventing and treating influenza. The development of off-the-shelf, effective, safe, low-cost drugs for prophylaxis against respiratory viral infections is a major unmet medical need. Disclosed herein are compositions and methods for preventing and / or treating a disease or condition, such as a respiratory disease, in a subject (e.g., an animal, such as a mammal). In some embodiments, preventing a disease or condition includes preventing the establishment of a disease or condition in a subject. In some embodiments, preventing a disease or condition includes preventing the establishment of a disease or condition in a subject (e.g., after the subject is exposed to a pathogen). In some embodiments, preventing a disease or condition includes preventing the onset (e.g., progression) of a disease or condition in a subject (e.g., from a first condition to a second condition, to a more severe or advanced condition) (e.g., after the establishment of a disease in a subject, e.g., after the establishment via infection by a pathogen). In some embodiments, preventing a disease or condition includes preventing the transmission of a disease or condition from a first subject to a second subject.

[0017]

[0048] A composition 10 useful for preventing or treating a disease or condition of a subject may include a particle 12 (e.g., a liposome as described herein), a modulator 16 (e.g., a STING agonist, e.g., cGAMP), and / or an antigen 14 (e.g., as shown in FIG. 1). In some cases, a composition described herein may include a modulator (e.g., a STING agonist, e.g., cGAMP) without including a particle or an antigen. In some cases, a composition described herein may include a modulator (e.g., a STING agonist, e.g., cGAMP) that is encapsulated within, in contact with, bound to, and / or not in contact with a particle as described herein but is delivered (e.g., intranasally with the particle). In some cases, encapsulation of one or more modulators within a particle described herein (or, in some cases, binding to or incorporating one or more modulators into a particle) can increase the uptake efficiency, tissue targeting, stability, and / or efficacy of a composition or method of use thereof, for example, by controlling (e.g., maintaining) the spatial concentration of one or more modulators and / or by presenting a recognition signal (e.g., an antigen) to the target cells. In some cases, a composition comprising one or more modulators and not a particle or antigen (or comprising one or more modulators outside of, and not associated with, the particles of the composition) can elicit a faster response from a target tissue or cell and / or reduce the cost and / or complexity of producing the composition.In some cases, the compositions described herein may include one or more modulators (e.g., one or more STING agonist molecules, e.g., one or more cGAMP molecules) encapsulated within a particle (e.g., a liposome) and one or more modulators (e.g., one or more STING agonist molecules, e.g., one or more cGAMP molecules) that are delivered to a subject but are not encapsulated by, bound to, or incorporated into a particle. In some cases, a composition comprising one or more modulators (e.g., one or more STING agonist molecules, e.g., one or more cGAMP molecules) encapsulated within a particle (e.g., liposome) and one or more modulators (e.g., one or more STING agonist molecules, e.g., one or more cGAMP molecules) that are delivered to a subject but are not encapsulated by, associated with, or incorporated into a particle may provide the benefits of both the "naked" modulator (e.g., faster response and / or different target cells or tissues than an encapsulated modulator) and the modulators encapsulated within, associated with, or incorporated into a particle (e.g., liposome) as described herein. In some cases, the compositions described herein may comprise a lipid-based liposomal particle, a modulator (e.g., a pattern recognition receptor agonist, e.g., a STING agonist), and an application-specific antigen (e.g., a coronavirus spike protein ("S-protein"), a nucleocapsid protein ("N-protein"), or a chimeric antigen protein) associated with the liposomal particle. In some embodiments, the association of the application-specific antigen with the liposomal particle (e.g., its outer surface) encapsulating the modulator payload can significantly increase targeting and / or delivery of the modulator payload to a target tissue of interest (e.g., the nasal and / or pulmonary compartments of a subject).In some embodiments, association of application-specific antigens with liposomal particles (e.g., direct association, e.g., via incorporation into the liposomal membrane, or via coupling to the membrane surface of the liposome) can increase the efficacy of the composition in preventing and / or treating a disease or condition in a subject, e.g., by spatially concentrating the antigens and modulators of the composition (e.g., in a target tissue). Disclosed herein are compositions (e.g., NanoSTING compositions, which may be immunotransmitters encapsulated in liposomes) that can rapidly activate the body's innate immune system to promote a broad-spectrum antiviral response against influenza and SARS-CoV-2 mutant strains in mice and hamsters. In some cases, a single nasal dose of NanoSTING can (1) treat infection and minimize symptoms throughout the respiratory tree, (2) reduce transmission by reducing infectious virus in the nasal passages, (3) protect against oseltamivir-resistant influenza and highly infectious strains of SARS-CoV-2 (alpha and delta), and / or (4) provide durable protection against reinfection by stimulating adaptive immunity. In some cases, administration of NanoSTING can upregulate interferon-stimulated and / or antiviral pathways, such as those pathways in the nasal turbinates and / or lungs. In some embodiments, the NanoSTING composition can be a stable, low-cost, broad-spectrum antiviral therapeutic for prophylaxis or early post-exposure treatment.

[0018]

[0049] In some cases, the compositions and methods disclosed herein (e.g., including NanoSTING, which may include nanoparticle formulations of cGAMP) allow for sustained release of cGAMP to both the nasal compartment and the lungs for 48 hours or longer after administration to a subject. The compositions disclosed herein (e.g., including NanoSTING formulations) may be comprised of lipids and a STING activator such as cGAMP, but may also contain one or more divalent metal ions, such as Mg. 2+ or Mn 2+ cGAMP (e.g., delivered to a subject according to the compositions and or methods disclosed herein) can activate multiple antiviral pathways and facilitate responses mediated by type I interferon (IFN-I). Quantitative modeling using SARS-CoV-2 infection in humans can be used to confirm that pre-exposure or early post-exposure prophylaxis with extremely low doses of the compositions disclosed herein (e.g., NanoSTING compositions) can provide clinically observable benefit. The compositions disclosed herein (e.g., NanoSTING) can be used to prevent clinical disease (e.g., SARS-CoV-2 and / or multiple mutant strains of influenza A, such as oseltamivir-resistant influenza A), reduce viral load (e.g., by several orders of magnitude), reduce pathogen and / or disease transmission, and / or engage adaptive immunity to confer durable protection from reinfection. Due to their stability and ease of administration, as well as the comprehensive nature of the immune response elicited, the compositions disclosed herein (e.g., NanoSTING) provide ideal non-invasive, broad-spectrum antiviral therapeutic compositions against respiratory viruses.

[0019] composition

[0050] A composition for preventing or treating a disease or condition in a subject (e.g., a therapeutic composition) may include a delivery vehicle (e.g., a particle). The delivery vehicle of a composition useful in preventing or treating a disease or condition in a subject may be a particle, e.g., a lipid-based particle. For example, a composition useful in preventing or treating a disease or condition in a subject may include a lipid-based particle, e.g., a liposome (e.g., having a membrane with an outer surface and an internal space). In some embodiments, a composition includes one or more modulators (e.g., one or more types of modulators). In some embodiments, a modulator may include a small molecule, a protein, a fragment thereof, and / or a polynucleotide or a fragment thereof. In some embodiments, a modulator (e.g., a STING agonist) or a combination of modulators may be selected for use in the compositions described herein for its ability to induce activation or inhibition of a signal transduction pathway and / or a systemic response pathway (e.g., a stimulator of interferon genes (STING) pathway) in a subject. The composition useful in preventing or treating a disease or condition in a subject (or the transmission of the disease to a second subject) may comprise one or more antigens. The composition useful in preventing or treating a disease or condition in a subject (or the transmission of the disease to a second subject) may comprise multiple antigens. In some embodiments, the antigen of the composition described herein can elicit an immune response in the subject, which may be useful in preventing or preventing a disease (e.g., respiratory disease) or condition (e.g., cancer) in the subject. In some embodiments, the composition for preventing or treating a disease or condition in a subject may comprise a modulator (e.g., a STING agonist, e.g., cGAMP) encapsulated in a particle (e.g., liposome), and the antigen is associated with the liposome or a part thereof (e.g., via incorporation into the membrane of the liposome).

[0020]

[0051] In some embodiments, the compositions described herein can be used in a method for treating or preventing a disease or condition in a subject (e.g., a mammal, e.g., a human). In some embodiments, the compositions described herein can be administered to a subject prior to exposure to a pathogen (e.g., a pathogen that causes respiratory disease or cancer), thereby, for example, preventing the subject from acquiring a disease or condition associated with exposure to (e.g., infection by) the pathogen (e.g., as illustrated in FIG. 2A). In some embodiments, the compositions disclosed herein can be administered in the form of a vaccine. In some embodiments, the compositions described herein can be administered to a subject after exposure to a pathogen (e.g., a pathogen that causes respiratory disease or cancer), thereby, for example, treating (e.g., alleviating, or in some embodiments curing) a disease or condition associated with exposure to (e.g., infection by) the pathogen after the subject acquires a disease or condition associated with the pathogen through exposure to the pathogen (e.g., as illustrated in FIG. 2B).

[0021] particle

[0052] The composition of the present disclosure may comprise a delivery vehicle, such as a particle. The particle may comprise a means for carrying a payload (e.g., a modulator payload) and / or one or more antigens (e.g., an antigen associated with a part of the particle, such as a membrane or an outer surface). In some embodiments, the particle may comprise a membrane or a wall. In some embodiments, the membrane or wall of the particle may define an interior space. In some embodiments, the interior space of the particle may comprise one or more modulators. In some embodiments, one or more antigens may be associated with the membrane or wall of the particle (e.g., the outer surface of the membrane or wall of the particle). The particle may comprise a lipid-based particle, a carbon-based particle, a metal-based particle, or a combination thereof.

[0022]

[0053] The particles of the compositions described herein may be lipid-based particles. In some embodiments, the lipid-based particles may be liposomes. The compositions described herein may include pulmonary surfactant-biomimetic particles. The particles of the compositions disclosed herein (e.g., lipid-based particles) may include a plurality of molecules, e.g., a plurality of molecules assembled in a membrane. In some embodiments, the particles (e.g., lipid-based particles) or a portion thereof may be anionic (e.g., may include an anionic membrane or one or more anionic lipids). For example, the particles (e.g., lipid-based particles) may include anionic lipids (e.g., DPPE, DPPE-PEG2000, DPPC, DPPG) or neutral lipids (e.g., cholesterol). In some embodiments, the particles of the compositions described herein (e.g., lipid-based nanoparticles comprising NanoSTING compositions) may not include cationic lipids. In some embodiments, the particles (e.g., lipid-based particles) or a portion thereof may be cationic. In some cases, the particle (e.g., lipid-based particle) may include cationic (e.g., positively charged) lipid. For example, the particle (e.g., lipid-based particle) may include lipid selected from 1,2-dipalmitoyl-3-trimethylammonium-propane chloride (DPTAP) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In some embodiments, the particle (e.g., lipid-based particle) may be zwitterionic. In some embodiments, the particle (e.g., lipid-based particle) or a portion thereof may have a net zero charge. In some embodiments, the particle (e.g., lipid-based particle) or a portion thereof may be uncharged. In some embodiments, the particle (e.g., liposome) of the composition described herein may include dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, 1,2-bis(diphenylphosphino)ethane (DPPE), cholesterol, or a combination thereof.In some embodiments, the particles (e.g., liposomes) of the compositions described herein may comprise poly(ethylene glycol)-lipids (e.g., PEG-lipids).In some embodiments, the particles (e.g., liposomes) of the compositions described herein may comprise DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPE-PEG2000 (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), cholesterol, 1,2-dipalmitoyl-3-trimethylammonium-propane chloride (DPTAP), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), or combinations thereof. In some embodiments, the particles may include a combination of DPPC and DPPG, for example, in a molar ratio of about 10:1. In some embodiments, the particles may include a combination of DPPC and cholesterol, for example, in a molar ratio of about 10:1. In some embodiments, the particles may include a combination of DPPC and DPPE-2000, for example, in a molar ratio of about 10:1. In some embodiments, the particles may include a combination of DPPG and cholesterol, for example, in a molar ratio of about 1:1. In some embodiments, the particles may include a combination of cholesterol and DPPE-PEG2000, for example, in a molar ratio of about 1:1. In some embodiments, the particles may include a combination of DPPG and DPPE-PEG2000, for example, in a molar ratio of about 1:1. In some embodiments, the particles may include DPPC, DPPG, cholesterol, and DPPE-PEG2000. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000, respectively, in a molar ratio of 10:1:1:1. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 20:1:1:1, respectively.In some embodiments, the particles may be comprised of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 5:1:1:1, respectively. In some embodiments, the particles may be comprised of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:2:1:1, respectively. In some embodiments, the particles may be comprised of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:1:2:1, respectively. In some embodiments, the particles may be comprised of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:1:1:2, respectively. In some embodiments, the particles may be comprised of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:2:2:1, respectively. In some embodiments, the particles may be comprised of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:1:2:2, respectively. In some embodiments, the particle may be comprised of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:2:1:2, respectively. In some embodiments, the molar ratio of a first molecule that comprises the particle (e.g., a lipid-based particle) to a second molecule that comprises the particle may be 1:100 to 1:1, 1:50 to 1:2, 1:25 to 1:3, 1:10 to 1:5, 1:50 to 1:1, 1:25 to 1:1, 1:10 to 1:1, 1:5 to 1:1, 1:3 to 1:1, 1:25 to 1:10, 1:50 to 1:25, 1:100 to 1:50, or may be greater than 1:100. In some embodiments, the molar ratio of the first molecule that constitutes the particle (e.g., lipid-based particle) of the present disclosure to the second molecule that constitutes the particle can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:50, 1:100, or any range therebetween. In some embodiments, the molar ratio of the first molecule that constitutes the particle (e.g., lipid-based particle) can be 1:5.In some embodiments, the molar ratio of the first molecule that constitutes the particle (e.g., lipid-based particle) may be 1:10. In some embodiments, the molar ratio of the first molecule that constitutes the particle (e.g., lipid-based particle) may be 1:20. In some embodiments, the molar ratio of the first molecule that constitutes the particle (e.g., lipid-based particle) may be 1:50. In some embodiments, the molar ratio of the first molecule that constitutes the particle (e.g., lipid-based particle) may be 1:100. In some embodiments, the first molecule that constitutes the particle (e.g., lipid-based particle) of the composition described herein may be dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol, 1,2-bis(diphenylphosphino)ethane (DPPE), cholesterol, or a combination thereof. In some embodiments, the second molecule constituting a particle (e.g., a lipid-based particle) of a composition described herein may be DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPE-PEG2000 (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), or cholesterol.

[0023]

[0054] The particles of the compositions described herein may be nanoparticles.Maintaining a small particle size (e.g., average hydrodynamic particle size of less than 300 nanometers (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) and / or low polydispersity (e.g., polydispersity index of less than 0.25, less than 0.24, less than 0.23, less than 0.22, less than 0.21, or less than 0.20) can improve the stability and / or delivery efficiency of the particles.In some embodiments, the particles may have an outer diameter of 1 nm to 500 nm, 1 nm to 750 nm, or 1 nm to 1000 nm.In some embodiments, the particles are from 1 nm to 10 nm, 1 nm to 15 nm, 1 nm to 20 nm, 1 nm to 30 nm, 1 nm to 50 nm, 1 nm to 75 nm, 1 nm to 100 nm, 1 nm to 150 nm, 1 nm to 200 nm, 1 nm to 250 nm, 1 nm to 300 nm, 1 nm to 400 nm, 1 nm to 500 nm, 10 nm to 15 nm, 10 nm to 20 nm, 10 nm to 30 nm, 10 nm to 50 nm, 10 nm to 75 nm m, 10nm~100nm, 10nm~150nm, 10nm~200nm, 10nm~250nm, 10nm~300nm, 15nm~20nm, 15nm~30nm, 15nm~50nm, 15nm~75 nm, 15nm~100nm, 15nm~150nm, 15nm~200nm, 15nm~250nm, 15nm~300nm, 20nm~30nm, 20nm~50nm, 20nm~75nm, 20nm~10 0nm, 20nm~150nm, 20nm~200nm, 20nm~250nm, 20nm~300nm, 30nm~50nm, 30nm~75nm, 30nm~100nm, 30nm~150nm, 30nm ~200nm, 30nm~250nm, 30nm~300nm, 50nm~75nm, 50nm~100nm, 50nm~150nm, 50nm~200nm, 50nm~250nm, 50nm~300nm, 7 The particles may have an outer diameter of 5 nm to 100 nm, 75 nm to 150 nm, 75 nm to 200 nm, 75 nm to 250 nm, 75 nm to 300 nm, 100 nm to 150 nm, 100 nm to 200 nm, 100 nm to 250 nm, 100 nm to 300 nm, 150 nm to 200 nm, 150 nm to 250 nm, 150 nm to 300 nm, 200 nm to 250 nm, 200 nm to 300 nm, or 250 nm to 300 nm. In some embodiments, the particles may have an outer diameter of 1 nm, 10 nm, 15 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, or 500 nm. In some embodiments, the particles may have an outer diameter of at least 1 nm, 10 nm, 15 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, or 500 nm.In some embodiments, the particles may have an outer diameter of at most 1 nm, 10 nm, 15 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 400 nm, 500 nm, 750 nm, or 1000 nm.

[0024]

[0055] In some embodiments, the population of particles may have an average (e.g., external) diameter of 1 nm to 1000 nm at 25 degrees Celsius (° C.), for example, 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days, and the temperature may be room temperature. In some embodiments, the population of particles may have an average (e.g., external) diameter of 1 nm to 100 nm at 25 degrees Celsius (° C.), for example, 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days. In some embodiments, the population of particles may have an average (e.g., external) diameter of greater than 100 nm and up to 200 nm at 25 degrees Celsius (° C.), e.g., for 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days. In some embodiments, the population of particles may have an average (e.g., external) diameter of greater than 200 nm and up to 300 nm at 25 degrees Celsius (° C.), e.g., for 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days.In some embodiments, the population of particles may be at 25 degrees Celsius (°C), e.g., for up to 30 days, between 1 nm and 20 nm, between 1 nm and 30 nm, between 1 nm and 50 nm, between 1 nm and 75 nm, between 1 nm and 100 nm, between 1 nm and 125 nm, between 1 nm and 150 nm, between 1 nm and 175 nm, between 1 nm and 200 nm, between 1 nm and 300 nm, between 1 nm and 1000 nm, between 20 nm and 30 nm, between 20 nm and 50 nm, between 20 nm and 75 nm, between 20 nm and 100 nm, between 20 nm and 125 nm. , 20nm~150nm, 20nm~175nm, 20nm~200nm, 20nm~300nm, 20nm~1000nm, 30nm~50nm, 30nm~75nm, 30nm~100nm, 30nm~125nm, 3 0nm~150nm, 30nm~175nm, 30nm~200nm, 30nm~300nm, 30nm~1000nm, 50nm~75nm, 50nm~100nm, 50nm~125nm, 50nm~150nm, 50n m~175nm, 50nm~200nm, 50nm~300nm, 50nm~1000nm, 75nm~100nm, 75nm~125nm, 75nm~150nm, 75nm~175nm, 75nm~200nm, 75n m~300nm, 75nm~1000nm, 100nm~125nm, 100nm~150nm, 100nm~175nm, 100nm~200nm, 100nm~300nm, 100nm~1000nm, 125nm~15 In some embodiments, the nanoparticles may have an average (e.g., external) diameter of 0 nm, 125 nm to 175 nm, 125 nm to 200 nm, 125 nm to 300 nm, 125 nm to 1000 nm, 150 nm to 175 nm, 150 nm to 200 nm, 150 nm to 300 nm, 150 nm to 1000 nm, 175 nm to 200 nm, 175 nm to 300 nm, 175 nm to 1000 nm, 200 nm to 300 nm, 200 nm to 1000 nm, or 300 nm to 1000 nm. In some embodiments, a population of particles may have an average (e.g., external) diameter of 1 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 300 nm, or 1000 nm at 25 degrees Celsius (°C), for example, for up to 30 days.In some embodiments, the particle population may have an average (e.g., external) diameter of at least 1 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 300 nm, or 1000 nm at 25 degrees Celsius (°C), for example, for up to 30 days.In some embodiments, the particle population may have an average (e.g., external) diameter of at most 1 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 300 nm, or 1000 nm at 25 degrees Celsius (°C), for example, for up to 30 days.In some cases, the average diameter may be the average hydrodynamic particle size.

[0025]

[0056] In some embodiments, the population of particles may have an average (e.g., external) diameter of 1 nm to 1000 nm, for example, for 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days at 37 degrees Celsius (°C), which may be body temperature. In some embodiments, the population of particles may have an average (e.g., external) diameter of 1 nm to 100 nm, for example, for 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days at 37 degrees Celsius (°C). In some embodiments, the population of particles may have an average (e.g., external) diameter of greater than 100 nm and up to 200 nm at 37 degrees Celsius (° C.), for example, for 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4-7 days, more than 7 days, 7-30 days, or more than 30 days. In some embodiments, the population of particles may have an average (e.g., external) diameter of greater than 200 nm and up to 300 nm at 37 degrees Celsius (° C.), for example, for 1 day (e.g., up to 24 hours), 2 days (e.g., up to 48 hours), 3 days (e.g., up to 72 hours), up to 96 hours, 4-7 days, more than 7 days, 7-30 days, or more than 30 days.In some embodiments, the population of particles may be at 37 degrees Celsius (°C), e.g., for up to 30 days, between 1 nm and 20 nm, between 1 nm and 30 nm, between 1 nm and 50 nm, between 1 nm and 75 nm, between 1 nm and 100 nm, between 1 nm and 125 nm, between 1 nm and 150 nm, between 1 nm and 175 nm, between 1 nm and 200 nm, between 1 nm and 300 nm, between 1 nm and 1000 nm, between 20 nm and 30 nm, between 20 nm and 50 nm, between 20 nm and 75 nm, between 20 nm and 100 nm, between 20 nm and 125 nm. , 20nm~150nm, 20nm~175nm, 20nm~200nm, 20nm~300nm, 20nm~1000nm, 30nm~50nm, 30nm~75nm, 30nm~100nm, 30nm~125nm, 3 0nm~150nm, 30nm~175nm, 30nm~200nm, 30nm~300nm, 30nm~1000nm, 50nm~75nm, 50nm~100nm, 50nm~125nm, 50nm~150nm, 50n m~175nm, 50nm~200nm, 50nm~300nm, 50nm~1000nm, 75nm~100nm, 75nm~125nm, 75nm~150nm, 75nm~175nm, 75nm~200nm, 75n m~300nm, 75nm~1000nm, 100nm~125nm, 100nm~150nm, 100nm~175nm, 100nm~200nm, 100nm~300nm, 100nm~1000nm, 125nm~15 In some embodiments, the nanoparticles may have an average (e.g., external) diameter of 0 nm, 125 nm to 175 nm, 125 nm to 200 nm, 125 nm to 300 nm, 125 nm to 1000 nm, 150 nm to 175 nm, 150 nm to 200 nm, 150 nm to 300 nm, 150 nm to 1000 nm, 175 nm to 200 nm, 175 nm to 300 nm, 175 nm to 1000 nm, 200 nm to 300 nm, 200 nm to 1000 nm, or 300 nm to 1000 nm. In some embodiments, a population of particles may have an average (e.g., external) diameter of 1 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 300 nm, or 1000 nm at 37 degrees Celsius (°C), for example, for up to 30 days.In some embodiments, the particle population may have an average (e.g., external) diameter of at least 1 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 300 nm, or 1000 nm at 37 degrees Celsius (°C), for example, for up to 30 days.In some embodiments, the particle population may have an average (e.g., external) diameter of at most 1 nm, 20 nm, 30 nm, 50 nm, 75 nm, 100 nm, 125 nm, 150 nm, 175 nm, 200 nm, 300 nm, or 1000 nm at 37 degrees Celsius (°C), for example, for up to 30 days.In some cases, the average diameter may be the average hydrodynamic particle size.

[0026]

[0057] The modulator and / or antigen of the present disclosure may be associated with the particle of the present disclosure. For example, in some embodiments, the antigen and STING modulator of the present disclosure may be located in different regions of the particle of the present disclosure. In some embodiments, the modulator of the present disclosure (e.g., STING modulator) may be encapsulated in the particle (e.g., STING modulator 16 encapsulated in particle 12 as illustrated in FIG. 1). In some embodiments, all or part of the antigen of the present disclosure may be associated with (e.g., attached to, attached to, adsorbed on, electrostatically interacted with, covalently bound to, non-covalently bound to, integrated with, or formulated on) the surface of the particle (e.g., antigen 14 on the surface of particle 12 as illustrated in FIG. 1). In some embodiments, all or part of the antigen is encapsulated within the lipid-based particle. In some embodiments, the association (e.g., adsorption) of the antigen with the surface of the particle can increase the stability of the particle and / or increase the delivery efficiency after administration (e.g., to the target tissue).

[0027]

[0058] In some embodiments, one or more antigens of the present disclosure may be encapsulated in a particle (e.g., lipid-based) of the present disclosure. In some embodiments, one or more modulators (e.g., including STING modulators) of the present disclosure may be associated with the outer surface of the particle. In some embodiments, one or more antigens of the present disclosure may be encapsulated in a particle (e.g., lipid-based) while one or more modulators (e.g., including STING modulators) of the present disclosure are associated with the outer surface of the particle. In some embodiments, one or more antigens of the present disclosure may be associated with the outer surface of a particle (e.g., lipid-based) of the present disclosure. In some embodiments, one or more modulators (e.g., including STING modulators) of the present disclosure may be encapsulated in a particle. In some embodiments, one or more antigens of the present disclosure may be associated with the outer surface of a particle (e.g., lipid-based) of the present disclosure while one or more modulators (e.g., including STING modulators) of the present disclosure are encapsulated in a particle. In some embodiments, one or more antigens and one or more modulators (including, for example, STING modulators) of the present disclosure may both be encapsulated in a (e.g., lipid-based) particle. In some embodiments, one or more antigens and one or more modulators (including, for example, STING modulators) of the present disclosure may both be associated with the surface of a (e.g., lipid-based) particle of the present disclosure. In some embodiments, one or more antigens of the present disclosure may be integrated into the membrane of a (e.g., lipid-based) particle of the present disclosure. In some embodiments, one or more modulators of the present disclosure (including, for example, STING modulators) of the present disclosure may be integrated into the membrane of a (e.g., lipid-based) particle of the present disclosure.In some embodiments, one or more antigens of the present disclosure may be encapsulated in a particle (e.g., lipid-based) of the present disclosure, while one or more modulators (e.g., including STING modulators) are integrated into the membrane of the particle (e.g., lipid-based) of the present disclosure. In some embodiments, one or more antigens of the present disclosure may be associated with the outer surface of a (lipid-based) particle, while one or more modulators (e.g., including STING modulators) are integrated into the membrane of a particle (e.g., lipid-based) of the present disclosure. In some embodiments, one or more modulators (e.g., including STING modulators) may be encapsulated in a particle (e.g., lipid-based) of the present disclosure, while one or more antigens of the present disclosure are integrated into the membrane of the particle. In some embodiments, one or more modulators (e.g., including STING modulators) may be associated with the outer surface of a particle (e.g., lipid-based) of the present disclosure, while one or more antigens of the present disclosure are integrated into the membrane of the particle. In some embodiments, incorporation of both the antigen and modulator (e.g., STING modulator) of the present disclosure into a (e.g., lipid-based) particle can facilitate coordinated cytosolic delivery.

[0028]

[0059] In some embodiments, the compositions described herein may include divalent ions, such as divalent cations. In some embodiments, the compositions include divalent cations encapsulated in, adsorbed on, covalently coupled to, electrostatically interacting with, or formulated on the membrane (e.g., membrane surface) of the particles (e.g., lipid-based nanoparticles) described herein. In some embodiments, the compositions include divalent cations encapsulated in, adsorbed on, covalently coupled to, electrostatically interacting with, or formulated on the membrane (e.g., membrane surface) of the particles (e.g., lipid-based nanoparticles) described herein. 2+ , Mg 2+ , Ca 2+ , and Zn 2+For example, the composition may comprise a modulator (e.g., a STING agonist), a particle (e.g., a lipid-based nanoparticle), and a divalent cation, such as Mn 2+ , Mg 2+ , Ca 2+ , or Zn 2+ may also include

[0029] antigen

[0060] The composition useful in preventing or treating respiratory disease or pathology in a subject may comprise an antigen. The antigen (or a part thereof) of the present disclosure may be associated with the surface of a particle (e.g., lipid-based particle, e.g., liposome) (e.g., attached to, attached to, adsorbed on, electrostatically interacted with, covalently bound to, non-covalently bound to, integrated with, or formulated on). In some embodiments, the antigen may be suitable for generating immunity against disease in a subject. For example, in some embodiments, one or more antigens in the composition of the present disclosure may be capable of eliciting an immune response against disease in a subject.

[0030]

[0061] The antigen of the compositions described herein may comprise an attenuated or killed pathogen or a portion thereof that causes a disease (e.g., one or more of the pathogens described herein). In some embodiments, the antigen may comprise a peptide or protein associated with the pathogen, or a portion thereof. In some embodiments, the antigen may comprise a surface protein (e.g., a receptor protein) of the pathogen, or a portion thereof. In some embodiments, the antigen may be in the form of a polynucleotide, e.g., a polynucleotide that can be used to express a second antigen (e.g., a plasmid DNA molecule that expresses a second antigen).

[0031]

[0062] The antigen of the compositions described herein may comprise a spike protein or a portion thereof. In some embodiments, the antigen of the compositions described herein may comprise at least one component of the coronavirus spike protein (S-protein). In some embodiments, the antigen of the compositions described herein may comprise a monomeric form of the coronavirus spike protein (S-protein). In some embodiments, the antigen of the compositions described herein may comprise a multimeric form of the coronavirus spike protein (S-protein).

[0032]

[0063] In some embodiments, the antigen of the present disclosure may comprise 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, the antigen of the composition may comprise a chimeric protein (e.g., a chimeric spike protein).

[0033]

[0064] In some embodiments, the antigen may comprise a monomeric or multimeric form of the SARS-CoV2 spike protein (S) containing a D614G mutation, an A222V mutation, an S477N mutation, a D80Y mutation, an S98F mutation, or a combination thereof.

[0034]

[0065] In some embodiments, the antigen may be a mixture of SARS-CoV2 spike proteins harboring different mutations. In some embodiments, the antigen may comprise a monomeric or multimeric form of the nucleocapsid protein. For example, the antigen of the present disclosure may comprise a monomeric or multimeric form of the SARS-CoV2 nucleocapsid (N) protein. In some embodiments, the antigen may be a monomeric or multimeric form of the SARS-CoV2 nucleocapsid (N) protein containing the A220V mutation.

[0035]

[0066] In some embodiments, the antigen may comprise an influenza virus antigen or a portion thereof, an influenza A virus antigen or a portion thereof, an influenza B virus antigen or a portion thereof, a parainfluenza virus antigen or a portion thereof, an adenovirus antigen or a portion thereof, an enterovirus antigen or a portion thereof, a coronavirus antigen or a portion thereof, a respiratory syncytial virus (RSV) antigen or a portion thereof, a rhinovirus antigen or a portion thereof, a DNA virus antigen or a portion thereof, an RNA virus antigen or a portion thereof, or a combination thereof. In some embodiments, the antigen may include an antigen of 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-related coronavirus 2 (SARS-CoV-2), a mutant strain of SARS-CoV-2 (e.g., 20A.EU1, or spike mutant D614G), or a combination thereof.In some embodiments, the antigen may comprise a (e.g., coronavirus) alpha mutant spike protein monomer, a (e.g., coronavirus) alpha mutant spike protein trimer, a (e.g., coronavirus) beta mutant spike protein monomer, a (e.g., coronavirus) beta mutant spike protein trimer, a (e.g., coronavirus) gamma mutant spike protein monomer, a (e.g., coronavirus) gamma mutant spike protein trimer, a (e.g., coronavirus) delta mutant spike protein monomer, a (e.g., coronavirus) delta mutant spike protein trimer, a receptor binding domain (RBD) portion of a (e.g., coronavirus) alpha mutant spike protein, an RBD portion of a (e.g., coronavirus) beta mutant spike protein, an RBD portion of a (e.g., coronavirus) gamma mutant spike protein, and / or an RBD portion of a (e.g., coronavirus) delta mutant spike protein.

[0036]

[0067] In some embodiments, an antigen of a composition or method described herein (e.g., an antigen comprising a nucleocapsid (N) protein) may have an amino acid sequence according to SEQ ID NO: 1. In some embodiments, an antigen of a composition or method described herein (e.g., an antigen comprising a nucleocapsid protein) may have an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, an antigen (e.g., an antigen comprising a nucleocapsid protein) may be encoded by a nucleotide sequence according to SEQ ID NO: 2. In some embodiments, an antigen may be encoded by a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, greater than 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, an antigen of a composition or method described herein (e.g., an antigen comprising a spike (S) protein) may have the amino acid sequence of SEQ ID NO: 3. In some embodiments, an antigen of a composition or method described herein (e.g., an antigen comprising a spike (S) protein) may have an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 3. In some embodiments, an antigen (e.g., an antigen comprising a spike (S) protein) may be encoded by a nucleotide sequence according to SEQ ID NO: 4. In some embodiments, an antigen (e.g., an antigen comprising a spike (S) protein) may be encoded by a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, greater than 99%, or 100% sequence identity to SEQ ID NO: 4.

[0037]

[0068] In some embodiments, it may be advantageous for the compositions described herein to include both N-protein and S-protein (e.g., where the compositions are utilized in the methods described herein).For example, the compositions described herein may include an N-protein having an amino acid sequence at least 90% identical to SEQ ID NO:1, and an S-protein having an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:3.For example, the compositions described herein may include an N-protein having an amino acid sequence at least 95% identical to SEQ ID NO:1, and an S-protein having an amino acid sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:3. For example, the compositions described herein may include an N-protein having an amino acid sequence that is 100% identical to SEQ ID NO:1, and an S-protein having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO:3.

[0038]

[0069] In some embodiments, it may be beneficial for the compositions described herein to contain equal amounts of N-protein and S-protein.In some embodiments, the compositions or methods described herein may benefit from adjusting the ratio of N-protein to S-protein, so that the compositions contain non-uniform amounts of N-protein and S-protein.For example, the compositions described herein may contain more N-protein than S-protein.In some embodiments, it may be beneficial for the compositions described herein to contain more S-protein than N-protein. In some embodiments, the compositions described herein are less than 1:1,000, 1:1,000 to 1:500, 1:500 to 1:200, 1:200 to 1:100, 1:100 to 1:50, 1:50 to 1:25, 1:25 to 1:20, 1:20 to 1:10, 1:10 to 1:5, 1:5 to 1:4, 1:4 to 1:3, 1:3 to 1:2, 1:2 to 2:3, 2:3 to 3:4, 3:4 to 4:5, 4:5 to 1:1, 1:1 to 5:4, 5:4 to 4:3, 4: The mass ratio of N-protein to S-protein (e.g., mass ratio of N-protein:S-protein) may be 3 to 3:2, 3:2 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 10:1, 10:1 to 20:1, 20:1 to 25:1, 25:1 to 50:1, 50:1 to 100:1, 100:1 to 200:1, 200:1 to 500:1, 500:1 to 1,000:1, or greater than 1,000:1. In some embodiments, the mass ratio of spike protein of a composition described herein to nucleocapsid protein of the composition is 1:2.In some embodiments, the compositions described herein are less than 1:1,000, 1:1,000 to 1:500, 1:500 to 1:200, 1:200 to 1:100, 1:100 to 1:50, 1:50 to 1:25, 1:25 to 1:20, 1:20 to 1:10, 1:10 to 1:5, 1:5 to 1:4, 1:4 to 1:3, 1:3 to 1:2, 1:2 to 2:3, 2:3 to 3:4, 3:4 to 4:5, 4:5 to 1:1, 1:1 to 5:4, 5:4 to 4:3, 4:4 to 4:5, 4:5 to 1:1, 4:4 to 4:3, 4:4 to 4:5 ... The molar ratio of N-protein to S-protein (e.g., molar ratio of N-protein:S-protein) may be from 1:3 to 3:2, 3:2 to 2:1, 2:1 to 3:1, 3:1 to 4:1, 4:1 to 5:1, 5:1 to 10:1, 10:1 to 20:1, 20:1 to 25:1, 25:1 to 50:1, 50:1 to 100:1, 100:1 to 200:1, 200:1 to 500:1, 500:1 to 1,000:1, or greater than 1,000:1.

[0039]

[0070] In some embodiments, the compositions or methods described herein may include an N-protein associated with the membrane of a (e.g., lipid-based) nanoparticle (e.g., embedded therein, covalently coupled thereto, or non-covalently coupled thereto) and an S-protein that is not associated with the membrane of a nanoparticle (e.g., contained within the nanoparticle or on the outside of the nanoparticle and not coupled thereto (e.g., co-treated)). In some embodiments, the compositions or methods described herein may include an S-protein associated with the membrane of a (e.g., lipid-based) nanoparticle (e.g., embedded therein, covalently coupled thereto, or non-covalently coupled thereto) and an N-protein that is not associated with the membrane of a nanoparticle (e.g., contained within the nanoparticle or on the outside of the nanoparticle and not coupled thereto (e.g., co-treated)). In some embodiments, the compositions or methods described herein may include an N-protein associated with the membrane of a (e.g., lipid-based) nanoparticle (e.g., embedded therein, covalently coupled thereto, or non-covalently coupled thereto) and an S-protein that is associated with the membrane of a nanoparticle. In some embodiments, the compositions or methods described herein may include an N-protein that is not associated with the membrane of a (e.g., lipid-based) nanoparticle (e.g., contained within the nanoparticle or on the outside of the nanoparticle and not coupled to the nanoparticle (e.g., by co-treatment)), and an S-protein that is not associated with the membrane of the nanoparticle.

[0040]

[0071] In some embodiments, the antigen may be a mixture of SARS-CoV2 spike and nucleocapsid proteins. In some embodiments, the antigen may be a mixture of SARS-CoV2 spike and nucleocapsid proteins harboring different mutations.

[0041]

[0072] In some embodiments, the antigen may be an influenza virus antigen or portion thereof, an influenza A virus antigen or portion thereof, an influenza B virus antigen or portion thereof, a parainfluenza virus antigen or portion thereof, an adenovirus antigen or portion thereof, an enterovirus antigen or portion thereof, a coronavirus antigen or portion thereof, a respiratory syncytial virus (RSV) antigen or portion thereof, a rhinovirus antigen or portion thereof, a DNA virus antigen or portion thereof, an RNA virus antigen or portion thereof, or a combination thereof.

[0042]

[0073] In some embodiments, the composition of the present disclosure is suitable for generating immunity against cancer in a subject.In some embodiments, the antigen of the composition of the present disclosure is suitable for generating immunity against cancer in a subject.For example, in some embodiments, the antigen in the therapeutic composition of the present disclosure can elicit an immune response against cancer (e.g., cancers described above) in a subject.

[0043]

[0074] In some embodiments, the antigen of the composition of the present disclosure may be an attenuated or killed tumor cell (or a part thereof) associated with cancer.In some embodiments, the antigen may comprise a peptide or protein associated with cancer.In some embodiments, the antigen may comprise a surface protein (e.g., a receptor protein) of a cancer cell.

[0044]

[0075] In some embodiments, the antigen may comprise a mutated protein of a cancer cell, hi some embodiments, the antigen may be a synthetic long peptide that targets a cancer mutation.

[0045]

[0076] The antigen of the composition disclosed herein may be in various forms.For example, in some embodiments, the antigen may be a recombinant peptide or protein that is recognized by T cells, or a peptide epitope.In some embodiments, the antigen of the composition disclosed herein may comprise a tandem minigene.In some embodiments, the antigen may be in the form of a nucleotide that expresses the antigen (e.g., a plasmid DNA molecule that expresses the antigen).

[0046] Modulator

[0077] The compositions described herein (e.g., comprising a particle, e.g., a lipid-based particle, and optionally an antigen) for preventing or treating a disease in a subject may include a modulator. The modulator of the compositions described herein may be a pattern recognition receptor modulator, such as a STING modulator (e.g., a STING pathway modulator). In some embodiments, the modulator of the compositions described herein may be a pattern recognition receptor agonist. In some embodiments, the modulator of the compositions described herein may be a pattern recognition receptor antagonist. The compositions of the present disclosure may include various types of STING modulators. For example, in some embodiments, the STING modulator is an antagonist of the STING pathway. In some embodiments, the STING modulator is an agonist of the STING pathway. In some embodiments, the modulator of the compositions described herein is capable of activating the STING pathway in a subject (e.g., a subject to which the composition is administered). In some embodiments, the modulator of the compositions described herein is capable of inhibiting the STING pathway in a subject (e.g., a subject to which the composition is administered).

[0047]

[0078] The STING modulators of the compositions described herein may include bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), amidobenzimidazole, derivatives of amidobenzimidazole, nucleotide modulators, plasmid DNA modulators, nucleic acid modulators, CF501, SHR1032, or combinations thereof.

[0048]

[0079] In some embodiments, STING modulator can be an antagonist of STING pathway.In some embodiments, STING antagonist can be particularly effective in treating and / or preventing disease, for example, when disease-causing pathogen (e.g., RNA virus such as rhinovirus) utilizes STING pathway to promote viral replication.In some embodiments, STING antagonist can be utilized to inhibit viral replication by reducing the accessibility of virus to STING pathway.In some embodiments, STING antagonist includes but is not limited to C-178, H-151, and combinations thereof.

[0049]

[0080] In some embodiments, the modulator of the composition described herein may be located in the inner space of the particle of the composition.For example, the modulator may be encapsulated in the particle of the composition described herein (e.g., lipid-based particle, e.g., liposome).In some embodiments, the modulator of the composition described herein may be associated with the surface of the particle of the composition (e.g., the outer surface of the membrane).In some embodiments, the modulator associated with the particle surface is associated by covalently or non-covalently binding to the particle surface.In some embodiments, the modulator associated with the particle surface is associated by being integrated into the particle surface (e.g., membrane).

[0050]

[0081] In some embodiments, the nucleic acid sequence of the antigen of the composition described herein and the nucleic acid sequence of the modulator of the composition may be at least 85%, at least 90%, at least 95%, or 100% identical. In some embodiments, a part of the nucleic acid sequence of the antigen of the composition described herein may be at least 85%, at least 90%, at least 95%, or 100% identical to the nucleic acid sequence of the modulator of the composition. In some embodiments, an antigen comprising a nucleic acid sequence at least 85%, at least 90%, at least 95%, or 100% identical to the nucleic acid sequence of the modulator described herein may be encapsulated in a particle (e.g., lipid-based particle) of the composition described herein (e.g., in this case, the modulator may be encapsulated in a lipid-based particle). In some embodiments, both the antigen and the modulator described herein (e.g., in this case, the antigen and the modulator comprise at least 85%, at least 90%, at least 95%, or 100% identical nucleic acid sequences) are encapsulated in a lipid-based particle described herein. In some embodiments, compositions comprising an antigen and a modulator that comprise sequences that are at least 85%, at least 90%, at least 95%, or 100% identical may be delivered intranasally.

[0051]

[0082] In some cases, it may be advantageous to formulate the compositions described herein within a desired pH range. For example, formulation of the compositions described herein (e.g., for nasal delivery) at pH 4.0 to 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 tissues in the nasal cavity that may occur when extremely acidic or basic formulations are used for nasal delivery (e.g., because the pH of the nasal cavity can be about 5.5 to 6.5). In some cases, the compositions described herein may have a pH of 4.0 to 7.5. In some cases, the compositions described herein may be of any of the following compositions: 4.0-4.5, 4.0-4.8, 4.0-5.0, 4.0-5.2, 4.0-5.5, 4.0-5.8, 4.0-6, 4.0-6.2, 4.0-6.5, 4.0-7, 4.0-7.5, 4.5-4.8, 4.5-5.0, 4.5-5.2, 4.5-5. .5, 4.5~5.8, 4.5~6.0, 4.5~6.2, 4.5~6.5, 4.5~7.0, 4.5~7.5, 4.8~5.0, 4.8~5.2, 4.8~5.5, 4.8~5.8, 4.8~6.0, 4.8~6.2, 4.8~6.5, 4.8~7.0, 4.8~7.5, 5.0~5.2, 5.0~5.5, 5 .0~5.8, 5.0~6.0, 5.0~6.2, 5.0~6.5, 5.0~7.0, 5~7.5, 5.2~5.5, 5.2~5.8, 5.2~6.0, 5.2~6.2, 5.2~6.5, 5.2~7.0, 5.2~7.5, 5.5~5.8, 5.5~6.0, 5.5~6.2, 5.5~6.5, 5.5~7.0 , 5.5-7.5, 5.8-6.0, 5.8-6.2, 5.8-6.5, 5.8-7.0, 5.8-7.5, 6.0-6.2, 6.0-6.5, 6.0-7, 6.0-7.5, 6.2-6.5, 6.2-7, 6.2-7.5, 6.5-7.0, 6.5-7.5, or 7.0-7.5. In some cases, the compositions described herein may have a pH of 4.0, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 7.0, or 7.5.In some cases, the compositions described herein may have a pH of at least 4.0, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 7.0, or 7.5. In some cases, the compositions described herein may have a pH of at most 4.0, 4.5, 4.8, 5.0, 5.2, 5.5, 5.8, 6.0, 6.2, 6.5, 7.0, or 7.5.

[0052]

[0083] In some cases, it may be advantageous to formulate the compositions described herein within a desired osmolarity range. For example, formulation of the compositions described herein (e.g., for nasal delivery) with an osmolarity of 50-900 mOsm / kg may improve absorption while avoiding possible epithelial damage (e.g., which may occur with compositions with very low osmolarity) and increased mucosal secretions (e.g., which may occur with compositions with very high osmolarity) (e.g., because the osmolarity of the nasal cavity may be about 280 mOsm / kg). In some cases, the compositions described herein may have an osmolarity of 50 mOsm / kg to 900 mOsm / kg. In some cases, the compositions described herein may be administered in a range of concentrations from 50mOsm / kg to 150mOsm / kg, 50mOsm / kg to 250mOsm / kg, 50mOsm / kg to 300mOsm / kg, 50mOsm / kg to 350mOsm / kg, 50mOsm / kg to 450mOsm / kg, 50mOsm / kg to 550mOsm / kg, 50mOsm / kg to 600mOsm / kg, 50mOsm / kg to 700mOsm / kg, 50mOsm / kg to 800mOsm / kg, 50mOsm / kg to 900mOsm / kg, 50mOsm / kg to 1000mOsm / kg, ...0mOsm / kg, 50mOsm / / kg~650mOsm / kg, 50mOsm / kg~750mOsm / kg, 50mOsm / kg~850mOsm / kg, 50mOsm / kg~900mOsm / kg, 1 50mOsm / kg~250mOsm / kg, 150mOsm / kg~300mOsm / kg, 150mOsm / kg~350mOsm / kg, 150mOsm / kg~450 mOsm / kg, 150mOsm / kg~550mOsm / kg, 150mOsm / kg~650mOsm / kg, 150mOsm / kg~750mOsm / kg, 150mO sm / kg~850mOsm / kg, 150mOsm / kg~900mOsm / kg, 250mOsm / kg~300mOsm / kg, 250mOsm / kg~350mOsm / kg, 250mOsm / kg~450mOsm / kg, 250mOsm / kg~550mOsm / kg, 250mOsm / kg~650mOsm / kg, 250mOsm / k g~750mOsm / kg, 250mOsm / kg~850mOsm / kg, 250mOsm / kg~900mOsm / kg, 300mOsm / kg~350mOsm / kg,300mOsm / kg~450mOsm / kg, 300mOsm / kg~550mOsm / kg, 300mOsm / kg~650mOsm / kg, 300mOsm / kg ~750mOsm / kg, 300mOsm / kg~850mOsm / kg, 300mOsm / kg~900mOsm / kg, 350mOsm / kg~450mOsm / kg , 350mOsm / kg~550mOsm / kg, 350mOsm / kg~650mOsm / kg, 350mOsm / kg~750mOsm / kg, 350mOsm / kg ~850mOsm / kg, 350mOsm / kg~900mOsm / kg, 450mOsm / kg~550mOsm / kg, 450mOsm / kg~650mOsm / kg , 450mOsm / kg~750mOsm / kg, 450mOsm / kg~850mOsm / kg, 450mOsm / kg~900mOsm / kg, 550mOsm / kg ~650mOsm / kg, 550mOsm / kg~750mOsm / kg, 550mOsm / kg~850mOsm / kg, 550mOsm / kg~900mOsm / kg , 650 mOsm / kg to 750 mOsm / kg, 650 mOsm / kg to 850 mOsm / kg, 650 mOsm / kg to 900 mOsm / kg, 750 mOsm / kg to 850 mOsm / kg, 750 mOsm / kg to 900 mOsm / kg, or 850 mOsm / kg to 900 mOsm / kg. In some cases, the compositions described herein may have an osmolality of 50 mOsm / kg, 150 mOsm / kg, 250 mOsm / kg, 300 mOsm / kg, 350 mOsm / kg, 450 mOsm / kg, 550 mOsm / kg, 650 mOsm / kg, 750 mOsm / kg, 850 mOsm / kg, or 900 mOsm / kg. In some cases, the compositions described herein may have an osmolality of at least 50 mOsm / kg, 150 mOsm / kg, 250 mOsm / kg, 300 mOsm / kg, 350 mOsm / kg, 450 mOsm / kg, 550 mOsm / kg, 650 mOsm / kg, 750 mOsm / kg, 850 mOsm / kg, or 900 mOsm / kg.It may have an osmolality of 150 mOsm / kg, 250 mOsm / kg, 300 mOsm / kg, 350 mOsm / kg, 450 mOsm / kg, 550 mOsm / kg, 650 mOsm / kg, 750 mOsm / kg, 850 mOsm / kg, or 900 mOsm / kg.

[0053]

[0084] In some cases, it may be advantageous to formulate the compositions described herein within a desired viscosity range. For example, formulations of the compositions described herein (e.g., for nasal delivery) with a viscosity of, for example, 1.1 cP (centipoise) to 50 cP (e.g., 1.5 cP to 50 cP) can increase residence time in the nasal cavity, but have a detrimental effect on droplet size (e.g., this can affect the spray pattern and / or distribution in the nasal cavity). In some cases, the compositions described herein may have a viscosity of 1 cP (centipoise) to 100 cP. In some cases, the compositions described herein may comprise a dilution of 1 cP to 1.1 cP, 1 cP to 1.5 cP, 1 cP to 2 cP, 1 cP to 5 cP, 1 cP to 7 cP, 1 cP to 10 cP, 1 cP to 15 cP, 1 cP to 25 cP, 1 cP to 50 cP, 1 cP to 100 cP, 1.1 cP to 1.5 cP, 1.1 cP to 2 cP, 1.1 cP to 5 cP, 1.1cP~7cP, 1.1cP~10cP, 1.1cP~15cP, 1.1cP~25cP, 1.1cP~50cP, 1.1cP~100cP, 1.5 cP~2cP, 1.5cP~5cP, 1.5cP~7cP, 1.5cP~10cP, 1.5cP~15cP, 1.5cP~25cP, 1.5cP~50cP, 1. 5cP~100cP, 2cP~5cP, 2cP~7cP, 2cP~10cP, 2cP~15cP, 2cP~25cP, 2cP~50cP, 2cP~100cP, 5cP~7cP, 5cP~10cP, 5cP~15cP, 5cP~25cP, 5cP~50cP, 5cP~100cP, 7cP~10cP, 7cP~15cP, 7 In some cases, the compositions described herein may have a viscosity of 1 cP, 1.1 cP, 1.5 cP, 2 cP, 5 cP, 7 cP, 10 cP, 15 cP, 25 cP, 50 cP, or 100 cP.In some cases, the compositions described herein may have a viscosity of at least 1 cP, 1.1 cP, 1.5 cP, 2 cP, 5 cP, 7 cP, 10 cP, 15 cP, 25 cP, 50 cP, or 100 cP. In some cases, the compositions described herein may have a viscosity of at most 1.0 cP, 1.1 cP, 1.5 cP, 2 cP, 5 cP, 7 cP, 10 cP, 15 cP, 25 cP, 50 cP, or 100 cP.

[0054] How to use

[0085] In additional embodiments, the present disclosure relates to a method of treating or preventing a disease in a subject by administering to the subject a therapeutic composition of the present disclosure. In a more specific embodiment illustrated in Figure 2A, the method of the present disclosure includes a step of administering to the subject a therapeutic composition (step 20), thereby treating or preventing the disease in the subject (step 22).

[0055]

[0086] As described in more detail herein, the therapeutic compositions and methods of the present disclosure may have multiple embodiments. For example, the therapeutic compositions of the present disclosure may include various types of STING modulators and antigens. Furthermore, the methods of the present disclosure may be utilized to administer the therapeutic compositions of the present disclosure to multiple subjects to treat or prevent various diseases in various ways.

[0056] Purpose

[0087] In some embodiments, the compositions and / or methods described herein may be useful in preventing or treating respiratory diseases or conditions, such as influenza, coronavirus, respiratory syncytial virus, rhinovirus, or combinations thereof.In some embodiments, the compositions and / or methods described herein may be useful in preventing or treating severe acute respiratory syndrome (SARS), acute respiratory distress syndrome (ARDS), and / or hypercytokinemia (e.g., cytokine storm).In some embodiments, the compositions and / or methods described herein may be useful in preventing and / or treating cancer, such as lung cancer.

[0057]

[0088] There is an urgent need for safe and effective vaccines against respiratory diseases and cancer.In some embodiments, the compositions described herein may comprise vaccines or therapeutic treatments (e.g., delivered intranasally) that contain liposomes that contain agonist of stimulator of interferon genes (STING) pathway to enable humoral immunity, T cell immunity, systemic immunity and / or mucosal immunity.

[0058]

[0089] Innate immunity is the first line of defense against invading pathogens. Innate immunity (unlike adaptive immunity, which can be customized for each pathogen) can be triggered by pattern recognition receptors (PRRs) on host cells that recognize conserved pathogen-associated molecular patterns (PAMPs). This mode of recognition can ensure the presence of an immediate response that does not require customization.

[0059]

[0090] In the context of RNA viruses, recognition and activation of virus-specific RNA molecules can result in the activation of interferon regulatory factors (IRFs) and nuclear factor kappa B (NF-κB). Collectively, these transcriptional regulators execute a broad antiviral program that includes the synthesis and secretion of type I and type III interferons and the subsequent upregulation of IFN-stimulated genes (ISGs).

[0060]

[0091] This comprehensive antiviral program can apply strong selection pressure on viral replication. Viruses have evolved sophisticated countermeasures to interfere with interferon signaling. The interplay between interferon-mediated responses and viral countermeasures is heterogeneous, which may explain the heterogeneity of morbidity and mortality seen in humans.

[0061]

[0092] During pathogen infection (e.g., coronavirus or other respiratory pathogens), type 1 interferon responses may be suppressed, and the balance between ISGs and proinflammatory responses mediated by NF-κB may then be dysregulated. As a result, patients with advanced disease upon pathogen infection (e.g., respiratory pathogen infection) may exhibit low interferon signaling but intensify tumor necrosis factor (TNF) and interleukin-6 (IL-6) secretion. Indeed, humans with autoantibodies against interferon that neutralize interferon function may have a high risk of developing advanced disease.

[0062]

[0093] Similarly, humans with inborn errors of type I IFN immunity may develop life-threatening COVID-19 at a faster rate than those without such errors. In some embodiments, pretreatment of cell lines with type I interferon can inhibit viral replication in otherwise susceptible cells. Without being bound by theory, these considerations may help explain why the lack of a robust type I IFN response may be the basis of advanced COVID-19.

[0063]

[0094] The stimulator of interferon genes (STING) pathway is a PRR that senses circular DNA dinucleotides, activates IRF3 and NF-κB, and leads to the synthesis of ISGs. It is thought to be mainly important for sensing bacteria and DNA viruses, but the role of STING in RNA virus-mediated type I IFN and cytokine production requires more detailed study. As shown herein, the role of STING in RNA virus-mediated type I IFN and cytokine production may be virus- and cell type-specific.

[0064]

[0095] Sensing of double-stranded RNA from viruses such as coronaviruses in the cytoplasm of human cells can be achieved by RIG-I-like receptors, such as retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation gene 5 (MDA5). In some embodiments, sensing of pathways downstream of these receptors leads to the activation of two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi). In some embodiments, these kinases are also activated by the activated STING pathway, suggesting that PRRs have evolved to sense various molecules but may converge in their downstream responses.

[0065]

[0096] By utilizing STING agonists as therapeutic agents, this conserved downstream effector response can be exploited to promote balanced activation of type I interferons even in the context of SARS-CoV2 infection. By allowing release of type I interferons by STING agonists independent of RIG-1-like receptors, we increase the likelihood that this will not be targeted by countermeasures evolved by the virus.

[0066]

[0097] The release of interferon can enable a broad antiviral program that limits viral replication, thus reducing the severity of the disease. On the other hand, RNA viruses such as rhinoviruses can hijack the STING pathway to promote viral replication. In this situation, it may be useful to inhibit viral replication by utilizing STING antagonists to quell inflammation.

[0067]

[0098] Without being bound by theory, the therapeutic compositions of the present disclosure can be used to treat or prevent disease through various mechanisms of action. For example, in some embodiments, the therapeutic compositions of the present disclosure can be used to treat disease in a subject. In some embodiments, the therapeutic compositions of the present disclosure can be used to prevent disease in a subject. In some embodiments, the therapeutic compositions of the present disclosure can be used to treat and prevent disease in a subject. In some embodiments, the therapeutic compositions of the present disclosure can be used to treat or prevent disease in a subject by generating immunity to the disease in the subject.

[0068]

[0099] For example, in some embodiments, the therapeutic composition of the present disclosure can induce immune response against disease in subject.In some embodiments, the therapeutic composition of the present disclosure can induce such immunity through at least one of natural immunity, mucosal immunity, systemic immunity, cellular immunity, humoral immunity, T cell immunity, systemic neutralizing antibody production, induction of IgG response, induction of IgA response, induction of IgM response, induction of T cell response, induction of mucosal IgA response in lung and nasal compartment, induction of Th1 T cell response, induction of CD8+ T cell response, induction of CD4+ T cell response, induction of NK cell response, activation or inhibition of stimulator of interferon genes (STING) pathway, or combination thereof.

[0069]

[0100] In some embodiments, the compositions disclosed herein can elicit a mean dilution titer (e.g., of IgG or IgA specific for a pathogen antigen or a portion thereof) in the serum of a subject of at least 1:25, greater than 1:25. In some embodiments, the compositions disclosed herein can elicit a mean dilution titer (e.g., of IgG or IgA specific for a pathogen antigen or a portion thereof) in the bronchoalveolar lavage fluid (BALF) of a subject of 1:25 to 1:50, 1:30 to 1:50, 1:40 to 1:50, 1:50 to 1:60, or at least 1:25, greater than 1:25, or less than 1:60.

[0070]

[0101] In some embodiments, the composition disclosed in the present invention can cause or help develop immunity against disease in a subject, for example by eliciting a natural immune response against disease in the subject.In some embodiments, the elicited natural immune response can lead to the activation of interferon regulator factor (IRF), nuclear factor kappa B (NF-kappa B), or a combination thereof.In some embodiments, the elicited natural immune response can lead to the synthesis and secretion of type I and type III interferon (IFN), followed by the upregulation of IFN-stimulated genes (ISGs).

[0071]

[0102] In some embodiments, the therapeutic composition of the present disclosure can be used to generate immunity against disease in a subject, for example, via mucosal immunity and / or systemic immunity. In some embodiments, the therapeutic composition of the present disclosure can be useful for generating immunity in a subject via mucosal immunity, systemic immunity, and cellular immunity. In some embodiments, systemic immunity can be generated via the production of neutralizing antibodies against antigens. In some embodiments, cellular immunity can be generated in spleen cells and / or lung cells. In some embodiments, mucosal immunity can be generated via the production of IgA in IgA-secreting cells in the nasal compartment and lungs, as well as in the spleen.

[0072]

[0103] The methods and therapeutic compositions of the present disclosure can provide a number of advantages in various embodiments, for example, in some embodiments, the therapeutic compositions of the present disclosure can be administered to large populations without the need for large clinical facilities.

[0073]

[0104] Furthermore, in some embodiments, the therapeutic compositions of the present disclosure may prevent the establishment of early viral reservoirs (e.g., in the nasal compartment) and may help control transmission of the virus between and / or within individuals.

[0074]

[0105] In some embodiments, the therapeutic composition of the present disclosure can correct the lack of interferon activation, which may be one of the main escape mechanisms mediated by respiratory viruses.In addition, for example, the therapeutic composition of the present disclosure can be utilized in some embodiments to induce one or more of IgM, IgG, IgA and T cell responses in subjects, so that the therapeutic composition of the present disclosure can provide a universal mucosal adjuvant for developing broad-spectrum therapeutic compositions against multiple pathogens, such as coronavirus or other respiratory pathogens (such as other viral respiratory pathogens).

[0075]

[0106] In some embodiments, the methods and therapeutic compositions of the present disclosure may have veterinary uses, including the treatment or prevention of diseases in various types of animals and other similar uses.

[0076] Formulation and Administration

[0107] The compositions disclosed herein may be in various forms. In some embodiments, the compositions described herein may be delivered as solubilized liquid. In some embodiments, the therapeutic compositions disclosed herein are suitable for nasal and / or inhalation administration to subjects. In some embodiments, nasal delivery of the compositions disclosed herein can be used to target tissues in the nasal compartment. In some embodiments, inhalation administration of the compositions disclosed herein can be used to target tissues in the pulmonary compartment.

[0077]

[0108] In some embodiments, syringe or liquid dropper can be used to administer the compositions described herein to subject's nasal cavity.In some embodiments, administering the compositions described herein to subject can include using spray nozzle, nebulizer, and / or atomizer.In some embodiments, the therapeutic composition of the present disclosure can be self-administered.

[0078]

[0109] In some embodiments, the compositions of the present disclosure also include one or more stabilizers. In some embodiments, the stabilizers include, but are not limited to, antioxidants, sequestering agents, UV stabilizers, or combinations thereof.

[0079]

[0110] In some embodiments, the composition of the present disclosure also comprises one or more surfactants.In some embodiments, the surfactants include, but are not limited to, anionic surfactants, sugars, cationic surfactants, zwitterionic surfactants, nonionic surfactants, or combinations thereof.

[0080]

[0111] In some embodiments, the composition of the present disclosure also comprises one or more excipients.In some embodiments, excipients include but are not limited to lactose, sucrose, starch powder, cellulose ester of alkanoic acid, trehalose, cellulose alkyl ester, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, gelatin, gum arabic, trehalose, sodium alginate, polyvinylpyrrolidone, polyvinyl alcohol, or combinations thereof.

[0081]

[0112] In some embodiments, the compositions of the present disclosure may be lyophilized (e.g., freeze-dried). Lyophilization of the compositions of the present disclosure (or components thereof) may increase the storage stability (e.g., storability) of the compositions described herein. In some embodiments, one or more components of the compositions of the present disclosure may be lyophilized. In some embodiments, lyophilization of the compositions of the present disclosure (or components thereof) may allow for easy preparation of the compositions, for example, for use (e.g., administration to a subject) in areas where material preparation facilities are not easily accessible. For example, the modulators, lipid-based particles, and / or antigens described herein may be lyophilized, and then rehydrated and mixed (e.g., as described herein) to formulate the compositions described herein at the site of administration to a subject that is away from a regular medical or pharmaceutical facility. In some embodiments, the lyophilized compositions described herein (or portions thereof, such as lyophilized particles or lyophilized antigens or lyophilized modulators) may be stored at 4°C. In some embodiments, the lyophilized compositions described herein (or portions 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.

[0082]

[0113] 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.

[0114] The compositions of the present disclosure can be administered by a variety of methods. For example, in some embodiments, administration is by methods including, but not limited to, 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, inhalation administration, or a combination thereof.

[0083]

[0115] In some embodiments, the composition disclosed herein can be administered in a single dose (e.g., via intranasal administration).In some embodiments, the method disclosed herein can include administering (e.g., via intranasal administration) a dose of the composition described herein to a subject (e.g., an animal 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 after suspected exposure) to a pathogen that can cause disease, such as the respiratory disease described herein. For example, a method for treating a subject exposed to, at risk of being exposed to, or suspected of being exposed to a pathogen that may cause a disease, such as a respiratory disease, may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to the subject (e.g., an animal 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 after suspected exposure) to the pathogen.In some cases, a method for treating a subject suffering from a disease, such as a respiratory disease, due to a pathogen (e.g., selected from the pathogens described herein) may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to a subject (e.g., an animal 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 after suspected exposure) to the pathogen. In some cases, a method for treating a subject suffering from a disease, such as a respiratory disease caused by a pathogen (e.g., selected from a pathogen described herein), may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to a subject (e.g., an animal 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 clinical diagnosis of the disease in the subject.

[0084]

[0116] In some cases, a method for slowing or halting the progression of a disease, such as a respiratory disease, caused by a pathogen (e.g., selected from a pathogen described herein) in a subject may include administering (e.g., via intranasal administration) to a subject (e.g., an animal subject) one or more doses of a composition described herein 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 after suspected exposure) to the pathogen. In some cases, a method for slowing or halting the progression of a disease, such as a respiratory disease caused by a pathogen (e.g., selected from a pathogen described herein) in a subject may include administering (e.g., via intranasal administration) to a subject (e.g., an animal subject) one or more doses of a composition described herein 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 clinical diagnosis of the disease (or stage, severity, or change in its stage or severity) in the subject.

[0085]

[0117] In some cases, a method for reducing or preventing the risk of transmission of a pathogen (or a condition caused by a pathogen) from a first subject to a second subject may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to the first subject (e.g., an animal 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) of the first subject to the pathogen. In some cases, a method for reducing or preventing the risk of transmission of a pathogen (or a condition caused by a pathogen) from a first subject to a second subject may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to the second subject (e.g., an animal 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 clinical diagnosis of the disease (or stage, severity, or change in its stage or severity) in the first subject.In some cases, a method for reducing or preventing the risk of transmission of a pathogen (or a condition caused by a pathogen) from a first subject to a second subject may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to the second subject (e.g., an animal 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) of the first subject to the pathogen. In some cases, a method for reducing or preventing the risk of transmission of a pathogen (or a condition caused by a pathogen) from a first subject to a second subject may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to the first subject (e.g., an animal 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 clinical diagnosis of the disease (or stage, severity, or change in its stage or severity) in the first subject.

[0086]

[0118] In some cases, a method for reducing or preventing a subject's risk of contracting a pathogen (or a condition caused by a pathogen) may include administering (e.g., via intranasal administration) one or more doses of a composition described herein to a first subject (e.g., an animal 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 before the subject engages in an activity associated with an increased risk of exposure to the pathogen.

[0087]

[0119] In some embodiments, the therapeutic composition of the present disclosure can be administered in multiple doses. In some embodiments, the method of treating or preventing a disease in a subject (e.g., an animal subject) can include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15-20, 20-25, 25-30, 30-40, 40-50, or more than 50 doses. In some embodiments, the composition of the present disclosure can be administered to a subject (e.g., an animal subject) at multiple time points (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 time points). In some embodiments, the method of the present disclosure can include administering to the subject a first dose comprising a composition of the present disclosure and a second dose comprising a composition of the present disclosure. In some embodiments, the composition of the first dose is the same as the composition of the second dose. In some embodiments, the composition of the first dose is different from the composition of the second dose. In some embodiments, the disclosed method may include administering a first dose and a second dose to a subject (e.g., an animal subject) 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 apart. In some embodiments, the disclosed method may include administering a first dose and a second dose to a subject (e.g., an animal subject) at most 6 hours, at most 12 hours, at most 24 hours, at most 36 hours, at most 2 days, at most 3 days, at most 4 days, at most 5 days, at most 6 days, at most 7 days, at most 14 days, or at most 28 days apart. In some embodiments, the methods of the present disclosure may include administering a first dose and a second dose to a subject (e.g., an animal subject) at intervals of 6 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, or 28 days.In some embodiments, the methods of the present disclosure may include administering each dose after the first dose of the plurality of doses to a subject (e.g., an animal subject) 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 administration of the previous dose (e.g., where each dose of the plurality of doses comprises one or more compositions of the present disclosure). In some embodiments, the method of the present disclosure may include administering each dose of the plurality of doses after the first dose to a subject (e.g., an animal subject) at most 6 hours, at most 12 hours, at most 24 hours, at most 36 hours, at most 2 days, at most 3 days, at most 4 days, at most 5 days, at most 6 days, at most 7 days, at most 14 days, or at most 28 days after administration of the previous dose (e.g., where each dose of the plurality of doses comprises one or more compositions of the present disclosure). In some embodiments, the method of the present disclosure may include administering each dose of the plurality of doses after the first dose to a subject (e.g., an animal subject) at most 6 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 14 days, or 28 days after administration of the previous dose (e.g., where each dose of the plurality of doses comprises one or more compositions of the present disclosure). In some embodiments, the composition is administered to the subject in 2, 3, 4, 5, or more than 5 doses. In some embodiments, the second, third, and fourth dose (e.g., of a 4-dose treatment) is administered 12 to 36 hours after the previous dose. In some embodiments, the second, third, and fourth dose (e.g., of a 4-dose treatment) is administered 1, 2, 4, 6, 8, 10, 12, 18, 24, 30, or 36 hours after the previous dose.

[0088]

[0120] In some embodiments, the compositions described herein (e.g., "NanoSTING" compositions comprising a lipid-based particle, a modulator, and an antigen) may be formulated and / or administered as a monotherapy (e.g., to treat a disease or condition in a subject, e.g., a disease or condition caused by exposure to a pathogen).

[0089]

[0121] The compositions or methods described herein (e.g., for treating a disease or condition in a subject, for preventing a disease or condition in a subject, or for preventing the transmission of a disease or condition from a first subject to a second subject) may include a nanoparticle (e.g., a lipid-based nanoparticle) and a modulator (e.g., a pattern recognition receptor agonist protein, such as a STING agonist). In some embodiments, the compositions or methods that include a nanoparticle (e.g., a lipid-based nanoparticle) and a modulator may lack an antigen. In some embodiments, the modulator of the composition or method that includes a nanoparticle and a modulator but lacks an antigen may be encapsulated within the nanoparticle. In some embodiments, the compositions that include a nanoparticle and a modulator but lacks an antigen may be administered to a subject (e.g., a human subject or a non-human mammalian subject) in the doses, dosage forms, and / or time courses of treatment or vaccination described herein. For example, a composition comprising the nanoparticles and the modulator, but lacking the antigen, can be administered to a subject (e.g., a human subject or a non-human mammalian subject) before the subject is exposed to an infectious agent (e.g., a virus, e.g., a respiratory virus such as a coronavirus, influenza virus, parainfluenza virus, or rhinovirus). In some embodiments, a composition comprising the nanoparticles and the modulator, but lacking the antigen, can be administered to a subject (e.g., a human subject or a non-human mammalian subject) before the subject suffers from and / or develops a respiratory disease or condition (e.g., associated with a respiratory virus) or, in some embodiments, a cancer, such as lung cancer. In some embodiments, a composition comprising the nanoparticles and the modulator, but lacking the antigen, can be administered to a subject (e.g., a human subject or a non-human mammalian subject) after the subject is exposed to an infectious agent (e.g., a virus, e.g., a respiratory virus such as a coronavirus, influenza virus, parainfluenza virus, or rhinovirus).In some embodiments, the composition comprising nanoparticles and modulators but lacking antigens can be administered to a subject (e.g., a human subject or a non-human mammalian subject) after the subject has contracted and / or is symptomatic of a respiratory disease or condition (e.g., associated with a respiratory virus) or cancer, such as lung cancer in some embodiments. In some embodiments, the composition comprising nanoparticles and modulators but lacking antigens can be administered intranasally to a subject. In some embodiments, the composition and method comprising nanoparticles and modulators but lacking antigens may be relatively inexpensive or logistically less complicated to manufacture, store, and / or prepare. In some embodiments, the composition comprising nanoparticles and modulators but lacking antigens can be administered to a subject in combination with a composition comprising nanoparticles, modulators, and antigens, for example, as part of a single treatment or vaccination regimen, simultaneously, sequentially, or separately.

[0090]

[0122] The compositions or methods described herein (e.g., for treating a disease or condition in a subject, for preventing a disease or condition in a subject, or for preventing the transmission of a disease or condition from a first subject to a second subject) may include a modulator (e.g., a pattern recognition receptor agonist protein, such as a STING agonist) as described herein. In some embodiments, the compositions or methods that include a modulator may lack an antigen and a nanoparticle (e.g., a liposome). In some embodiments, the modulator can be administered to a subject (e.g., a human subject or a non-human mammalian subject) without an antigen or nanoparticle, for example, to treat or prevent a disease or condition in the subject (e.g., a disease or condition associated with cancer or a respiratory virus or respiratory disease). In some embodiments, the compositions that include a modulator but lack an antigen or nanoparticle can be administered to a subject (e.g., a human subject or a non-human mammalian subject) in the doses, dosage forms, and / or time courses of treatment or vaccination described herein. For example, a composition comprising a modulator but lacking the antigen or nanoparticles can be administered to a subject (e.g., a human subject or a non-human mammalian subject) before the subject is exposed to an infectious agent (e.g., a virus, e.g., a respiratory virus such as a coronavirus, influenza virus, parainfluenza virus, or rhinovirus). In some embodiments, a composition comprising a modulator but lacking the antigen or nanoparticles can be administered to a subject (e.g., a human subject or a non-human mammalian subject) before the subject suffers from and / or develops a respiratory disease or condition (e.g., associated with a respiratory virus) or, in some embodiments, a cancer, such as lung cancer. In some embodiments, a composition comprising a modulator but lacking the antigen or nanoparticles can be administered to a subject (e.g., a human subject or a non-human mammalian subject) after the subject is exposed to an infectious agent (e.g., a virus, e.g., a respiratory virus such as a coronavirus, influenza virus, parainfluenza virus, or rhinovirus).In some embodiments, the composition comprising the modulator but lacking the antigen or nanoparticles can be administered to a subject (e.g., a human subject or a non-human mammalian subject) after the subject has contracted and / or is symptomatic of a respiratory disease or condition (e.g., associated with a respiratory virus) or cancer, such as lung cancer in some embodiments. In some embodiments, the composition comprising the modulator but lacking the antigen or nanoparticles can be administered intranasally to a subject. In some embodiments, the composition and method comprising the modulator but lacking the antigen or nanoparticles may be relatively inexpensive or logistically less complicated to manufacture, store, and / or prepare. In some embodiments, the composition comprising the nanoparticles and the modulator but lacking the antigen can be administered to a subject in combination with a composition comprising the nanoparticles, the modulator, and the antigen, for example, as part of a single treatment or vaccination regimen, simultaneously, sequentially, or separately.

[0091]

[0123] In some embodiments, a composition of the present disclosure (e.g., for treating a subject having a disease or condition, such as a respiratory disease, or for preventing a subject from contracting said disease or condition, or for transmitting said disease or condition to a second subject) may comprise between 0.1 μg and 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, the compositions of the present disclosure comprise from 0.1 μg to 1 μg, 0.1 μg to 2 μg, 0.1 μg to 3 μg, 0.1 μg to 5 μg, 0.1 μg to 10 μg, 0.1 μg to 20 μg, 0.1 μg to 30 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 120 μg, 0.1 μg to 200 μg, 1 μg to 2 μg, 1 μg to 3 μg, 1 μg to 5 μg, 1 μg to 10 μg, 1 μg to 20 μg. g, 1μg~30μg, 1μg~50μg, 1μg~100μg, 1μg~120μg, 1μg~200μg, 2μg~3μg, 2μg~5μg, 2μg~10μg, 2μg~20μg, 2μg~30μg , 2μg~50μg, 2μg~100μg, 2μg~120μg, 2μg~200μg, 3μg~5μg, 3μg~10μg, 3μg~20μg, 3μg~30μg, 3μg~50μg, 3μg~100μg g, 3μg~120μg, 3μg~200μg, 5μg~10μg, 5μg~20μg, 5μg~30μg, 5μg~50μg, 5μg~100μg, 5μg~120μg, 5μg~200μg, 10μg ~20μg, 10μg~30μg, 10μg~50μg, 10μg~100μg, 10μg~120μg, 10μg~200μg, 20μg~30μg, 20μg~50μg, 20μg~100μg, 20

[0043] The present invention may comprise between 1 μg and 120 μg, 20 μg and 200 μg, 30 μg and 50 μg, 30 μg and 100 μg, 30 μg and 120 μg, 30 μg and 200 μg, 50 μg and 100 μg, 50 μg and 120 μg, 50 μg and 200 μg, 100 μg and 120 μg, 100 μg and 200 μg, or 120 μg and 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).In some embodiments, the compositions of the present disclosure may comprise 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, the compositions of the present disclosure may comprise at least 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, compositions of the present disclosure may contain up to 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).

[0092]

[0124] In some embodiments, a composition of the present disclosure (e.g., for treating a subject having a disease or condition, such as a respiratory disease, or for preventing a subject from contracting said disease or condition, or for transmitting said disease or condition to a second subject) may comprise between 0.1 μg and 200 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles such as . In some embodiments, the compositions of the present disclosure comprise from 0.1 μg to 1 μg, 0.1 μg to 2 μg, 0.1 μg to 3 μg, 0.1 μg to 5 μg, 0.1 μg to 10 μg, 0.1 μg to 20 μg, 0.1 μg to 30 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 120 μg, 0.1 μg to 200 μg, 1 μg to 2 μg, 1 μg to 3 μg, 1 μg to 5 μg, 1 μg to 10 μg, 1 μg to 20 μg. g, 1μg~30μg, 1μg~50μg, 1μg~100μg, 1μg~120μg, 1μg~200μg, 2μg~3μg, 2μg~5μg, 2μg~10μg, 2μg~20μg, 2μg~30μg , 2μg~50μg, 2μg~100μg, 2μg~120μg, 2μg~200μg, 3μg~5μg, 3μg~10μg, 3μg~20μg, 3μg~30μg, 3μg~50μg, 3μg~100μg , 3μg~120μg, 3μg~200μg, 5μg~10μg, 5μg~20μg, 5μg~30μg, 5μg~50μg, 5μg~100μg, 5μg~120μg, 5μg~200μg, 10μg~ 20μg, 10μg~30μg, 10μg~50μg, 10μg~100μg, 10μg~120μg, 10μg~200μg, 20μg~30μg, 20μg~50μg, 20μg~100μg, 20μg The composition may comprise between 100 and 120 μg, 20 μg to 200 μg, 30 μg to 50 μg, 30 μg to 100 μg, 30 μg to 120 μg, 30 μg to 200 μg, 50 μg to 100 μg, 50 μg to 120 μg, 50 μg to 200 μg, 100 μg to 120 μg, 100 μg to 200 μg, or 120 μg to 200 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles).In some embodiments, the compositions of the present disclosure may comprise 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles). In some embodiments, the compositions of the present disclosure may comprise at least 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles). In some embodiments, a composition of the present disclosure may contain up to 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles).

[0093]

[0125] In some embodiments, a composition of the present disclosure comprising 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) (e.g., for treating a subject having a disease or condition, such as a respiratory disease, or for preventing the subject from contracting said disease or condition, or for transmitting said disease or condition to a second subject) may comprise between 0.1 μg and 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).In some embodiments, compositions of the present disclosure comprising 20 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles) may be administered in amounts of 0.1 μg to 1 μg, 0.1 μg to 2 μg, 0.1 μg to 3 μg, 0.1 μg to 5 μg, 0.1 μg to 10 μg, 0.1 μg to 20 μg, 0.1 μg to 30 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 120 μg, 0.1 μg to 200 μg, ... μg, 1μg~3μg, 1μg~5μg, 1μg~10μg, 1μg~20μg, 1μg~30μg, 1μg~50μg, 1μg~100μg, 1μg~120μg, 1μg~200μg, 2μg~3μg, 2μg~5μg g, 2μg~10μg, 2μg~20μg, 2μg~30μg, 2μg~50μg, 2μg~100μg, 2μg~120μg, 2μg~200μg, 3μg~5μg, 3μg~10μg, 3μg~20μg, 3μg~30 μg, 3μg~50μg, 3μg~100μg, 3μg~120μg, 3μg~200μg, 5μg~10μg, 5μg~20μg, 5μg~30μg, 5μg~50μg, 5μg~100μg, 5μg~120μg, 5 μg~200μg, 10μg~20μg, 10μg~30μg, 10μg~50μg, 10μg~100μg, 10μg~120μg, 10μg~200μg, 20μg~30μg, 20μg~50μg, 20μg~100

[0043] The therapeutically effective amount of the ... In some embodiments, a composition of the present disclosure comprising 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) may comprise 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist, e.g., cGAMP)).In some embodiments, a composition of the present disclosure comprising 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) may comprise at least 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist, e.g., cGAMP)). In some embodiments, a composition of the present disclosure comprising 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) may comprise up to 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist, e.g., cGAMP)).

[0094]

[0126] In some embodiments, the compositions of the present disclosure may include multiple different antigens. For example, the compositions of the present disclosure may include nucleocapsid protein (N-protein) and S-protein (e.g., coronavirus spike protein). In some embodiments, the compositions of the present disclosure may include multiple antigens of a single class. For example, the compositions of the present disclosure may include monomeric antigens (e.g., monomeric S-protein), trimeric antigens (e.g., trimeric S-protein), chimeric antigens (e.g., chimeric S-protein), and / or mutants of antigens (e.g., mutant S-protein).

[0095]

[0127] In some embodiments, a composition of the present disclosure (e.g., for treating a subject having a disease or condition, such as a respiratory disease, or for preventing a subject from contracting the disease or condition, or for transmitting the disease or condition to a second subject) may contain between 0.1 μg and 200 μg of an antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or a RSV antigen protein). In some embodiments, the compositions of the present disclosure comprise from 0.1 μg to 1 μg, 0.1 μg to 2 μg, 0.1 μg to 3 μg, 0.1 μg to 5 μg, 0.1 μg to 10 μg, 0.1 μg to 20 μg, 0.1 μg to 30 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 120 μg, 0.1 μg to 200 μg, 1 μg to 2 μg, 1 μg to 3 μg, 1 μg to 5 μg, 1 μg to 10 μg, 1 μg to 20 μg, 1 μg to 30μg, 1μg~50μg, 1μg~100μg, 1μg~120μg, 1μg~200μg, 2μg~3μg, 2μg~5μg, 2μg~10μg, 2μg~20μg, 2μg~30μg, 2μg~50μg , 2μg~100μg, 2μg~120μg, 2μg~200μg, 3μg~5μg, 3μg~10μg, 3μg~20μg, 3μg~30μg, 3μg~50μg, 3μg~100μg, 3μg~120μg, 3μg~200μg, 5μg~10μg, 5μg~20μg, 5μg~30μg, 5μg~50μg, 5μg~100μg, 5μg~120μg, 5μg~200μg, 10μg~20μg, 10μg~30μg , 10μg~50μg, 10μg~100μg, 10μg~120μg, 10μg~200μg, 20μg~30μg, 20μg~50μg, 20μg~100μg, 20μg~120μg, 20μg~200μg The vaccine may contain 100 μg, 30 μg to 50 μg, 30 μg to 100 μg, 30 μg to 120 μg, 30 μg to 200 μg, 50 μg to 100 μg, 50 μg to 120 μg, 50 μg to 200 μg, 100 μg to 120 μg, 100 μg to 200 μg, or 120 μg to 200 μg of antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or RSV antigen protein).In some embodiments, the compositions of the present disclosure may comprise 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of an antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or RSV antigen protein). In some embodiments, the compositions of the present disclosure may comprise at least 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of an antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or RSV antigen protein). In some embodiments, the compositions of the present disclosure may contain up to 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or RSV antigen protein). In some embodiments, the compositions of the present disclosure (e.g., for treating a subject with a disease or condition, such as a respiratory disease, or for preventing the subject from contracting the disease or condition, or for transmitting the disease or condition to a second subject) containing 20 μg of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) may contain 0.1 μg to 200 μg of antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or RSV antigen protein).In some embodiments, compositions of the present disclosure comprising 20 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles) may be administered in amounts of 0.1 μg to 1 μg, 0.1 μg to 2 μg, 0.1 μg to 3 μg, 0.1 μg to 5 μg, 0.1 μg to 10 μg, 0.1 μg to 20 μg, 0.1 μg to 30 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 120 μg, 0.1 μg to 200 μg, 1 μg to 2 ... μg~3μg, 1μg~5μg, 1μg~10μg, 1μg~20μg, 1μg~30μg, 1μg~50μg, 1μg~100μg, 1μg~120μg, 1μg~200μg, 2μg~3μg, 2μg~5μg, 2μg~ 10μg, 2μg~20μg, 2μg~30μg, 2μg~50μg, 2μg~100μg, 2μg~120μg, 2μg~200μg, 3μg~5μg, 3μg~10μg, 3μg~20μg, 3μg~30μg, 3μg~ 50μg, 3μg~100μg, 3μg~120μg, 3μg~200μg, 5μg~10μg, 5μg~20μg, 5μg~30μg, 5μg~50μg, 5μg~100μg, 5μg~120μg, 5μg~200μg, 10μg~20μg, 10μg~30μg, 10μg~50μg, 10μg~100μg, 10μg~120μg, 10μg~200μg, 20μg~30μg, 20μg~50μg, 20μg~100μg, 20μg~12 The vaccine may contain 0 μg, 20 μg to 200 μg, 30 μg to 50 μg, 30 μg to 100 μg, 30 μg to 120 μg, 30 μg to 200 μg, 50 μg to 100 μg, 50 μg to 120 μg, 50 μg to 200 μg, 100 μg to 120 μg, 100 μg to 200 μg, or 120 μg to 200 μg of antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or RSV antigen protein).In some embodiments, a composition of the present disclosure comprising 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) may comprise 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or a RSV antigen protein). In some embodiments, a composition of the present disclosure comprising 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) may comprise at least 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or a RSV antigen protein). In some embodiments, a composition of the present disclosure comprising 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) may contain up to 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or a RSV antigen protein).

[0096]

[0128] In some embodiments, a composition of the present disclosure (e.g., for treating a subject having a disease or condition, such as a respiratory disease, or for preventing the subject from contracting said disease or condition, or for transmitting said disease or condition to a second subject) comprising at least 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) and at least 4 μg of an antigen may comprise between 0.1 μg and 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).In some embodiments, a composition of the present disclosure comprising at least 20 μg of particles (e.g., lipid-based particles (e.g., liposomes), e.g., lipid-based nanoparticles) and at least 4 μg of antigen is 0.1 μg to 1 μg, 0.1 μg to 2 μg, 0.1 μg to 3 μg, 0.1 μg to 5 μg, 0.1 μg to 10 μg, 0.1 μg to 20 μg, 0.1 μg to 30 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 120 μg, 0.1 μg to 1 μg, 0.1 μg to 2 μg, 0.1 μg to 30 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 120 μg, 0.1 μg to 200 μg, 0.1 μg to 200 μg, 0.1 μg to 300 μg, 0.1 μg to 50 μg, 0.1 μg to 100 μg, 0.1 μg to 200 μg, 0.1 μg to 300 μg, 0.1 μg to 400 μg, 0.1 μg to 500 μg, 0.1 μg to 100 μg, 0.1 μg to 1200 μg, 0.1 μg to 200 μg, 0.1 μg to 300 μg, 0.1 μg to 400 μg, 0.1 μg to 500 μg, 0.1 μg to 1000 μg, 0.1 μg to 200 μg, 0. .1μg~200μg, 1μg~2μg, 1μg~3μg, 1μg~5μg, 1μg~10μg, 1μg~20μg, 1μg~30μg, 1μg~50μg, 1μg~100μg, 1μg~120μg, 1μg~200μg , 2μg~3μg, 2μg~5μg, 2μg~10μg, 2μg~20μg, 2μg~30μg, 2μg~50μg, 2μg~100μg, 2μg~120μg, 2μg~200μg, 3μg~5μg, 3μg~10μg, 3 μg~20μg, 3μg~30μg, 3μg~50μg, 3μg~100μg, 3μg~120μg, 3μg~200μg, 5μg~10μg, 5μg~20μg, 5μg~30μg, 5μg~50μg, 5μg~100μg g, 5μg~120μg, 5μg~200μg, 10μg~20μg, 10μg~30μg, 10μg~50μg, 10μg~100μg, 10μg~120μg, 10μg~200μg, 20μg~30μg, 20μg~5 In some embodiments, the composition may comprise 0 μg, 20 μg to 100 μg, 20 μg to 120 μg, 20 μg to 200 μg, 30 μg to 50 μg, 30 μg to 100 μg, 30 μg to 120 μg, 30 μg to 200 μg, 50 μg to 100 μg, 50 μg to 120 μg, 50 μg to 200 μg, 100 μg to 120 μg, 100 μg to 200 μg, or 120 μg to 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).In some embodiments, a composition of the present disclosure comprising at least 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) and at least 4 μg of an antigen may comprise 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, a composition of the present disclosure comprising at least 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) and at least 4 μg of an antigen may comprise at least 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, a composition of the present disclosure comprising at least 20 μg of a particle (e.g., a lipid-based particle (e.g., a liposome), e.g., a lipid-based nanoparticle) and at least 4 μg of an antigen may comprise up to 0.1 μg, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 20 μg, 30 μg, 50 μg, 100 μg, 120 μg, or 200 μg of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).

[0097]

[0129] In some embodiments, the dose administered to a subject by a composition or method described herein may comprise 0.1 μg to 20 μg of a composition described herein (e.g., comprising a modulator and a particle, or comprising a modulator, a particle, and an antigen). In some embodiments, the dose administered to a subject by a composition or method described herein may comprise 0.1 μg to 0.5 μg, 0.1 μg to 1 μg, 0.1 μg to 2.5 μg, 0.1 μg to 5 μg, 0.1 μg to 7.5 μg, 0.1 μg to 9.3 μg, 0.1 μg to 10 μg, 0.1 μg to 15 μg, 0.1 μg to 20 μg, 0. 5μg~1μg, 0.5μg~2.5μg, 0.5μg~5μg, 0.5μg~7.5μg, 0.5μg~9.3μg, 0.5μg~10μg, 0.5μg~15μ g, 0.5μg~20μg, 1μg~2.5μg, 1μg~5μg, 1μg~7.5μg, 1μg~9.3μg, 1μg~10μg, 1μg~15μg, 1μg~2 0μg, 2.5μg~5μg, 2.5μg~7.5μg, 2.5μg~9.3μg, 2.5μg~10μg, 2.5μg~15μg, 2.5μg~20μg, 5μg ~7.5μg, 5μg~9.3μg, 5μg~10μg, 5μg~15μg, 5μg~20μg, 7.5μg~9.3μg, 7.5μg~10μg, 7.5μg~1 It may comprise 5 μg, 7.5 μg to 20 μg, 9.3 μg to 10 μg, 9.3 μg to 15 μg, 9.3 μg to 20 μg, 10 μg to 15 μg, 10 μg to 20 μg, or 15 μg to 20 μg of a composition described herein (e.g., including a modulator and a particle, or including a modulator, a particle, and an antigen). In some embodiments, the dose administered to a subject by a composition or method described herein may comprise 0.1 μg, 0.5 μg, 1.0 μg, 1.2 μg, 2.3 μg, 2.5 μg, 4.7 μg, 5.0 μg, 7.5 μg, 9.3 μg, 10 μg, 15 μg, or 20 μg of a composition described herein (e.g., including a modulator and a particle, or including a modulator, a particle, and an antigen).In some embodiments, the dose administered to a subject by the compositions or methods described herein may comprise at least 0.1 μg, 0.5 μg, 1.0 μg, 1.2 μg, 2.3 μg, 2.5 μg, 4.7 μg, 5.0 μg, 7.5 μg, 9.3 μg, 10 μg, 15 μg, or 20 μg of the compositions described herein (e.g., including modulators and particles, or including modulators, particles, and antigens). In some embodiments, the dose administered to a subject by the compositions or methods described herein may comprise at most 0.1 μg, 0.5 μg, 1.0 μg, 1.2 μg, 2.3 μg, 2.5 μg, 4.7 μg, 5.0 μg, 7.5 μg, 9.3 μg, 10 μg, 15 μg, or 20 μg of the compositions described herein (e.g., including modulators and particles, or including modulators, particles, and antigens).

[0098]

[0130] In some embodiments, the compositions disclosed herein can be administered in combination with other therapeutic treatments.In some embodiments, other therapeutic treatments include, but are not limited to, immunotherapy, virus therapy, targeted inhibition, radiation therapy, chemotherapy, or a combination thereof.In some embodiments, the compositions described herein can include and / or be administered together with a treatment regimen (e.g., administered simultaneously or not) that includes an adjuvant (e.g., one or more antibodies, one or more antiviral treatments, one or more vaccines, one or more small molecules, one or more nucleic acids, and / or one or more peptides or proteins, such as interleukins (e.g., IL-21)).

[0099] subject

[0131] The therapeutic composition of the present disclosure can be administered to a subject in need thereof. For example, in some embodiments, the subject is a mammal (e.g., human). In some embodiments, the subject is a livestock animal. For example, the subject can be a dog or a cat. In some embodiments, the 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. In some embodiments, the subject can be a human. In some embodiments, the subject can be susceptible to or suffering from a respiratory infection. In some embodiments, the subject can be susceptible to or suffering from a condition caused by a respiratory virus. For example, the composition or method can be administered to a subject exposed to or infected with a respiratory virus, such as a respiratory RNA virus, such as a coronavirus (e.g., an alpha variant, a delta variant, or an omicron variant), an influenza virus (e.g., influenza A), a respiratory syncytial virus, a metapneumovirus, a parainfluenza virus, or a rhinovirus. In some embodiments, the subject can be susceptible to or suffering from cancer. In some embodiments, subject may be selected for treatment as a result of one or more symptoms of infection by pathogen or disease (e.g., infection by respiratory pathogen or respiratory disease).For example, subject may be selected for treatment based on one or more symptoms such as persistent cough, elevated body temperature (e.g., higher than 100.4 degrees measured by skin on forehead), body chills, painful joints, difficulty in breathing or shortness of breath, fluid accumulation in lungs, fatigue, headache, loss of taste or smell, or positive diagnostic test, e.g., PCR test.In some embodiments, subject may be selected for treatment with the composition disclosed herein based on demographic risk factors, such as obesity, old age (e.g., over 65 years old), immune disorder, or pregnancy.In some embodiments, a subject may be selected for treatment based on risk of infection with a pathogen or suffering from a disease (e.g., infection with a respiratory pathogen or suffering from a respiratory disease), for example, if the subject has a job that involves close interaction with customers, frequent interaction with at-risk populations, handling biological samples, or close contact with potentially infected individuals.

[0100] Treatment or prevention of disease

[0132] Pathogens and cancer have raised significant health and economic concerns. For example, viral infection caused by severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2) has led to the COVID-19 pandemic, which is currently the world's most urgent health and economic crisis. Furthermore, cancers, such as lung cancer, have a high mortality rate.

[0101]

[0133] In addition, therapeutic options to effectively treat and prevent cancer and infections caused by pathogens are very limited. For example, there are currently no drugs available to treat or prevent viral infections caused by SARS-CoV-2.

[0102]

[0134] In addition, many vaccines aimed at preventing infections caused by pathogens are intramuscular vaccines designed to elicit systemic immunity without conferring natural immunity such as mucosal immunity or broad T cell immunity. Such intramuscular vaccines are limited because the nasal compartment is the first barrier that pathogens need to breach before dissemination to the lungs.

[0103]

[0135] Furthermore, there is a high level of unpredictability in the efficacy of vaccine formulations: only about one-third of vaccines that progress to Phase I clinical trials achieve final FDA approval. This success rate implies that even with existing technologies to stimulate the immune system, developing effective immunity against each pathogen requires the design and optimization of the correct antigens and adjuvants.

[0104]

[0136] Achieving FDA approval does not indicate that a vaccine is effective. Vaccine development is a lengthy process that faces numerous challenges, even in the context of the COVID19 pandemic.

[0105]

[0137] Even though the majority of platforms target the spike protein as an antigen, optimizing the antigen design is desirable to ensure vaccine efficacy. The difficulty in selecting an appropriate vaccine design is particularly strong for HIV-1, Hepatitis C and malaria, where vaccines remain difficult to obtain despite decades of efforts and multiple candidates.

[0106]

[0138] Moreover, for respiratory pathogens, the nasal compartment is the first barrier that pathogens need to breach before dissemination to the lungs. However, current intramuscular vaccines are designed to elicit systemic immunity without conferring natural immunity, such as mucosal immunity.

[0107]

[0139] Therefore, there is an urgent need for safe and durable therapeutic compositions for treating and preventing various diseases, such as infections caused by pathogens and various types of cancer. For example, there is an urgent need for safe and durable therapeutic compositions for treating or preventing pandemics caused by respiratory pathogens (e.g., viral infections caused by coronaviruses, such as SARS-CoV-2). Many embodiments of the present disclosure address the above needs.

[0108]

[0140] The therapeutic compositions and methods of the present disclosure can be utilized to treat or prevent a disease or condition (e.g., a disease or condition associated with a pathogen). For example, in some embodiments, the disease may be an infection caused by a pathogen. In some embodiments, the disease may be a respiratory disease. In some embodiments, the pathogen may be a respiratory pathogen. In some embodiments, the respiratory pathogen may be a virus. In some embodiments, the virus may be an influenza virus, an influenza A virus (e.g., California / 04 / 2009 (H1N1) or Hong Kong / 2369 / 2009 (H1N1)), an influenza B virus, a parainfluenza virus, an adenovirus, an enterovirus, a coronavirus, a respiratory syncytial virus, a rhinovirus, a DNA virus, an RNA virus, a mutant strain thereof, or a combination thereof.

[0109]

[0141] In some embodiments, the pathogen is a coronavirus. In some embodiments, the coronavirus includes, but is not limited to, 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-related coronavirus 2 (SARS-CoV-2), a mutant strain of SARS-CoV-2 (e.g., 20A.EU1, or spike mutant strain D614G), or a combination thereof. In some embodiments, the coronavirus is a SARS-CoV-2 virus, an alpha variant thereof (e.g., B.1.1.7), a delta variant thereof (e.g., B.1.617.2), an omicron variant thereof (e.g., B.1.1.529), or a combination thereof.

[0110]

[0142] In some embodiments, the pathogen is an influenza virus, hi some embodiments, the influenza virus includes an oseltamivir-susceptible strain, a treatment-resistant strain, or a combination thereof.

[0111]

[0143] In some embodiments of the compositions and methods, a subject (e.g., a mammalian subject, e.g., a human, mouse, hamster, dog, cat, or non-human primate) may be prophylactically treated to protect against viral infection (e.g., SARS-CoV-2 coronavirus) by intranasal delivery of a composition described herein (e.g., comprising a modulator, e.g., a STING agonist and a lipid-based nanoparticle) and an antigen (e.g., a spike protein associated with, embedded in, or coupled to the outer surface of a lipid-based nanoparticle) as part of a single dose or multiple dose regimen. In some embodiments, treatment with the composition can elicit an immune response in the subject in one or more body compartments, such as the blood, spleen, lungs, and / or nasal compartment. In some embodiments, the compositions and methods described herein can elicit an increase in anti-antigen IgG (e.g., anti-spike protein IgG) and / or anti-antigen IgA (e.g., anti-spike protein IgA) detectable in the subject's lung tissue (e.g., BALF) or blood (e.g., serum). In some embodiments, the compositions and methods described herein can increase the number of IgA-secreting B cells and / or the number of antigen-specific T cells (e.g., spike protein-specific T cells), for example, in the spleen.In some embodiments, the increase in antigen-specific T cells can be detected in the lung after administering the compositions described herein to a subject.In some embodiments, the increased anti-antigen IgA and / or the increased presentation and / or activity of germinal center (GC) B cells or T follicular helper (Tfh) cells can be detected in the nasal compartment after administering the compositions described herein to a subject.

[0112]

[0144] In some embodiments, the disease to be treated or prevented in the subject is cancer.In some embodiments, cancer can be 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 combination thereof.In some embodiments, cancer can be lung cancer. EXAMPLES

[0113] Example 1: Preparation, characterization, and stability of NanoSTING

[0145] This example describes the preparation, characterization, and stability of NanoSTING, a liposomal formulation containing the natural immunotransmitters, cyclic GMP-AMP, and cGAMP (Figure 3A).

[0114]

[0146] The nanoparticles enhanced stability and delivery to alveolar macrophages, promoting responses in the upper airways and lungs. Dynamic light scattering (DLS) analysis revealed that the mean particle size of NanoSTING was 98 nm with a polydispersity index of 0.25 (Figure 3E). The zeta potential of NanoSTING was -40 mV (Figure 3F). Administration of NanoSTING was able to induce an interferon response by using THP-1 monocytic cells modified to conditionally secrete luciferase downstream of an interferon regulatory factor (IRF)-responsive promoter (Figure 3B). THP-1 cells with NanoSTING were administered at doses ranging from 2.5 to 10.0 μg, and kinetic measurements were performed for 24 h by measuring luciferase activity in the supernatant. Low levels of luciferase activity were observed at 6 h, and secretion was maximal with NanoSTING at 24 h, 5, and 10 μg (Figure 4). The stability of NanoSTING nanoparticles over time was measured at two different temperatures, 25°C and 37°C. DLS was then used to track the instability, and zeta potential was used to assess changes in nanoparticle charge. The hydrodynamic diameter of NanoSTING remained virtually unchanged over 30 days at 25°C (Figure 3C), while there was a slight increase in hydrodynamic diameter after 2 weeks at 37°C (average: 114 nm at 25°C and 154 nm at 37°C) (Figure 3D). There was no significant change in zeta potential at both temperatures (-45 mV at 25°C and 37°C). These results demonstrated that NanoSTING was immunologically active and that the nanoparticles remained stable even without refrigeration.

[0115] Example 2: Sustained interferon-beta (IFNβ) secretion in the nasal compartment following NanoSTING delivery

[0147] This example demonstrates the ability of NanoSTING to deliver cGAMP and induce the secretion of effector cytokines in the nasal compartment of mice.

[0116]

[0148] Despite cGAMP being a potent natural activator of STING and thus acting as an immunotransmitter, its clinical utility is hampered by lack of cell permeability and rapid degradation by plasma ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), resulting in an in vivo half-life of only approximately 35 min. Various amounts of NanoSTING (10-40 μg) were delivered intranasally to groups of BALB / c mice. Nasal turbinates and lungs were then harvested and cGAMP levels were assayed using a quantitative ELISA (Figure 5A and 5B). A dose-dependent increase in the concentration of GAMP in nasal turbinates was observed; at low doses (10 μg), cGAMP concentrations were quantified for up to 12 h and returned to baseline by 24 h, whereas at higher doses (20-40 μg), cGAMP concentrations were quantified for 24 h and returned to baseline by 48 h (Figure 5B). In the lung, cGAMP was only detectable at higher concentrations (20 and 40 μg) (Figure 5C). In the same animals, serum profiling revealed that cGAMP was not detectable in the circulation at any time point, even at the highest dose (40 μg) (Figures 7D, 7E, and 7F). These data confirmed that NanoSTING can deliver cGAMP to cells of the nasal passages in a concentration- and time-dependent manner, even in the absence of systemic exposure.

[0117]

[0149] The biological relevance of NanoSTING's ability to deliver cGAMP and activate the STING pathway was assessed using a panel of 10 genes to comprehensively measure immune responses. The panel consisted of effector cytokines, C-X-C motif chemokine ligand 10 (Cxcl10) and interferon beta (Ifnb); interferon-stimulated genes (ISGs), such as Isg15, interferon regulatory factor 7 (Irf7), myxovirus resistance proteins 1 and 2 (Mx1 and Mx2), and interferon-inducible protein with tetratricopeptide repeats 1 (Ifit1); as well as nonspecific pro-inflammatory cytokines (Il6, Il10, and Tnf). BALB / c mice were given various doses of intranasal NanoSTING and quantitative qRT-PCR was performed on nasal turbinates (6–48 h) (Figure 5A). The effector cytokines Cxcl10 and Ifnb showed the greatest induction (7,000-20,000-fold induction) and remained elevated at 48 h (Figures 5D and 5E). Five ISGs demonstrated strong induction from 6 h (300-1,000-fold) to 24 h, followed by attenuation from 24 to 48 h (Figures 5F, 5G, 5H, 5-I, 5J, and 5N). The NanoSTING inflammatory response was linked to the IFN pathway, as the pro-inflammatory cytokine Il6 showed a brief induction at 6 h (5,000-fold), which attenuated significantly by 24 h and was at baseline at 48 h (Figure 5K). Furthermore, Tnf and Il10 showed only weak induction (15-60-fold) (Figures 5L and 5M). These results demonstrate that NanoSTING elicits a rapid and sustained inflammatory response that generates both effector cytokines and ISGs, while activating very minimal nonspecific pro-inflammatory cytokines.

[0118]

[0150] As qRT-PCR data suggested a strong induction of the effector cytokines, Ifnb and Cxcl10, we quantified IFN-β and CXCL10 protein concentrations in nasal turbinates. Consistent with the transcriptional data, quantitative ELISA confirmed that both IFNβ and CXCL10 were detectable in nasal turbinates and lungs for up to 24 hours (Figures 7A, 7B, and 7C). IFN-β and CXL10 were not observed in tested sera from the same animals (Figures 7D, 7E, and 7F), demonstrating that stimulation of innate immunity by nasal NanoSTING is local and not systemic.

[0119] Example 3: Identification of a robust IFN-I signature in hamster lungs following NanoSTING administration

[0151] This example describes experimental confirmation of induction of interferon-dependent and interferon-independent pathways following intranasal administration of NanoSTING in hamsters.

[0120]

[0152] Hamsters are a well-characterized model for SARS-CoV-2 challenge that mimics severe disease in humans; animals demonstrate an easily quantifiable clinical disease characterized by rapid weight loss, very high viral loads in the lungs, and extensive pulmonary pathology. In addition, unlike the K18-hACE2 transgenic model, hamsters recover from the disease (as do most humans), thus offering the opportunity to study the effects of treatments in both the lungs (disease) and nasal passages (transmission).

[0121]

[0153] Biodistribution was investigated by varying the transport volume of intranasally delivered NanoSTING. Intranasal administration of Evans Blue dye at low and high volumes (40 μl and 120 μl) stained the nasal turbinates, lungs, and stomach in hamsters (Figures 8C, 8D, and 8E). However, at both volumes, significant amounts of dye were delivered to the nasal turbinates and lungs (intended target organs) (Figure 8C), and the normalized ratios of distribution to these tissues were independent of administration volume (Figures 8D and 8E). These results suggested that in the hamster model, biodistribution following intranasal delivery of liquid formulations was not affected by inoculum volume.

[0122]

[0154] To evaluate the pulmonary effects of intranasal NanoSTING, groups of hamsters received a daily dose of either NanoSTING (60 μg) or PBS (control) for four consecutive days. Both groups of animals showed no differences in clinical signs such as temperature or body weight between the two groups (Figures 8A and 8B). On day 5, lungs from hamsters were isolated for unbiased whole-transcriptome profiling using RNA-sequencing (RNA-seq). A total of 2,922 differentially expressed genes (DEGs) were identified between the two groups with a false positive rate (FDRq value <0.25) (Figure 10A). Type I IFN responses were induced in NanoSTING-treated lungs, including canonical ISGs such as Mx1, Isg15, Uba7, Ifit2, Ifit3, Ifit35, Irf7, Adar, and Oas2 (Figure 10B). Effector cytokines, Cxcl9-11 and Ifnb, were also induced in treated hamsters (Figure 10C), indicating robust induction of direct antiviral proteins such as Ddx60 and Gadd45g (Figure 11A). Next, gene-set enrichment analysis was performed to compare pathways that were differentially induced upon treatment with NanoSTING. Changes in these populations were interrogated against the Molecular Signatures Database (Hallmark, C2, and C7 curated gene sets). Distinct clusters of pathways for both type I and type III interferons were observed in the lungs of NanoSTING-treated animals. Specificity of the response was confirmed by qRT-PCR analysis by quantifying Mx1-2, Isg15, Irf7, Cxcl11, Ifnb, Il6, and Il10 (Figure 9A). Since the gene signature of the interferon-independent activity of STING is known, we performed gene set enrichment analysis (GSEA) to identify NanoSTING-activated interferon-independent pathways and associated proteins (Figures 11A and 11B).

[0123]

[0155] Collectively, these results demonstrate that cGAMP-mediated activation of STING by NanoSTING efficiently activates both interferon-dependent and interferon-independent antiviral pathways in the lung.

[0124] Example 4: Predicting the effect of early treatment NanoSTING on viral replication using quantitative modeling

[0156] This example describes the results of modeling experiments performed to analyze the levels of SARS-CoV-2 replication following NanoSTING administration.

[0125]

[0157] In vivo mechanistic experiments demonstrated that NanoSTING induced a broad antiviral program by engaging the innate immune system. Using a mathematical modeling approach combined with human viral load data, we identified a treatment window and quantified the relative amount of type I IFN (or related pathways) elicited by NanoSTING required for therapeutic benefit. To simplify the modeling framework, we performed tests under the hypothesis that in vivo cGAMP functions solely to stimulate an interferon response. With this hypothesis, we modeled the range of relative interferon ratios (RIR, 0-1) required to elicit via NanoSTING compared to the population-level peak interferon response observed during SARS-CoV-2 infection (Figures 11C and 11D) and investigated the impact on viral clearance. Based on this model, an RIR of just 0.27 (27% of natural infection) is deemed sufficient to achieve a 50% reduction in viral load (based on area under the curve, AUC), and an RIR value of at least 0.67 is expected to achieve a 100% reduction in viral load (Figure 11E). Next, the window for treatment initiation was modeled, revealing that intervention is most effective when initiated within 2 days after infection (Figure 11F). In contrast, when treatment is initiated after the peak of viral replication, even an RIR of 1 does not readily result in improvements in outcome (Figures 11F, 11G, 11H, 11-I, 11J, and 11K).

[0126]

[0158] Collectively, results from these quantitative modeling predicted that (a) a single dose of NanoSTING was required to elicit even modest amounts of IFN, which may have been within reach given that our data support extensive IFNβ induction (Figures 5E, 5M, and 11B) and that natural infection with viruses such as SARS-CoV-2 and influenza A are known to suppress interferon production, and (b) the optimal treatment window was both as a prophylaxis and shortly after infection.

[0127] Example 5: Treatment with NanoSTING protects against SARS-CoV-2 delta strains and induces protection against SARS-CoV-2 reinfection

[0159] This example describes the level of protection against the SARS-CoV-2 Delta (B.1.617.2) strain after a single dose of NanoSTING in hamsters.

[0128]

[0160] The Delta strain (B.1.617.2) was chosen because it causes both upper and lower respiratory tract disease, and thus increased disease severity compared to previous variants (Wuhan and Beta strains). Groups of 12 animals were treated with a single intranasal dose of 120 μg NanoSTING, followed 24 hours later by immunization with 10 μg of NanoSTING via the intranasal route. 4Animals in the placebo-treated (PBS) group exhibited severe weight loss, with a mean peak weight loss of 8.3%. In contrast, the majority of animals treated with NanoSTING were protected from weight loss (mean peak weight loss of only 2.0%) (Figure 12C). This small loss in body weight was similar to the results obtained with adenovirus-vectored vaccines challenged with either the Wuhan or Beta strains. Half of the animals were sacrificed on day 2 (peak of viral replication) to quantify infectious viral load. Even with the highly infectious Delta strain, NanoSTING reduced infectious viral load in the lungs 2 days after infection by 300-fold compared to placebo-treated animals (Figure 12D). This reduction in viral load in the lungs was strictly associated with the prevention of weight loss in these animals. Quantification of viral load in the nasal compartment revealed that NanoSTING treatment reduced infectious viral load in the nasal compartment 2 days after infection by 1,000-fold compared to placebo-treated animals (Figure 12D). Reduction of viral replication in the nasal compartment models the nature of human transmission and confirmed that treatment with NanoSTING reduced the likelihood of transmission. To map the duration of efficacy of prophylactic NanoSTING treatment, hamsters were then administered a single intranasal dose of NanoSTING, followed by 10-fold administration of NanoSTING 72 hours later. 4 Mice were challenged with TCID50 of the delta strain (Figure 13A). NanoSTING showed modest protection from weight loss and a significant reduction in infectious viral load, even when administered 72 hours prior to challenge (Figures 13A, 13B, and 13C). To test the post-viral effect of NanoSTING, intranasal NanoSTING was delivered 6 hours after exposure to delta virus (Figure 14A). There was a 340-fold and 13-fold reduction in infectious virus in the nasal passages and lungs, respectively (Figures 14B and 14C).

[0129]

[0161] These results demonstrated that a single-dose treatment with NanoSTING effectively minimized clinical symptoms, protected the lungs, and reduced infectious virus in the nasal passages.

[0162] One advantage of activating the innate immune system to clear a viral infection is that this process mimics natural infection while minimizing the risk of clinical symptoms. To test whether adaptive immunity is activated, hamsters were intranasally administered NanoSTING and 24 hours later, the animals were then cultured for 10 min. 4 NanoSTING-treated animals were challenged with TCID50 of the delta mutant strain (Figure 12A). Administration of NanoSTING reduced weight loss during the primary challenge. Unlike placebo-treated animals, NanoSTING-treated animals regained weight, and by day 4, their weights were not significantly different from those of unchallenged animals. The reduced weight loss was due to a reduction in infectious viral load in lung and nasal tissues (Figures 12C, 12D, and 12E). On day 28, animals were rechallenged with the delta mutant strain. NanoSTING-treated animals were completely protected from weight loss during the secondary challenge, and the weights of the animals were identical to those of previously unchallenged animals (Figure 12F).

[0130]

[0163] These results established that a single intranasal treatment with NanoSTING activated innate antiviral programs and protected against clinical disease during primary infection while promoting adaptive immunity that provided durable protection from reinfection.

[0131] Example 6: Effect of NanoSTING treatment on IFN-evasive SARS-CoV-2 alpha mutant (B.1.1.7)

[0164] This example explores the effect of different doses of NanoSTING and different doses of treatment on SARS-CoV-2 alpha strains.

[0132]

[0165] The alpha (B.1.1.7) mutant provides an interesting model for testing the efficacy of NanoSTING, as it is known to be resistant to IFN-1 signaling in vitro. Hamsters were pretreated with two intranasal doses of NanoSTING (30 μg and 120 μg) and 24 hours later, 10 4The hamsters were challenged with TCID50 of the alpha mutant strain of NanoSTING (Figure 16A). Treatment with either dose of NanoSTING protected the hamsters from severe weight loss (Figure 16B). A combined scoring rubric (ranging from 1 to 12) was used to account for both pathology and disease to analyze lung tissue at day 6 after viral challenge. Animals treated with NanoSTING had significantly reduced total pathology scores and minimal evidence of inflammatory cell infiltration or alveolar damage (Figures 17A and 17B). In addition, we quantified the viral load in the lung and nasal compartments and found a significant reduction in viral load in both compartments, even at day 2 (Figures 17C and 17D).

[0133]

[0166] Thus, these results established that treatment with nasal NanoSTING reduced SARS-CoV-2 replication in vivo by orders of magnitude and conferred protection against IFN-I-evasive strains of SARS-CoV-2.

[0134] Example 7: Assessing transmission of SARS-CoV-2 Omicron mutant strains following NanoSTING treatment

[0167] This example describes experimental confirmation that treatment with NanoSTING can block transmission and prevent transmission of the more infectious SARS-CoV-2 omicron variant.

[0135]

[0168] The Omicron variant (B.1.1.529) has the highest infectiousness of any known strain of SARS-CoV-2. The use of the Omicron variant sets the highest standard for NanoSTING in terms of preventing the spread of the virus. To evaluate whether prophylactic treatment of infected hamsters would prevent transmission to contact hamsters and whether treatment of contact hamsters after exposure would reduce viral replication, experiments were performed with three groups of 16 hamsters. In each group, eight hamsters were infected intranasally with 104 TCID50 of the SARS-CoV-2 Omicron variant, and one day after infection, each hamster was paired with a sentinel hamster that was housed with them (Figure 22A). None of the infected animals showed weight loss (Figure 22D). Sentinel hamsters were either (a) housed with index hamsters treated with NanoSTING (120 μg) (group 2) or (b) housed with infected but untreated hamsters and then treated with NanoSTING (group 3). As with other strains of SARS-CoV-2 tested, pretreatment of infected hamsters with NanoSTING blocked transmission in nearly all animals (7 of 8 treated animals were virus-free and 1 of 8 untreated animals was virus-free). Importantly, post-exposure treatment of sentinel hamsters was also effective in preventing infection (6 of 8 treated animals were virus-free), with all animals demonstrating a reduction in viral load (Figures 22B and 22C).

[0136]

[0169] These results demonstrate that NanoSTING is highly effective in blocking transmission of even the highly infectious Omicron strain. Example 8: Effect of NanoSTING treatment on protection from influenza similar to oseltamivir

[0170] This example reviews numerous experiments confirming that administration of NanoSTING is an effective treatment against influenza virus, as measured by weight loss, survival, and viral titers.

[0137]

[0171] Influenza viruses have evolved multiple mechanisms to reduce the host's innate immunity, including attenuation of the interferon response by the NS1 protein. One of the first-line treatment options for influenza involves post-exposure prophylaxis using oseltamivir, which inhibits the influenza neuraminidase protein. Therefore, oseltamivir (Tamiflu) may serve as a benchmark for evaluating the therapeutic efficacy of NanoSTING.

[0138]

[0172] Groups of 10 mice were treated with a single intranasal dose of NanoSTING and challenged 24 hours later with 4×LD50 (lethal dose 50) of a susceptible strain of influenza A / California / 04 / 2009 (H1N1) (FIG. 21A). Animals were evaluated for 14 days, with weight loss and survival used as primary endpoints. One group of mice was treated twice daily for 5 days with a clinically relevant dose of oseltamivir (30 mg / kg / day) as a positive control. Significant weight loss began in untreated animals on day 3 (peak of viral infection), and only one animal survived on day 11 with a 23% weight loss (FIG. 21B). On day 3, mice treated with NanoSTING did not experience significant weight loss, which was similar to unchallenged animals and a marked improvement over animals treated with oseltamivir (FIG. 21B). The effect of a single dose of NanoSTING (40 μg) diminished after day 3. However, even taking this reduction into account, the mean peak weight loss and the kinetics of weight loss were not significantly different from animals treated with oseltamivir (FIGS. 21A and 21B). Taken together, these results illustrated that NanoSTING protected animals from influenza comparably to clinically relevant doses of oseltamivir.

[0139]

[0173] However, even taking this reduction into account, the mean peak weight loss, kinetics of weight loss, and survival rate in treated animals were not significantly different from animals treated with oseltamivir (Figure 18B). Animals were pretreated with oseltamivir (30 mg / kg / day) or NanoSTING (40 μg) and then challenged with 4×LD50 (lethal dose 50) of a susceptible strain of influenza A / California / 04 / 2009 (H1N1) (Figure 18A). NanoSTING protected the animals, as evidenced by mean peak weight losses of −14%, −27%, and −32% in NanoSTING, placebo, and oseltamivir-treated animals, respectively (Figure 18B).

[0174] A single amino acid mutation (His275Tyr) in neuraminidase resulted in oseltamivir-resistant influenza virus in humans. Because NanoSTING relies on the host's innate immune response, experiments were performed to evaluate its efficacy against oseltamivir-resistant influenza A in mice. Groups of 10 mice were treated with a single intranasal dose of NanoSTING (40 μg) and challenged 24 hours later with 4×LD50 (lethal dose 50) of a resistant strain of influenza A (Figure 19A). Animals were evaluated for 14 days, with weight loss and survival as primary endpoints. One group of mice was treated twice daily for 5 days with a clinically relevant dose of oseltamivir (30 mg / kg / day) as a positive control. Mice treated with oseltamivir showed a mean peak weight loss of -32% compared to -8.2% in NanoSTING-treated animals (Figure 19B). Survival data for animals treated with NanoSTING was significantly different from animals treated with oseltamivir (FIG. 19C). In summary, NanoSTING demonstrated protective efficacy compared to oseltamivir, as evidenced by reduced weight loss and enhanced survival.

[0140]

[0175] Next, an experiment was designed to test the impact of NanoSTING treatment on the viral load in lung tissue. Groups of 10 mice were treated with a single intranasal dose of NanoSTING (40 μg) and challenged 24 hours later with 4×LD50 (lethal dose 50) of a resistant strain of influenza A (FIG. 20A). Animals were evaluated for 7 days for weight change and viral titer, which were used as primary endpoints. In this experiment, oseltamivir treatment had no significant effect and 90% of the mice died from disease. Single-dose treatment with NanoSTING protected animals from weight loss (mean peak weight loss at day 7 was 5.2% vs. 32% for placebo) (FIG. 20B). Seven days after virus challenge, there was a 500-fold reduction in infectious particles in the lungs compared to the placebo-treated group, illustrating the ability of NanoSTING to help prevent disease and death (FIG. 20C).

[0141]

[0176] Taken together, these results illustrated that NanoSTING treatment was effective against oseltamivir-resistant strains of influenza A. These experiments confirmed that NanoSTING functions as a broad-spectrum antiviral agent against influenza, including therapy-resistant strains.

[0142] Example 9: Preparation of STING-loaded liposomes and vaccine formulations

[0177] 2'-3" cyclic guanosine monophosphate adenosine monophosphate (cGAMP) was purchased from Chemietek (Indianapolis, IN). 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene-glycol)-2000] (DPPEPEG2000) were obtained from Avanti Polar Lipids (Alabaster, AL). We obtained cholesterol from Sigma Aldrich (St. Louis, MO).

[0143]

[0178] The liposomes were composed of DPPC, DPPG, cholesterol (Chol), and DPPE-PEG2000 in a molar ratio of 10:1:1:1. To prepare the liposomes, we mixed the lipids in CHCl3 and CH3OH, and we used a vacuum rotary evaporator at 45°C for approximately 80 minutes to evaporate the solution. We dried the resulting lipid thin film until all the organic solvent had evaporated. We then hydrated the lipid film by adding pre-warmed cGAMP solution (0.3 mg / ml in PBS buffer at pH 7.4). We mixed the hydrated lipids at an elevated temperature of 65°C for an additional 30 minutes, and then subjected it to a freeze-thaw cycle. We then sonicated the mixture for 60 minutes using a Brandson ultrasonicator (40 kHz). Next, we removed free untrapped cGAMP by Amicon ultrafiltration unit (MW cutoff 10 kDa). Then, we washed the cGAMP-liposomes three times using PBS buffer. We measured the cGAMP concentration in the filtrate against a standard curve of cGAMP at 260 nm by Take3 microabsorbance analyzer in Cytation 5 (BioTek). We calculated the final concentration and encapsulation efficiency of cGAMP encapsulated in the liposomes by subtracting the concentration of free drug in the filtrate. We purchased SARS-CoV-2 (2019-nCoV) nucleocapsid recombinant protein (VA, USA, #NR-53797) from BEI Resources and tested it in combination with STING-liposome suspension at two different doses (10 μg and 20 μg). We kept the vaccine formulation at room temperature to allow the proteins to adsorb onto the liposomes. We stored the formulated vaccine at 4° C. and used it for up to 2 months. The mean particle size, polydispersity index, and zeta potential were characterized by a Litesizer 500 (Anton Paar) at room temperature.

[0144]

[0179] The THP-1 dual cell line (Invivogen) was cultured at 37° C. and 5% CO2 in a humidified incubator and grown in RPMI / 10% FCS (Corning, NY, USA). In addition, we supplemented the THP-1 dual cell line with the respective selection agents (100 μg / ml Zeocin + 10 μg / ml Blasticidin) and the corresponding selection cytostatic agent from Invivogen.

[0145] Example 10: Preparation of STING-loaded liposomes and vaccine formulations

[0180] Cell stimulation experiments with luciferase reporter enzyme detection

[0181] We performed cell stimulation experiments using the manufacturer's instructions (Invivogen, CA, USA). First, we seeded cells in 96-well plates at 1×105 cells / well in 180 μl growth medium. Next, we performed serial dilutions of NanoSTING in growth medium. Then we incubated the cells at 37° C. for 24 hours. For detection of IRF activity, we collected 10 μl of culture supernatant per well at 6, 12, and 24 hours time points and transferred to a white (opaque) 96-well plate. We then read the plates on a Cytation 7 (Cytation 7, Bio-Tek Instruments, Inc.) after adding 50 μl of QUANTI-Luc™ (Invivogen) substrate solution per well, followed by instantaneous luminescence measurements, which were obtained as relative light units (RLU).

[0146] Example 11: Formulations for testing cytotoxicity

[0182] The cytotoxic activity of the NanoSTING compositions was evaluated by MTT assay. First, THP1-dual cells were incubated with NanoSTING at concentrations ranging from 46 μg / ml to 5.75 μg / ml for a period of 24 h. The cells were then treated with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO). After 4 h, all the medium containing the MTT solution (5 mg / ml) was aspirated from the wells. The remaining formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm using a 96-well microplate reader (Cytation 7, Biotek Instruments, Inc.). The data in Figures 27A and 27B are expressed as percentage of viability compared to untreated cells (considered as negative control - 100% viability) and cells treated with Triton X-100 (considered as positive control - 100% cell death).

[0147] Example 12: DNA binding assay

[0183] DNA binding studies were performed to check the suitability of the assay to detect DNA aggregation, and we used branched PEI as a positive control (Sigma Chemical Co., St. Louis, MO, #408727). DiYO-1 (AAT Biorequest #17579) and plasmid (pMB57.6)-DNA complexes were mixed in equal amounts of DNA and DiYO-1 (in 20 mM HEPES, 100 mM NaCl, pH=7.4) to achieve final concentrations of 400 nM and 8 nM, respectively. The solution was left at room temperature for 5 hours before use. PEI was then added to the DNA-DiYO-1 solution at different concentrations (R=0, 1, 2, 5, where R is the molar ratio of PEI nitrogen to DNA phosphate), mixed by vortexing for 1 min, and left to equilibrate for 2 hours. The fluorescence intensity of the solution was measured at excitation and emission wavelengths of 470 nm and 510 nm, respectively. The same procedure was performed with SARS-CoV2 N protein instead of PEI, which was added to the DNA-DiYO-1 solution at concentrations of 0.1 and 0.5 μM.

[0148] Example 13: Mice and Immunization

[0184] Female 7-9 week old BALB / c mice from Charles River Laboratories. Prior to compound administration, mice were anesthetized by intraperitoneal injection of ketamine and xylazine. Animals were administered compositions with one of two different concentrations of nucleocapsid protein (10 μg and 20 μg) and 20 μg of liposome-STING adjuvant.

[0149] Example 14: Weight monitoring and sample collection

[0185] After administration of the NanoSTING composition or control treatment, the body weight of the animals was monitored every 7 days for 4 weeks. For detection of humoral immune responses, serum was collected on days 7, 14, 21, and 27 after administration. Blood was kept at room temperature (RT) for 10 minutes to facilitate clotting, and then centrifuged at 2000g for 5 minutes. Serum was collected and stored at -80°C before being used for ELISA. Bronchoalveolar lavage fluid (BALF), lungs, and spleens were collected on day 27 after intranasal administration. For long-term storage, serum and other biological fluids (including protease inhibitors) were kept at -80°C. After dissociation, splenocytes and lung lymphocytes were frozen in FBS+10% DMSO and stored in the vapor phase of liquid nitrogen until further use.

[0150] Example 15: ELISA Assay

[0186] Anti-N protein antibody titers in serum or other biological fluids were determined using ELISA. Briefly, 1 μg / ml of N protein (Sino Biological, PA, USA) was coated onto ELISA plates (Corning, NY, USA) in PBS overnight at 4°C and 2 h at 37°C. Plates were then blocked with PBS + 1% BSA (Fisher Scientific, PA, USA) + 0.1% Tween 20™ (Sigma-Aldrich, MD, USA) for 2 h at room temperature. After washing, samples were added at different dilutions. Captured antibodies were detected by HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1:5,000; PA, USA), anti-mouse IgA (Bethyl Laboratories, 1:10,000; TX, USA), and detection antibody against mouse IgA (1:250) from a Mouse Total IgA ELISA kit from Invitrogen (CA, USA). A positive control (anti-NIgG) was obtained from Abeomics (CA, USA).

[0151] Example 16: Processing of spleens and lungs for ELISPOT and flow cytometry

[0187] For the isolation of pulmonary lymphocytes, the pulmonary vasculature was perfused by injecting 5 ml of 1 mM EDTA in Ca2+Mg2+-free PBS into the right ventricle. Each lung was cut into 100–300 mm sections using a scalpel. 2 The minced tissue was cut into 100 mm pieces. The minced tissue was transferred to a tube containing 5 ml of digestion buffer containing collagenase D (2 mg / ml, Roche #11088858001) and DNase (0.125 mg / ml, Sigma #DN25) in 5 ml of RPMI and left for 1 h, followed by 30 min at 37 °C in a water bath by vortex mixing every 10 min. The remaining intact tissue was disrupted by passing through a 21-gauge needle (6-8 times). The sample was incubated at room temperature for 90 min and the reaction was stopped by adding 500 μl of ice-cold stop buffer (1× PBS, 0.1 M EDTA). Tissue debris and dead cells were removed through a 40 μm disposable cell strainer (Falcon), and cells were collected after centrifugation at 300 g. The cell pellet was resuspended in 3 ml of ACK lysis buffer (Invitrogen) and incubated at room temperature for 3 min to lyse red blood cells, followed by centrifugation at 300 g. The supernatant was discarded and the cell pellet was resuspended in 5 ml of complete RPMI medium (Corning, NY, USA). The spleen in RPMI medium was then homogenized through a 40 μm cell strainer using the stiff end of a syringe plunger. The splenocytes were then incubated in 3 ml of ACK lysis buffer at room temperature for 3 min to remove red blood cells (RBCs) and then passed through a 40 μm strainer to obtain a single cell suspension. Lung lymphocytes and splenocytes were counted by trypan blue exclusion method.

[0152] Example 17: ELISPOT Assay

[0188] IFN-γ and IL-4 ELISpot assays were performed using the Mouse IFN-γ ELISPOT Basic Kit (ALP) and Mouse IL-4 ELISPOT Basic Kit according to the manufacturer's instructions (Mabtech, VA, USA). For cell activation control, cultures were treated with 10 ng / ml phorbol 12-myristate 13-acetate PMA (Sigma, St. Louis, MI, USA) and 1 μg / ml ionomycin (Sigma, St. Louis, MI, USA). Complete medium (RPMI supplemented with 10% FBS) was used as a negative control. Splenocytes and lung lymphocytes (3 × 10 5) were stimulated in vitro for 16-18 hours at 37°C with a pool of peptides consisting of 15-mer sequences with 11 amino acid overlaps covering the complete sequence of the nucleocapsid phosphoprotein ("N") of SARS-coronavirus 2 at a concentration of 1.5 μg / ml / peptide (Miltenyi Biotec; 130-126-699, Germany) in pre-coated ELISpot plates (MSIPS4W10 from Millipore) coated with AN18 IFN-γ (1 μg / ml, Mabtech #3321-3-250;) and 11B11 IL-4 (1 μg / ml, Mabtech #3311-3-250) coating antibodies. The following day, cells were washed and plates were developed using biotinylated R4-6A2 anti-IFNγ (Mabtech #3321-6-250) and BVD6-24G2 anti-IL-4 (Mabtech #3311-6-250) detection antibodies, respectively. Wells were washed and then treated with 1:30,000 diluted Extravidin-ALP antibody (Sigma, St. Louis, MI, USA) for 1 h at room temperature. After washing, spots were developed by adding 70 μL / well of BCIP / NBT-Plus substrate (Mabtech #3650-10) to the wells. Plates were incubated for 20–30 min for color development and then washed with water. Spots were quantified using Cytation 7 (Biotech Instruments, Inc.). Each spot corresponds to a cell secreting an individual cytokine. Values ​​were analyzed as the background-subtracted mean of triplicates measured.

[0153] Example 18: Cell surface staining, intracellular cytokine staining for flow cytometry

[0189] Spleen and lung lymphocytes from immunized and control animals were stimulated 18 h after stimulation with nucleocapsid protein-peptide pools at a concentration of 1.5 μg / ml / peptide (Miltenyi Biotec; 130-126-699, Germany) for 16–18 h at 37 °C to detect nucleocapsid protein-specific CD8+ T cell responses, followed by the addition of Brefeldin A (5 μg / ml BD Biosciences #BD555029) for the final 5 h of incubation. 10 ng / ml PMA (Sigma, St. Louis, MI, USA) and 1 μg / ml ionomycin (Sigma, St. Louis, MI, USA) were used as positive controls. Stimulation without peptide served as background control. Cells were harvested and stained with Live / Dead Aqua (Thermo Fisher #L34965) in PBS followed by blocking of Fc-receptors with anti-CD16 / CD32 (Thermo Fisher #14-0161-85) and then stained with the following antibodies in flow cytometry staining buffer (FACS): anti-CD4 The cells were stained with AF589 (clone GK1.5; Biolegend #100446), anti-CD8b (clone YTS156.7.7; Biolegend #126609), anti-CD69 (clone H1.2F3; Biolegend #104537), anti-CD137 (clone 1AH2; BD; #740364), anti-CCR7 (clone 4B12; Biolegend #120124), and anti-CD45 (clone 30-F11; BD; #564279) for 30 min on ice. The cells were washed twice with FACS buffer, and then they were fixed with 100 μl of IC fixation buffer (eBioscience) for 30 min at room temperature. The cells were permeabilized with 200 μl of permeabilization buffer (BD Cytofix lysis kit) for 10 min. Intracellular staining was performed overnight at 4°C using antibodies Alexa Fluor 488 interferon (IFN) gamma (clone XMG1.2; BD; #557735) and granzyme B (clone GB11; Biolegend; #515407).Cells were then washed with FACS buffer and they were analyzed on an LSR-Fortessa flow cytometer (BD Biosciences) using FlowJo™ software version 10.8 (Tree Star Inc, Ashland, OR, USA). Results were calculated as total background-subtracted cytokine-positive cells. The amount of antibody used was optimized by titration.

[0154] Example 19: Preparation and Characterization and Composition

[0190] This example shows that NanoSTING is a liposomal adjuvant that enables mucosal immunity. Liposomal nanoparticles were shown to increase stability and facilitate delivery to alveolar macrophages to promote responses in the lung. Dynamic light scattering (DLS) analysis showed that the mean particle size of NanoSTING was 100 nm with a polydispersity index of 0.21. The zeta potential of NanoSTING was -50 mV. The ability of NanoSTING to induce an interferon (IRF) response was confirmed by using THP-1 monocytic cells stably expressing secreted luciferase downstream of an IRF-responsive promoter. THP-1 dual cells were stimulated with NanoSTING at concentrations ranging from 1.2 to 9.3 μg and kinetic measurements were performed for 24 hours by measuring luciferase activity in the supernatant. Although low levels of luciferase secretion were observed at 6 hours, secretion was maximal at 24 hours, and at this time point, all of the concentrations tested showed similar responses. Taken together, these results illustrated that optimal activation of the STING pathway by these NanoSTING compositions was observed at 24 hours.

[0155]

[0191] As immunogen, trimeric S protein based on SARS-CoV-2B.1.351(beta) was used, containing additional proline and alanine substitutions to confer stability. A single-step "mix and immunize" approach was utilized to allow for protein adsorption onto liposomes. Using a standard S-protein quantitative ELISA, it was confirmed that 61.5% of the S-protein was adsorbed onto liposomes. The adsorbed trimeric S-protein (NanoSTING-S) exhibited a mean particle size of 104 nm and a mean zeta potential of 133 mV, with a polydispersity index of 0.24. Compared to NanoSTING (81 nm, -35 mV), NanoSTING-S is more negatively charged as expected based on the isoelectric point (pI) of the protein. Unlike trimeric S protein, which is known to aggregate in solution, testing of NanoSTING-S after 6 months of storage at 4 °C did not reveal any evidence of aggregation or change in zeta potential.

[0156] Example 20: Single-dose immunization of mice with NanoSTING-S vaccine results in cross-reactive humoral and cellular immunity

[0192] Mice were immunized with a single intranasal dose of NanoSTING-S, and no clinical symptoms such as weight loss were observed throughout the observation period. 100% seroconversion was observed on day 7, with the response peaking on day 21. ELISA was performed on day 28 to quantify binding to both mutant full-length and receptor-binding domain (RBD) proteins instead of neutralization. High serum IgG titers were observed against the alpha (B.1.1.7), gamma (P.1), and delta (B.1.617.2) full-length proteins as well as the beta. High serum IgG titers were also observed against the RBD of both the beta and alpha mutant strains. SARS-CoV-2-specific antibody responses were assessed in bronchoalveolar lavage fluid (BALF), confirming robust IgG titers against full-length beta. IgA-mediated protection is an essential component of mucosal immunity against respiratory pathogens. To confirm the role of nasal NanoSTING as a mucosal adjuvant, IgA responses in serum were tested. Strong IgA responses were detected against all three variants tested. Collectively, these results established that a single-dose immunization with NanoSTING-S resulted in robust IgG and IgA responses that were cross-reactive against different spike variants, including the delta variant.

[0157]

[0193] To evaluate vaccine-induced S-specific T cell responses, splenocytes and lung lymphocytes were harvested 28 days after immunization. Spleen- and lung-derived T cells were stimulated with a pool of overlapping 15mer peptides, and antigen-specific T cells were quantified using IFN-γ (Th1 / Tc1 responses) and IL-4 (Th2 responses) ELISPOT assays. NanoSTING-S-immunized mice demonstrated robust and significant lung and splenic T cell responses, with over 1000 cells, respectively. 6The mean number of IFN-γ spots per cell was 500 and 176, respectively. Lung and spleen cells were also stimulated with the Wuhan S protein and a pool of peptides containing different mutations in the S protein. A significant Th1 response to these mutation-specific S peptides was observed, confirming a broad T cell response targeting both conserved and mutated regions of the S protein. In contrast to the IFN-γ (Th1 / Tc1) response, no measurable IL-4 (Th2) response was observed when immunized with NanoSTING-S. Collectively, these results established that intranasal vaccination elicited a strong cross-reactive Th1 / Tc1 response with no evidence of a Th2 response.

[0158] Example 21: Immune responses elicited by NanoSTING-S confer protection against highly infectious Delta strains

[0194] To test the protective efficacy of NanoSTING-S, a golden hamster challenge model was used. This animal model recapitulates more severe disease in humans, with animals demonstrating easily quantifiable clinical disease characterized by rapid weight loss, very high viral load in the lungs, and extensive lung lesions. In addition, unlike the K18-hACE2 model, hamsters recover from disease (like humans), thus offering the opportunity to study the impact of treatment in both the lungs (disease) and nasal passages (transmission). Animals were challenged with the delta strain for two reasons: (1) the delta strain is highly infective, causes severe lung damage, and has become the predominant strain in humans, and (2) delta-specific S-mutations, including L452R and T478K within the RBD, are not present in the immunogen, thus challenge with delta provides an opportunity to evaluate cross-protection.

[0159]

[0195] Based on findings with vaccines targeting other respiratory pathogens, intranasal immunization of groups of 10 hamsters with NanoSTING-S at two doses was used. Immunized hamsters were then administered 10 4The animals were challenged via the intranasal route with a delta variant strain with a TCID50 of 1. Animals in the sham-vaccinated group showed severe weight loss, with a mean peak weight loss of 8.3%. In contrast, the majority of animals vaccinated with NanoSTING-S were protected from weight loss (mean peak weight loss of 2.3%), similar to the results obtained with adenovirus vector vaccines challenged with either the Wuhan or beta strains. Half of the animals were killed on day 2 (peak of viral replication) and the other half on day 6 (peak of weight loss in non-immunized animals) to quantify viral load. Even with the highly infectious delta strain, NanoSTING-S reduced infectious viral load in the lungs by 300-fold compared to sham-vaccinated animals on day 2; infectious virus was undetectable in all animals on day 5. Viral replication in the animal lungs models clinical human disease and death, while viral replication in the nasal compartment models human transmission. Immunization with NanoSTING-S reduced infectious viral load 380-fold in the nasal compartment compared to non-immunized animals on day 2. Vaccinated animals showed a further significant reduction in infectious virus on day 5. This reduction in viral load in the nasal compartment was superior to an intramuscularly delivered adenovirus vaccine, suggesting an advantage of mucosal vaccination.

[0160]

[0196] Lung tissue collected on day 6 post-challenge was analyzed by using a combined scoring rubric (ranging from 1 to 12) to account for both pathology and disease severity. Significant immune cell infiltration and widespread viral pneumonia in the lungs of sham-vaccinated hamsters was observed. It was observed that vaccinated and challenged animals had significantly reduced gross pathology scores with minimal evidence of inflammatory cell infiltration or alveolar damage. Collectively, the results from NanoSTING-S immunized hamsters challenged with these delta strain coronavirus challenges demonstrate that the NanoSTING composition is cross-protective and can effectively protect the lungs, minimizing infectious virus in the nasal passages.

[0161] Example 22: Single-dose immunization of mice with NanoSTING-N vaccine results in durable humoral and cellular immunity

[0197] Because the N protein is largely conserved across the various SARS-CoV-2 variants, we investigated an N protein-based vaccine. Independent studies using K18-hACE2 mice immunized with a viral vector-based N protein and challenged with early lineage variants (Wuhan and alpha) show mixed results with either partial protection or a complete lack of protection.

[0162]

[0198] The SARS-CoV-2 N protein is predicted to consist of a nucleic acid (RNA) binding domain, a C-terminal dimerization domain, and three intrinsically disordered domains that can promote phase separation with nucleic acids. When recombinant N protein from insect cells was used as an immunogen, a predominant band with a size of 47 kD was identified by SDS-PAGE. To confirm the functional capacity of the recombinant N protein, the interaction of the N protein with plasmid DNA was studied. The DNA aggregation probe DiYO-1, a bis-intercalating fluorophore whose quantum efficiency increases by several orders of magnitude when bound to ds-DNA, was used. The N protein was able to quench the fluorescence of the DNA-DiYO-1 complex in a concentration-dependent manner. At a concentration of 0.5 μM, the N protein reduced the fluorescence intensity to the same extent (96.3%) as polyethyleneimine (PEI) R=5, a known synthetic polycation DNA aggregation agent. These results confirmed that the recombinant N protein is a potent binder of dsDNA. To formulate the vaccine, the recombinant N protein, NanoSTING-N, was gently mixed with NanoSTING to adsorb the protein onto the liposomes. Although the N protein exhibited strong aggregation properties upon storage at 4°C, NanoSTING-N was stable and showed no changes in size or zeta potential. These results suggested that the NanoSTING-N vaccine exists in the form of stable nanoparticulate colloids.

[0163]

[0199] Two groups of mice were immunized intranasally with liposomes containing cGAMP and either 10μg or 20ug of Wuhan N protein (NanoSTING-N10 and NanoSTING-N20, respectively). Similar to the NanoSTING-S vaccine, animals vaccinated with NanoSTING-N10 and NanoSTING-N20 reported no weight loss or significant abnormalities over 28 days. Fourteen days after immunization (day 14), 100% of mice receiving NanoSTING-N seroconverted, with robust anti-N IgG levels detected with a mean dilution titer of 1:640, and the response increased to a mean dilution titer on day 27. On day 15, serum concentrations of anti-S IgG antibodies increased, with a mean dilution titer of 1:4,400 detected. IgG responses at both doses were similar at all of the time points tested, with IgG titers elicited by NanoSTING-N20 being higher than those elicited by NanoSTING-N10, although the difference was not significant. In contrast to vaccination with trimeric NanoSTING-S (early response on day 7), the kinetics of the IgG response was delayed, with responses observed only on day 14. We assessed SARS-CoV-2-specific antibody responses in BALF; NanoSTING-N10 and NanoSTING-N20 showed mean IgG titers of 1:15 and 1:86, respectively. Consistent with mucosal vaccination, serum IgA was observed with NanoSTING-N10 (titer of 1:40) and Nano-STING-N20 (titer of 1:53). Taken together, these results established that immunization with the N protein resulted in robust humoral immunity in serum and lungs.

[0164]

[0200] One concern with N protein-based vaccines is that Th2 responses to the N protein may mediate antibody-dependent enhancement (ADE) of viral infection. Therefore, we quantified both Th1 and Th2 responses elicited in the lungs and spleens of immunized animals.

[0165]

[0201] Mice immunized with Nano-STING-N10 and NanoSTING-N20 demonstrated robust and significant splenic T cell responses, with 10 cells / mL of immunized mice, respectively. 6 Animals immunized with NanoSTING-N10 and NanoSTING-N20 showed elevated T cell responses in the lungs, with an average of 143 and 176 IFN-γ spots per individual. 6 There was an average of 102 and 154 IFN-γ spots per mouse. In contrast to the IFN-γ (Th1 / Tc1) responses, no measurable IL-4 (Th2) responses were observed upon immunization with NanoSTING-N10 and NanoSTING-N20. Taken together, these results established that nasal vaccination elicited a strong Th1 response with no evidence of a Th2 response.

[0166]

[0202] CD8+ T cell responses can complement antibody-mediated responses and provide protection independent of antibody responses. Activation and function of N protein-specific memory CD8+ T cells in the lung airways, lung parenchyma, and spleen were observed. We utilized the effector molecule granzyme B and the activation-induced marker CD137 to define N protein-reactive CD8+ T cells in the spleen and lung. Ex vivo restimulation with pools of N peptides resulted in a significant increase in the frequency of activated (CD8+CD137+) and cytotoxic (CD8+GzB+) T cells in the spleen, and to a lesser extent in the lung, of both NanoSTING-N10 and NanoSTING-N20 vaccinated mice. The overall frequency of lung-resident CD103+CD69+CD8+ T cells did not differ between immunized animals and the control group. Taken together, these results established that NanoSTING-N elicits cytotoxic T cell responses in the lung and spleen.

[0167]

[0203] The immunogenicity of the N protein based on the alpha variant was also evaluated. Two major mutations in the N protein, R203K and G204R, are fixed in all subsequent variants. The composition was formulated based on lyophilized N protein, and mice immunized with NanoSTING-N showed robust serum IgG and IgA titers at day 28. The durability of the response was also assessed by measuring serum IgG titers at day 62, and no reduction in either IgG or IgA was observed. Similarly, N-reactive Th1 responses were confirmed to be preserved in the lung and spleen at day 62. Collectively, these mouse studies confirmed that immunization with NanoSTING-N elicits robust cellular and humoral immune responses.

[0168]

[0204] Based on the immunogenicity data in mice, the protective efficacy of NanoSTING-N in hamsters was evaluated. Hamsters were vaccinated with two doses of NanoSTING-N, and immunized hamsters were incubated for 10 min at 4°C for 10 min. 4 Vaccinate-vaccinated hamsters were challenged via the intranasal route with TCID50 of the delta variant. Animals in both vaccinated (6.9% mean peak weight loss) and sham-vaccinated groups exhibited severe weight loss (8.3% mean peak weight loss). Consistent with the lack of protection from weight loss, infectious virus titers were not different in the lungs or nasal passages at either day 2 or day 5 in both vaccinated and sham-vaccinated animals. Collectively, results from these NanoSTING-N hamster experiments demonstrate that the N protein as a single antigen is insufficient to confer protection against delta strains.

[0169] Example 23: Quantitative modeling of the combined immune response to both proteins predicts synergistic protection

[0205] Results from the NanoSTING-S experiments demonstrated that immune responses in the lungs protect against disease but are insufficient to eliminate viral replication in the nasal passages as a surrogate for transmission. On the other hand, results from NanoSTING-N suggested that in all cases the N protein, while highly immunogenic, may not have a predominant role in protecting against SARS-CoV-2 infection. Mathematical models were used in conjunction with viral load data to quantify the opportunity for synergistic protection in the nasal compartment by vaccinating against both proteins.

[0170]

[0206] A model was constructed to track viral load in the nasal passages by fitting parameters to reflect viral titers over time from infected patients. The vaccine induced a neutralizing antibody response against the spike protein, which serves as a de novo blocker of viral entry and also blocks viral production via effector mechanisms. A range of vaccine efficacy (40-100%) was modeled to account for differences in protection, particularly in the nasal compartment, and to explore the impact on viral clearance.

[0171]

[0207] Cytotoxic T cell responses directed against the N protein directly kill virus-infected cells and thus directly reduce the number of cells capable of producing / propagating virus. In the case of a NanoSTING-N vaccine that does not elicit neutralizing antibodies and cannot prevent viral entry, our model predicted that T cells would be expected to be efficacious. These estimates are beyond the known efficacy of T cell immunity, so it is not surprising that a single antigen, N-based vaccine would not confer protection. We modeled combined protection by including an S-directed vaccine that provides partial protection in the nasal compartment along with a cytotoxic T cell response against the N protein. When a range of S-protein vaccine efficacy (40-100%) was tested in combination with a cytotoxic N response in the nasal compartment, the model predicted that 1-3 cells would need to be eliminated in a day, an estimate well within the known range of T cell killing frequencies. Indeed, studies in humans infected with COVID19 demonstrated robust and long-lived cytotoxic T cell responses in the nasal compartment, and that CD8+ T cells specific for the N protein can directly inhibit viral replication. Using virus reduction data from our hamster challenge model with NanoSTING-S, the model predicted that an N-directed cytotoxic response would be sufficient to kill 1-3 cells / day in the nasal compartment. Collectively, results from these quantitative modeling predict that combination vaccines targeting the S and N proteins can mediate synergistic protection in eliminating viral replication in the nasal compartment.

[0172] Example 24: Single-dose immunization of mice with NanoSTING-NS vaccine results in balanced humoral and cellular immunity targeting both proteins resulting in sterilizing immunity

[0208] To test the prediction from the quantitative model that immune responses to both S and N proteins may be synergistic, a vaccine containing both antigens was formulated. Initially, immunogenicity experiments were performed in mice with 10 μg each of N and S proteins adjuvanted with NanoSTING, and it was observed that 100% of the animals seroconverted, but showed IgG responses to the S protein, with variable seroconversion to the N protein (40-80%). The mass ratio of alphaN:betaS proteins (2:1) was varied and NanoSTING-NS was formulated with NanoSTING adjuvanted. Single-dose intranasal vaccination with NanoSTING-NS resulted in strong serum IgG titers against alphaN; full-length alphaS, betaS, and deltaS; as well as betaS RBD. Strong antigen-specific cross-reactive IgG responses were recorded in BALF; and cross-reactive IgA responses were observed in serum. In both lung and spleen, Th1 responses were predominant against the N protein, but also against the S protein. Consistent with all other experiments, no evidence of a Th2 response was observed in either tissue.

[0173]

[0209] Based on these encouraging immunogenicity data in mice, the protective efficacy of NanoSTING-NS was evaluated in hamsters. Hamsters were vaccinated with two doses of NanoSTING-NS, and immunized hamsters were then treated with 10 4The animals were challenged via the intranasal route with a delta mutant strain at TCID50 of 1. NanoSTING-NS immunized animals were fully protected from weight loss (mean peak weight loss of 0.8%). Just like the NanoSTING-S vaccine, NanoSTING-NS eliminated viral replication in the lungs at day 6 post-challenge, suggesting that the S-specific immune response was the predominant factor in providing immunity in the lungs. Pathology also confirmed that there was minimal evidence of inflammatory cell infiltration or alveolar damage in vaccinated and challenged animals. In the nasal compartment, NanoSTING-NS showed a significant reduction in infectious viral particles at day 2 compared to NanoSTING-S, with complete elimination of infectious viral particles in animals vaccinated with NanoSTING-NS at day 6. Collectively, these results illustrated that sterilizing immunity can be achieved by synergistic protection from immune responses against the N protein in combination with immunity directed against the S protein.

[0174]

[0210] Unless otherwise specified, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the claimed subject matter.

[0175]

[0211] The chapter headings used herein are for organizational purposes and shall not 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, papers, books, and treatises, are expressly incorporated herein by reference in their entirety for all purposes.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 shall control.

[0176]

[0212] The singular forms "a", "an" and "the" as used herein are intended to include the plural forms as well, unless the context clearly dictates otherwise. For example, unless specifically stated otherwise, the phrases "a" or "an" mean "at least one" and the use of "or" means "and / or". The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items. It is further understood that the terms "comprise" and / or "comprising", or "including" and / or "comprising", as used herein, specify the presence of stated features, regions, integers, steps, operations, elements and / or components, but do not preclude the presence of one or more other features, regions, integers, steps, operations, elements, components and / or groups thereof. Terms such as "element" or "component" also encompass both elements or components that contain one unit and elements or components that contain more than one unit, unless specifically stated otherwise.

[0177]

[0213] A and / or B, as used herein, includes one or more of A or B, as well as combinations thereof, such as A and B. It is expected that the terms "first", "second", "third", etc., may be used herein to describe various elements, components, regions and / or sections, but these elements, components, regions and / or sections should not necessarily be limited by these terms. These terms may be used merely to distinguish one element, component, region or section from another element, component, region or section. Thus, a first element, component, region or section discussed herein may in some cases be referred to as a second element, component, region or section without departing from the teachings of the present disclosure.

[0178]

[0214] The term "subject" as used herein includes mammals, including primates, such as rats, mice, non-human primates, and humans.

[0215] The term "NanoSTING" is used herein to describe various embodiments of the compositions described herein. For example, "NanoSTING" can refer to a composition comprising a modulator (e.g., a pattern recognition receptor agonist, such as a STING agonist) and a particle (e.g., a lipid-based particle, such as a lipid-based nanoparticle). In some cases, "NanoSTING" refers to a composition comprising a modulator and a particle that is not associated with an antigen molecule. In some cases, the term NanoSTING refers to a composition comprising a modulator, a divalent cation (e.g., but not limited to, Mn2+, Mg2+, Ca2+, Zn2+) and a particle. In some cases, the term "NanoSTING" is used herein to refer to a composition described herein that includes an antigenic molecule, e.g., when described in the context of inclusion of an antigenic molecule and / or when the term is modified to reflect an embodiment that includes an antigenic molecule (e.g., "NanoSTING-monomer," "NanoSTING-trimer," "NanoSTING-RSV," "NanoSTING-S," "NanoSTING-varS," "NanoSTING-ChimS," NanoSTING-N," "NanoSTING-N1O," or "NanoSTING-N20").

[0179]

[0216] Throughout this disclosure, various embodiments are presented in the form of ranges. It is to be understood that the description in the form of ranges is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​within that range to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. For example, the description of a range such as 1-6 should be considered to include specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, as well as individual values ​​within that range, such as 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of the ranges that exist between them may be independently included in the smaller ranges and may also be included within the scope of the invention, subject to any specifically excluded limitations within the stated ranges. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention unless the context clearly indicates otherwise.

[0180]

[0217] As used herein and in the claims, unless otherwise specified, the terms "about," "approximately," or "substantially" refer to a variation of less than or equal to ±0.1%, ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, ±10%, ±11%, ±12%, ±14%, ±15%, or ±20% of a numerical value, depending on the embodiment, including increments therein. As an example, in some cases, about 100 nanometers (nm) represents a range of 95 nm to 105 nm (which is ±5% of 100 nm), 90 to 110 nm (which is ±10% of 100 nm), or 85 nm to 115 nm (which is ±15% of 100 nm), depending on the embodiment.

[0181]

[0218] While preferred embodiments of the present invention have been shown and described herein, it is expected that it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is to be understood that various modifications to the embodiments of the invention described herein can be employed in carrying out the invention. It is intended that the following claims define the scope of the invention, and further that methods and structures within the scope of these claims and their equivalents are covered thereby.

[0182]

[0219] All publications, patents, and patent applications mentioned herein are incorporated by reference for all purposes. All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The subject matter of Appendix A and B of U.S. Provisional Patent Application US63 / 301,918 and the subject matter of Appendix A and Appendix B of U.S. Provisional Patent Application US63 / 329,261 are incorporated by reference for all purposes, including all embodiments and combinations thereof described therein. In particular, embodiments of compositions and / or methods including one or more particles, modulators, antigens, subjects, and / or combinations thereof, and / or experimental examples disclosed in U.S. Provisional Patent Application US63 / 301,918 (including Annex A or Annex B thereto) and / or U.S. Provisional Patent Application US63 / 329,261 (including Annex A or Annex B thereto), are hereby incorporated by reference for all purposes.

[0183]

[0220] Although specific embodiments and examples are provided in the foregoing description, the subject matter of the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses, as well as modifications and equivalents thereof. In any method or process disclosed herein, the acts or operations of the method or process may be performed in any suitable order, and are not necessarily limited to any particular disclosed order. Although various operations may be described in sequence as multiple separate operations in a manner that may be useful in understanding a particular embodiment, the order of description should not necessarily be construed to imply that these operations are order-dependent. In addition, the structures, systems, and / or devices described herein may be embodied as integrated elements or as separate elements.

[0184]

[0221] For the purpose of comparing various embodiments, the specific aspects and advantages of these embodiments are described. Not all such aspects or advantages are necessarily achieved by any particular embodiment. Thus, for example, various embodiments may be performed in a manner that achieves or optimizes one or a group of advantages as taught herein, without necessarily achieving other aspects or advantages that may be taught or suggested herein.

Claims

Claim 1: A composition for use in treating a respiratory disorder in a subject, comprising: lipid-based particles; a modulator encapsulated within the particle; a modulator that is not encapsulated in, bound to, or incorporated into the particle; wherein the modulator is an agonist of the STING pathway.

2. The composition of claim 1 , wherein the lipid-based particle comprises an antigen.

3. The composition of claim 1 , wherein the lipid-based particle comprises a first antigen and a second antigen.

4. 4. The composition of claim 3, wherein the first antigen is a spike protein molecule or a portion thereof.

5. The composition of claim 4 , wherein the second antigen is a nucleocapsid protein molecule or a portion thereof.

6. 6. The composition of claim 5, wherein the lipid-based particle comprises a greater amount of nucleocapsid protein molecules than spike protein molecules.

7. 7. The composition of claim 6, wherein the ratio of nucleocapsid protein molecules to spike protein molecules is at least 1:

1.

8. The composition of claim 1 , wherein the composition is formulated for nasal delivery.

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

1.

10. The composition of claim 2 , wherein the antigen is associated with the outer surface of the lipid-based particle.

11. The composition of claim 1 , wherein the composition is lyophilized.

12. The composition of claim 1 , wherein the composition is in liquid form.

13. 2. The composition of claim 1, wherein the modulator is selected from the group consisting of bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), amidobenzimidazole, a derivative of amidobenzimidazole, a nucleotide modulator, a plasmid DNA modulator, a divalent cation, or a combination thereof.

14. 2. The composition of claim 1, wherein the modulator comprises cyclic guanosine monophosphate-adenosine monophosphate (cGAMP).

15. A kit comprising a composition, the kit including instructions for use, the composition comprising: lipid-based particles; a modulator encapsulated within the particle; a modulator that is not encapsulated in, bound to, or incorporated into the particle; wherein the modulator is an agonist of the STING pathway.

16. The composition of claim 1 , wherein the subject does not exhibit symptoms of respiratory disease.

17. 10. The composition of claim 1, wherein the sample obtained from the subject has a detectable level of a pathogen associated with the respiratory disease.

18. 10. The composition of claim 1, wherein the sample obtained from the subject does not have detectable levels of pathogens associated with the respiratory disease.

19. The composition of claim 1 , wherein the subject exhibits symptoms of the respiratory disease.

20. The composition of claim 1 , wherein the composition is administered to a subject in at least one dose.

21. The composition of claim 1 , wherein the composition is administered to a subject in one dose.

22. The composition of claim 1 , wherein the composition is administered to a subject in two doses.

23. 10. The composition of claim 1, wherein the composition is administered to the subject before the subject is exposed to a pathogen associated with the respiratory disease.

24. 24. The composition of claim 23, wherein the composition is administered to the subject at least one day before the subject is exposed to a pathogen associated with the respiratory disease.

25. 24. The composition of claim 23, wherein the composition is administered to the subject at least 3 days before the subject is exposed to a pathogen associated with the respiratory disease.

26. The composition of claim 1 , wherein the composition is administered to a subject via intranasal administration.

27. The composition of claim 1 , wherein the composition is administered to a subject via inhalation administration.

28. 10. The composition of claim 1, used to prevent the establishment in the subject, to prevent the progression in the subject, to prevent the transmission of the disease to a second subject, or a combination thereof.

29. The composition of claim 1, which initiates an innate immune response in a subject that results in associated adaptive immunity.

30. 10. The composition of claim 1, wherein the respiratory disease comprises an infection caused by a pathogen.

31. 31. The composition of claim 30, wherein the pathogen is a respiratory pathogen.

32. 31. The composition of claim 30, wherein the respiratory pathogen is a virus.

33. 33. The composition of claim 32, wherein the virus is selected from influenza virus, parainfluenza virus, adenovirus, enterovirus, coronavirus, respiratory syncytial virus, rhinovirus, DNA virus, RNA virus, mutant strains thereof, or combinations thereof.

34. 34. The composition of claim 33, wherein the virus is an influenza virus.

35. 35. The composition of claim 34, wherein the influenza virus comprises an oseltamivir-susceptible strain, a treatment-resistant strain, or a combination thereof.

36. 34. The composition of claim 33, wherein the virus is a coronavirus.

37. 37. The composition of claim 36, wherein the coronavirus is SARS-CoV-2 virus, its alpha variant, its delta variant, its omicron variant, or a combination thereof.

Images