Intranasal vaccines and therapies for respiratory diseases

Abstract

Described herein are compositions and methods for reducing the risk of infecting a subject with a respiratory disease or reducing the risk of spreading a respiratory disease from a first subject to a second subject. In some cases, the compositions or methods described herein may comprise modulators (e.g., pattern recognition receptors, such as STING agonists, such as cGAMP), in some cases, the compositions or methods described herein may comprise liposomes that may be used to encapsulate one or more STING agonists. In some cases, the liposomes of the compositions or methods described herein may comprise one or more antigens attached to, integrated into, or associated with a liposomal membrane of the liposomes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 301,918, filed on January 21, 2022, and U.S. Provisional Patent Application No. 63 / 329,261, filed on April 8, 2022. The entire contents of each of the above applications are incorporated herein by reference for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy created on January 23, 2023 is named AF23853.P179WO.xml and is 52,000 bytes in size. Applicants hereby incorporate by reference the materials in the sequence listing. Background of the Invention

[0006] There is an urgent need for safe and durable prophylactic and therapeutic compositions to address a variety of diseases, such as infections caused by respiratory pathogens. Many embodiments of the present disclosure address the above needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 A description is provided of a composition for treating or preventing a disease according to various embodiments of the present disclosure, the composition comprising a liposome, a payload, and an antigen.

[0008] Figure 2A and 2B Methods of treating or preventing a subject disease are described.

[0009] Figure 3A An overall schematic diagram of the formulation and intranasal delivery of a composition (eg, a NanoSTING composition) to an animal according to embodiments described herein is shown. Figure 3B Signaling pathways that may be involved in (e.g., activated by) embodiments of methods and compositions (e.g., including NanoSTING delivery) are described. In some cases, THP1-Lucia TM Cells can be used to stably express a secreted form of luciferase under the control of a synthetic interferon-responsive promoter, e.g., to demonstrate type I interferon (IFN) induction (e.g., via IRF pathway activation) in an in vitro setting by embodiments of the compositions and methods described herein.

[0010] Figure 3C and Figure 3DThe distribution of NanoSTING liposome particle sizes at 25 degrees Celsius and 37 degrees Celsius, respectively, as measured by dynamic light scattering, according to an embodiment, is illustrated.

[0011] Figure 3E The distribution of NanoSTING liposome particle sizes measured by dynamic light scattering according to an embodiment is illustrated. Figure 3F Shown are the zeta potentials of NanoSTING compositions measured by electrophoretic light scattering (ELS), according to embodiments described herein.

[0012] Figure 3G A table showing particle size (DH), polydispersity index (PDI), and zeta potential of compositions described herein (eg, NanoSTING particles) at 25 degrees Celsius and 37 degrees Celsius.

[0013] Figure 4 Illustrated are the kinetics of luciferase induction in THP1-bi cells by different concentrations of NanoSTING according to an embodiment. RLU: Relative Light Unit.

[0014] Figure 5A The experimental design is described for 3-4 Balb / c mice treated with a single dose of NanoSTING (10 μg, 20 μg, or 40 μg). Blood, nasal turbinates, and lungs of euthanized animals were analyzed at 6, 12, 24, 36, and 48 hours. cGAMP ( Figure 5B ), cGAMP from lungs was analyzed by ELISA ( Figure 5C ), serum cGAMP 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 concha). Figure 5E , 5F , 5G, 5H, 5-I, 5J, 5K, 5L, 5M and 5N show the fold change of gene expression respectively), IFNβ analyzed by ELISA and CXCL10 analyzed by ELISA. The results show a method for treating or preventing the subject disease. The analysis was performed using the Mann-Whitney test. The bars and columns show the median. Each point represents a single mouse. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0015] Figure 6 Shown is the method for obtaining Figure 5E-5NDNA sequences of primers for the indicated qRT-PCR experimental data.

[0016] Fig. 7A , 7B and 7C illustrate the Figure 5A The experimental data, according to the embodiment, detects IFNβ ( Fig. 7A ), and nasal concha ( Figure 7B ) and lungs ( Figure 7C ) of CXCL10. Fig.7D Shown is the quantification of cGAMP in mouse serum after treatment with NanoSTING according to an embodiment. Fig. 7E The detection of IFNβ concentration in mouse serum using quantitative ELISA according to the embodiment is shown. Figure 7F Detection of CXCL10 levels in mouse serum using quantitative ELISA according to the embodiments is shown. Fold changes in gene expression were analyzed using the Mann-Whitney test. Bars and columns show median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0017] Fig. 8A and 8B Shown are changes in body temperature and body weight in a NanoSTING vs. control (PBS) study in hamster subjects, respectively, where groups of animals were given a daily dose of 60 μg of NanoSTING (n=4 / group) or PBS (n=4 / group) intranasally for four consecutive days. After the last dose was given on day 4, the hamsters were euthanized on day 5 and lungs were subsequently collected. Figure 8C Representative images of hamster nasal conchae, lungs and stomach analyzed in the Evans blue dye assay are shown.Hamsters (n=4 / group) were intranasally administered 0.125% Evans blue dye in PBS (40 μL and 120 μL). Fig.8D and 8E Quantified distribution of Evans blue dye after intranasal administration is shown. In these experiments, supernatants from homogenized lung and stomach were treated with trichloroacetic acid and analyzed for absorbance at 620 nm. Dye concentrations were interpolated from a standard curve.

[0018] Fig. 9A Real-time qRT-PCR of fold induction of Cxcl11, Ccl5, Ifnb1, Isg15, Irf7, Mx1, Mx2, Il6, and Il10 mRNA from lungs of hamsters treated with 60 μg NanoSTING compared to control hamster lungs is shown (n=4). Fig. 9B Shows the collection Fig. 9A Data are the DNA sequences of the primers.

[0019] Fig. 10A , 10B and 10C show activation of IFN-dependent and IFN-independent pathways in the lungs of NanoSTING-treated hamsters using RNA sequencing. Fig. 10A Shown is a heat map of the top 50 differentially expressed genes (DEGs) between NanoSTING-treated lungs (marked in green) and control lungs (marked in black). Fig. 10B Shown is a volcano plot of DEGs comparing NanoSTING-treated and PBS-treated control animals. Fig. 10C A GSEA of C2 and C7 curated pathways visualized using Cytoscape is shown. Nodes (red and blue circles) represent pathways, and edges (green lines) represent genes that overlap between pathways. The size of the nodes represents the number of genes enriched within the pathway, and the thickness of the edges represents the number of overlapping genes. The color of the nodes is adjusted for FDR q values ​​ranging from 0 to 0.25. Pathway clusters are labeled as groups with similar themes.

[0020] Fig.11A GSEA of IFN-independent activity of the activated STING pathway in the lungs of NanoSTING treated animals is shown. This schematic represents the comparisons made between samples collected in the GSE149744 dataset to generate pathway gene sets. Fig. 11B Shown are the gene expressions related to IFN-dependent and IFN-independent antiviral pathways in the lung between the NanoSTING group and the control group. Fig. 11C and 11D A schematic diagram is shown, which indicates that ( Fig. 11C ) during natural infection and ( Fig.11D ) Rate constants and equations governing viral kinetics in the presence of NanoSTING treatment. Fig.11E The viral area under the curve (AUC) reduction for different NanoSTING efficacies (RIR) compared to natural infection is shown. Treatment started on day 0, assuming that the effect of NanoSTING treatment lasts only 24 hours. Fig.11F A heat map of viral AUC with different NanoSTING efficacies and treatment start times is shown. The red box indicates a combination with a viral AUC reduction close to 100%. Fig.11G The peak spontaneous response was shown to be independent of the initial viral load. Fig.11H The results show that after NanoSTING treatment, viral kinetics can be independent of initial viral titer. E0 is the initial number of infected cells after viral infection, which is a surrogate value for viral titer. Figure 11-I , 11Jand 11K show the evolution of viral kinetics at different treatment start times and NanoSTING efficacy. Fig.11L Shown is a heat map of viral AUC with varying NanoSTING potency and therapeutic treatment initiation time, as NanoSTING effects persisted for 48 hours after treatment initiation.

[0021] Fig. 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 NanoSTING treatment. For viral titers, Mann-Whitney test was used for analysis. Fig. 12A The experimental design is shown, where each group of 12 hamsters received a single dose of 120 μg NanoSTING followed by 10 4 TCID50 of SARS-CoV-2 delta variant challenge. Virus titers in lung and nasal tissues of half of the hamsters (n=6) were determined on day 6. The remaining six hamsters were re-challenged on day 28, and body weight changes were followed until day 35. Fig. 12B and 12C The percent weight change compared to baseline over the indicated time intervals is shown. Fig.12F Shown are the percent body weight changes from day 28 to day 35 post-challenge monitoring. Fig.12D and 12E Virus titers measured by plaque assay of nasal tissue and lungs 6 days after infection are shown. Body weight data were compared by mixed effects model for repeated measures analysis. Each point is a single 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.

[0022] Fig.13A , 13B 13C shows that, according to an embodiment, a single dose of the NanoSTING composition can prevent challenge with the SARS-CoV-2 delta variant (B.1.617.2). Hamsters were treated with NanoSTING (120 micrograms) 24 hours (e.g., "-1 day") or 72 hours (e.g., "-3 days") prior to challenge with the SARS-CoV-2 delta variant. The treatment reduced viral replication and prevented weight loss. Fig.13A , 13Band 13C show the quantification of viral titers in lung and nasal tissues by plaque assay on the second day after challenge. For viral titers, Mann-Whitney test was used for analysis. Body weight data were compared by mixed effects model for repeated measures analysis. Each point is a single 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.

[0023] Fig.14A , 14B 14C shows that according to an embodiment, a single dose of NanoSTING composition administered post-exposure can protect against challenge with SARS-CoV-2 delta variant (B.1.617.2). Each group of 6 hamsters was challenged with SARS-CoV2 virus (delta variant-B.1.617) and treated with a single dose of NanoSTING (120 μg) 6 hours later ( Fig.14A ). Animals were euthanized 2 days after infection and analyzed to determine viral loads in lung and nasal tissues. Fig. 14B and 14C Quantification of viral titers in lung and nasal tissues by plaque assay 22 days after challenge is shown. For viral titers, analysis was performed using the Mann-Whitney test. Body weight data were compared by a mixed effects model for repeated measures analysis. Each point is a single hamster and the bars represent the median. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0024] Fig.15A , 15B , 15C and 15D illustrate that a single dose of the NanoSTING composition, according to embodiments disclosed herein, helps protect against the SARS-CoV-2 delta variant. Hamsters were treated with NanoSTING (120 micrograms) 24 hours prior to challenge with the SARS-CoV-2 delta variant. The treatment reduced viral replication and prevented weight loss in the treated animals.

[0025] Fig.16A , 16B , 17A, 17B, 17C, and 17D illustrate the protective efficacy of NanoSTING against the interferon (IFN)-escaping SARS-CoV-2 alpha variant (B.1.1.7). Each group of 12 hamsters received two different doses of NanoSTING (30 μg and 120 μg) and were challenged with the SARS-CoV-2 alpha variant (B.1.1.7) 24 hours later ( Fig.16A Monitor body weight changes daily ( Fig. 16BAnimals were euthanized on day 5 for histopathology ( Fig.17A and 17B ), and the virus titers in lung and nasal tissues were measured on day 2 (n=6). Fig. 17B ) and representative hematoxylin and eosin (H&E) images ( Fig.17A ) shows lung histopathological changes in Syrian hamsters treated with NanoSTING; all images were acquired at 20×; scale bar, 100 μm. Virus titers in lung and nasal tissues were quantified by plaque assay on day 2 after challenge ( Fig. 17C and 17D ). For viral titers, the Mann-Whitney test was used for analysis. Body weight percentages were compared by mixed effects model for repeated measures analysis. Each point is a single 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.

[0026] Fig.18A and 18B Prophylactic administration of a composition disclosed herein (eg, a NanoSTING composition) prior to challenge with influenza A virus is shown. Fig.18A The experimental design is shown in which pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) treatment were compared to placebo-treated mice, followed by challenge with 4 times the median lethal dose (LD50) of a susceptible strain and monitoring for 14 days. Fig.18B Shows Fig.18A Body weight changes of subjects in the experiments shown. For viral titers, Mann-Whitney test was used for analysis. Body weight data were compared by mixed effects model for repeated measures analysis. We compared survival percentage using log-rank test (Mantel-Cox). Each point is a single mouse. Bars and columns show median. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0027] Fig.19A , 19B and 19C show prophylactic administration of a composition disclosed herein (eg, a NanoSTING composition) prior to challenge with influenza A virus. Fig.19A The experimental design is shown in which pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) treatment were compared to unchallenged mice, followed by challenge with 4 times the median lethal dose (LD50) of a resistant strain of influenza A / Hong Kong / 2369 / 2009 (H1N1) and monitoring for 14 days (n=10 / group). Fig.19B Shows Fig.19A Body weight changes of subjects in the indicated experiments. Fig.19C Shows Fig.19A Meyer Kaplan plot of subject survival during the experimental period shown. For viral titers, the Mann-Whitney test was used for analysis. Body weight data were compared by a mixed effects model for repeated measures analysis. Survival percentages were compared using the log-rank test (Mantel-Cox). Each point is a single mouse. Bars and columns show median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0028] Fig. 20A , 20B 20C show prophylactic administration of a composition disclosed herein (eg, a NanoSTING composition) prior to challenge with influenza A virus. Fig. 20A The experimental design is shown in which pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) treatment were compared to unchallenged mice, followed by challenge with 4 times the median lethal dose (LD50) of a resistant strain of influenza A / Hong Kong / 2369 / 2009 (H1N1) and monitoring for 7 days (n=10 / group). Fig. 20B Shows Fig. 20A Body weight changes of subjects in the indicated experiments. Fig. 20C It shows that Fig. 20A Infectious virus titers measured by lung plaque assay 7 days after challenge in the experiments shown. For virus titers, Mann-Whitney test was used for analysis. Body weight data were compared by mixed effects model for repeated measures analysis. Survival percentages were compared using log-rank test (Mantel-Cox). Each point is a single mouse. Bars and columns show median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0029] Fig.21A and 21B Prophylactic administration of a composition disclosed herein (eg, a NanoSTING composition) prior to challenge with influenza A virus is shown. Fig.21A The experimental design is shown in which pre-challenge NanoSTING (40 μg) and pre-challenge oseltamivir (30 mg / kg / day) treatment were compared to unchallenged mice (placebo) (n=10 / group), followed by challenge with 4 times the median lethal dose (LD50) of a resistant strain of Influenza A / California / 04 / 2009 (H1N1) and monitoring for 14 days (n=10 / group). Fig.21B Shows Fig.21A Body weight changes of subjects in the indicated experiments. Body weight data were compared by mixed effects model for repeated measures analysis. Each point is an individual hamster and bars represent median values. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0030] Fig.22A , 22B , 22C, and 22D show that intranasal administration of NanoSTING limits transmission and viral replication in the nasal passages of contact hamsters exposed to the SARS-CoV-2 Omicron (B.1.1.529) variant. Fig.22A The experimental design is shown, where on day 0, each group of 8 hamsters were vaccinated with 10 4 TCID50 of SARS-CoV-2 Omicron variant (B.1.1.529), and 24 hours later, index hamsters were paired with contact hamsters (n=8) and co-housed in clean cages for 4 days. In group II, hamsters were prophylactically pretreated with 120 μg NanoSTING 24 hours before infection, and in group III, contact hamsters were treated with NanoSTING 12 hours after the start of co-housed. Contact hamsters and index hamsters were euthanized on the 4th day of co-housed. Infectious virus particles in nasal tissues on days 2 and 5 after post-infectious virus administration were measured by plaque assay ( Fig.21B and 22C ). Fig.22D Longitudinal measurements of body weight of index hamsters infected intranasally with the SARS-CoV-2 Omicron variant are shown. For viral titers, analysis was performed using the Mann-Whitney test. Bars show 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 by a mixed effects model for repeated measures analysis. Each point is a single hamster and the bars represent the median. Mann-Whitney test: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant.

[0031] Fig.23A and 23B Shown is a schematic diagram of a NanoSTING-S composition according to embodiments disclosed herein and a timeline of immunogenicity data according to embodiments disclosed herein.

[0032] Fig.24A , 24B, 24C, 24D, 24E, 24F, 24G and 24H show the results of the NanoSTING-S composition used in mice according to the embodiments. The amount of lipid NanoSTING-S used was 10 μg S protein and 20 μg NanoSTING. This resulted in cross-reactivity against multiple SARS-CoV-2 strains including the Omicron variant.

[0033] Fig.25A , 25B , 25C and 25D show the effect of the NanoSTING-S vaccine in a hamster challenge model. NanoSTING-S: 20 μg S protein and 40 μg NanoSTING. Weight loss and infectious virus titers in lung and nasal tissues are shown. As shown, the weight loss and infectious virus titer data suggest favorable results for human immunity using a composition comprising NanoSTING-S. In some cases, the dose in humans is 50-200 μg S protein and 200-500 μg NanoSTING.

[0034] Fig.26A , 26B , 26C, 26D, 26E, 26F, 26G, 26H, 26-I, 26J, 26K, 26L, 26M, 26N and 26-O show the effect of NanoSTING-N vaccine on mice. These support the use of NanoSTING-N to achieve human immunity relying on antibody response and T cell response. The expected dose in humans is 50-200 μg N protein and 200-500 μg NanoSTING.

[0035] Figure 26P and 26Q IRF response of THP-1 bi-cells to poly(dA:dT) / LV and MTT assay on THP1-bi-cells are shown, respectively. According to an embodiment, the dose response of NanoSTING was performed using Triton X-100 as a cytotoxicity control.

[0036] Fig.27A , 27B , 27C, 27D, 27E, 27F and 27G show NanoSTING-NS vaccines in mice (20 μg N protein, 10 μg S protein and 20 μg NanoSTING containing liposome particles and cGAMP modulators). The ratio of N protein to S protein is optimized to be a 2:1 mass ratio. An N:S ratio of at least 1:1 (mass:mass) or higher provides a balance of immune responses against the two proteins. This results in cross-reactivity against multiple SARS-CoV-2 strains.

[0037] Fig.28A , 28B , 28C and 28D show the effect of NanoSTING-NS (NanoSTING-NS: 30μg N protein, 20μg S protein and 40μg NanoSTING containing liposomal particles and cGAMP modulator) vaccine in a hamster challenge model. Weight loss and infectious virus titers in lung and nasal tissues are shown. These support the use of NanoSTING-NS to achieve sterilizing immunity in humans. The expected dose in humans is 50-200μg N protein, 50-200μg S protein and 200-500μg NanoSTING.

[0038] Fig.29A and 29B Shown is the effect of NanoSTING-N vaccine (30 μg N protein and 40 μg NanoSTING containing liposomal particles and cGAMP modulator) in a hamster challenge model. Fig.29C and 29D Weight loss and infectious virus titers in lung and nasal tissues are shown. Summary of the invention

[0039] The alarming rate at which the SARS-CoV-2 pandemic has spread has highlighted that existing therapeutics for RNA viruses are woefully inadequate. Vaccines are the preferred means of preventing SARS-CoV-2, but they have three drawbacks. First, while the current generation of vaccines is being developed at an extraordinary pace (approximately 10 months), such development is still slow and requires vaccines to be customized for each emerging virus. Second, the mutational plasticity of RNA viruses such as SARS-CoV-2 facilitates their evolution and the emergence of new variants with the potential for immune escape. Conversely, even if the entire human population is not fully vaccinated, booster shots are required to fully prevent disease. As human experience with influenza has shown, requiring additional booster shots reduces human compliance and thus promotes disease transmission. Third, although existing vaccines are effective in preventing disease, they do not prevent transmission. The evolution of the SARS-CoV-2 Omicron (B.1.1.529) variant shows that viruses can adapt rapidly to facilitate rapid transmission. Therefore, while vaccines are necessary, they are insufficient against RNA viruses, and the availability of pre- or post-exposure prophylactics that can prevent disease and reduce transmission is an urgent and unmet clinical need. As disclosed herein, a single dose of intranasal NanoSTING can serve as a prophylactic against multiple respiratory viruses (and address drug-resistant variants).

[0040] Monoclonal antibodies (such as vaccines) can effectively prevent diseases, but they also have the same disadvantages as the above vaccines. The SARS-CoV-2 Omicron variant is almost completely resistant to neutralization by antibodies. In addition, monoclonal antibodies are expensive therapies and are administered in clinical settings, which further limits their widespread use. If only the efficacy is compared, prophylactic administration of antibodies in hamsters (12 hours before challenge) can prevent clinical disease (weight loss of about 2-5%, lung virus titer reduced by about 300 times), and has no effect on transmission. The compositions disclosed herein (e.g., NanoSTING compositions) are easy to administer (e.g., intranasally) and provide a wider administration window (24-72 hours), with comparable efficacy in reducing clinical disease while also reducing transmission.

[0041] Oral antiviral drugs that directly inhibit one or more viral proteins have been developed for SARS-CoV-2 (e.g., Molnupiravir and Paxlovid) and tested in humans, but they are susceptible to viral evolution and drug resistance. In addition, oral antiviral drugs are designed as post-exposure prophylaxis to prevent clinical disease and have no effect on viral transmission. Unlike these pathogen-specific antiviral drugs, the compositions disclosed herein (e.g., NanoSTING compositions) target multiple respiratory viruses. In addition, the favorable efficacy profiles comparing the compositions disclosed herein to these antiviral drugs in small animal models and the fact that these antiviral drugs have efficacy in humans (reducing clinical disease of SARS-CoV-2 by 30-89%) fully demonstrate the clinical translation potential of the compositions disclosed herein (e.g., NanoSTING compositions).

[0042] 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. Although these particles have demonstrated disease-modifying efficacy against both SARS-CoV-2 and influenza in small animal models, DIPs must be generated individually for each virus. Defective viral genomes (DVGs) based on poliovirus induce broad IFN-I responses and can help protect against a variety of viruses. Limited replication is critical for the efficacy of DVGs, but their broad applicability is limited by safety and the presence of pre-existing antibodies in the vaccinated population. Lipid nanoparticles complexed with defective genomes can alleviate these concerns and have shown efficacy against SARS-CoV-2 variants in K18-hACE2 mice. However, the generalizability of this approach to other viruses in the absence of viral replication has not been demonstrated.

[0043] Direct administration of aerosolized interferon has been tested in animals and humans to mobilize antiviral innate immunity. In hamsters challenged with SARS-CoV-2, prophylactic or early administration of universal interferon reduced lung damage, provided modest protection against weight loss (10% in treated animals compared with 20% in untreated animals), and reduced infectious viral particles (100-fold). In humans, postexposure prophylaxis with aerosolized IFN-α2b reduced in-hospital mortality compared with no IFN-α2b administration. In contrast, administration of IFN-α2b for more than five days after hospital admission delayed recovery and increased mortality, suggesting that the timing of IFN-α2b administration is critical for benefit. The limited effect of IFN-α on COVID19 reflects its modest efficacy as a preventive against influenza in humans.

[0044] Other synthetic pattern recognition receptor (PRR) small molecule agonists including stem-loop RNA 14 (SLR14), which is the smallest RIG-I agonist, and the STING agonist diAbzl have been tested against SARS-CoV-2 in K18-hACE2 mice. As with all small molecule drugs, their safety, off-target activity, and pharmacokinetics need to be fully evaluated before transformation. In some cases, the compositions disclosed herein (e.g., some NanoSTING compositions disclosed herein) contain naturally occurring lipids and cGAMP that have been tested in humans, which is an immunotransmitter of danger signals and is conserved in mammals (including humans). These compositions can be delivered safely and continuously, and can therefore be used as broad-spectrum antiviral drugs.

[0045] The present disclosure shows that the compositions disclosed herein (e.g., NanoSTING compositions) are safe, stable, low-cost and effective antiviral drugs that engage the STING pathway to initiate a broad-spectrum antiviral response that is effective against a variety of respiratory pathogens. The advantage of using the natural immune transmitter cGAMP is that STING activation can lead to interferon-dependent and interferon-independent activities to control viral replication. The broad-spectrum compositions (e.g., NanoSTING compositions) disclosed herein ensure that the activity is directed against a variety of variants, including variants that are resistant to other antiviral drugs. The ability to participate in adaptive immunity ensures that treatment with the compositions disclosed herein (e.g., NanoSTING compositions) can also prevent reinfection and produce lasting immunity. The availability of broad-spectrum drugs that are easy to store for a long time ensures that we are prepared to deal with future respiratory viruses.

[0046] Disclosed herein are compositions, methods, kits and systems for treating objects in need thereof, such as objects with pathogens that can cause respiratory diseases. In some cases, compositions, methods, kits and / or systems disclosed herein can be used to prevent objects (e.g., uninfected objects) from being infected with pathogens and / or diseases that can be caused by infectious pathogens, for example, by preventing objects from being infected by pathogens or by preventing the progression (e.g., diffusion or increased severity) of conditions (e.g., respiratory diseases) caused by pathogens in objects. In some cases, compositions, methods, kits and / or systems disclosed herein can be used to prevent objects from being reinfected by pathogens (e.g., wherein the pathogen can cause respiratory diseases). In some cases, compositions, methods, kits and / or systems disclosed herein can be used to prevent pathogens (e.g., pathogens that can cause respiratory diseases) from being spread from a first (e.g., infected) object to a second (e.g., uninfected) object. In some cases, pathogens are viruses. In some cases, pathogens are coronaviruses. In some cases, pathogens are influenza viruses.

[0047] In various embodiments, the composition comprises: lipid-based particles and a modulator, wherein the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof. In various embodiments, a composition for reducing the risk of a subject contracting a respiratory disease after the subject is exposed to a pathogen capable of causing a respiratory disease, comprising: lipid-based particles and a modulator, wherein the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof. In various embodiments, a composition for reducing the risk of a subject contracting a respiratory disease before the subject is exposed to a pathogen capable of causing a respiratory disease, comprising: lipid-based particles and a modulator, wherein the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof. In various embodiments, a composition for treating a respiratory disease in a subject, comprising: lipid-based particles and a modulator, wherein the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof. In various embodiments, a composition for reducing the rate of progression of a respiratory disease in a subject suffering from a respiratory disease, comprising: lipid-based particles and a modulator, wherein the modulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof. In various embodiments, a composition for reducing the risk of respiratory disease from a subject with respiratory disease to a subject without respiratory disease comprises: lipid-based particles and a regulator, wherein the regulator is a pattern recognition receptor agonist, an immune system activator, or a combination thereof. In some cases, the regulator is encapsulated in a lipid-based particle. In some cases, the subject has previously been infected with a respiratory disease. In some cases, the lipid-based particle comprises an antigen. In some cases, the lipid-based particle comprises 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 larger amount of nucleocapsid protein molecules than the spike protein molecule. 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 intranasal delivery. In some cases, the regulator is an agonist of the STING pathway. In some cases, the lipid-based particle comprises DPPC, DPPG, cholesterol, and DPPE-PEG2000 at 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 includes 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, its variant or combination thereof. In some cases, the virus is influenza virus. In some cases, influenza virus includes oseltamivir sensitive strains, treatment resistant strains or combinations thereof. In some cases, the virus is a coronavirus.In some cases, coronavirus is SARS-CoV-2 virus, its alpha variant, its delta variant, its Omicron variant or its combination. In some cases, the composition is freeze-dried. In some cases, the composition is in liquid form. In some cases, the regulator is selected from the following groups: double (3', 5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), aminobenzimidazole, derivatives of aminobenzimidazole, nucleotide regulators, plasmid DNA regulators, divalent cations CF501, SHR1032 or its combination. In some cases, the regulator includes cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). In various aspects, the kit includes the composition described in any one of the aspects or embodiments disclosed herein, including instructions for use. In various aspects, the composition described in any embodiment or aspect disclosed herein is used to manufacture a medicine for treating respiratory diseases.

[0048] In various aspects, a method comprising: administering a composition according to any one of the aspects and embodiments disclosed herein to a subject, wherein the subject does not show symptoms of respiratory disease. In some cases, a sample obtained from the subject has a detectable level of a pathogen associated with the respiratory disease. In some cases, a sample obtained from the subject does not have a detectable level of a pathogen associated with the respiratory disease. In various aspects, a method comprising: administering a compound according to any one of the aspects and embodiments disclosed herein to a subject, wherein the subject shows symptoms of respiratory disease. In some cases, a sample obtained from the subject has a detectable level of a pathogen associated with the 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 the 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 the respiratory disease. In some cases, the composition is administered to the subject at least three days before the subject is exposed to a pathogen associated with the respiratory disease. In some cases, the composition is administered by intranasal administration. In some cases, the composition is administered by inhalation. In some cases, the method is used to prevent the establishment of a disease in a subject. In some cases, the method is used to prevent the development of a disease in a subject. In some cases, the method is used to prevent the spread of a disease to a second subject. In some cases, the method initiates an innate immune response that results in an associated adaptive immunity. DETAILED DESCRIPTION

[0049] Treatments and vaccines for respiratory diseases have many limitations. For example, vaccines provide short-term relief for SARS-CoV2. However, the rapid evolution of drug-resistant viral variants requires additional supporting strategies, including broad-spectrum antivirals with both preventive and therapeutic properties.

[0050] SARS-CoV2 and influenza are among the leading infectious diseases causing death and morbidity worldwide. Drug resistance and viral mutations are major challenges in influenza prevention and treatment. The development of readily available, effective, safe and low-cost drugs for preventing respiratory viral infections is a major unmet medical need. Disclosed herein are compositions and methods for preventing and / or treating a disease or pathological condition (such as a respiratory disease) in a subject (e.g., an animal, such as a mammal). In some embodiments, preventing a disease or pathological condition includes preventing the establishment of a disease or pathological condition in a subject. In some embodiments, preventing a disease or pathological condition includes preventing the establishment of a disease or pathological condition in a subject (e.g., after the subject is exposed to a pathogen). In some embodiments, preventing a disease or pathological condition includes preventing the development (e.g., progression) of a disease or pathological condition in a subject (e.g., from a first state to a second, more severe or progressive state) (e.g., establishing a disease in a subject, e.g., by infection with a pathogen). In some embodiments, preventing a disease or pathological condition includes preventing the spread of a disease or pathological condition from a first subject to a second subject.

[0051] Compositions 10 for preventing or treating a disease or pathological condition in a subject may include particles 12 (e.g., liposomes as described herein), modulators 16 (e.g., STING agonists such as cGAMP), and / or antigens 14 (e.g., Figure 1As shown). In some cases, the compositions described herein may include a modulator (e.g., a STING agonist, such as cGAMP) without particles or antigens. In some cases, the compositions described herein may include a modulator (e.g., a STING agonist, such as cGAMP) that is encapsulated in, in contact with, bound to, and / or not in contact with particles but delivered (e.g., intranasally delivered) within the particles described herein. In some cases, encapsulating one or more modulators within particles described herein (or, in some cases, binding one or more modulators to particles or incorporating one or more modulators into particles) can increase the uptake efficiency, tissue targeting, stability, and / or efficacy of the composition or its method of use, 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 a target cell. In some cases, a composition comprising one or more modulators without particles or antigens (or comprising one or more modulators that are located outside the particles of the composition and are not associated with the particles of the composition) can elicit a more direct response from a target tissue or cell and / or reduce the cost and / or complexity of the composition manufacturing. In some cases, the compositions described herein may include one or more modulators (e.g., one or more STING agonist molecules, such as 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, such as one or more cGAMP molecules) delivered to a subject with the particle, but not encapsulated in, bound to, or incorporated into the particle. In some cases, compositions comprising one or more modulators (e.g., one or more STING agonist molecules, such as one or more cGAMP molecules) encapsulated within a particle (e.g., a liposome) and compositions comprising one or more modulators (e.g., one or more STING agonist molecules, such as one or more cGAMP molecules) delivered to a subject with the particle, but not encapsulated in, bound to, or incorporated into the particle can provide the benefits of a "naked" modulator (e.g., a more direct response and / or different target cells or tissues than an encapsulated modulator) and one or more modulators encapsulated, bound to, or incorporated into a particle (e.g., a liposome) described herein. In some cases, the compositions described herein may include lipid-based liposome particles, a modulator (such as a pattern recognition receptor agonist, e.g., a STING agonist), and an application-specific antigen (such as a coronavirus spike protein ("S-protein"), a nucleocapsid protein ("N-protein"), or a chimeric antigen protein) associated with the liposome particles. In some embodiments, association of the application-specific antigen with the liposome particles encapsulating the modulator cargo (e.g., the outer surface thereof) can significantly increase the targeting and / or delivery of the modulator cargo to a target tissue of interest (e.g., an intranasal compartment and / or a pulmonary compartment of a subject).In some embodiments, association of an application-specific antigen with a liposome particle (e.g., direct association, such as by incorporation into a liposome membrane or by coupling to a liposome membrane surface) can increase the efficacy of the composition in preventing and / or treating a disease or pathological condition in a subject, for example by spatially concentrating the antigen and modulator of the composition (e.g., at a target tissue). Disclosed herein are compositions (e.g., NanoSTING compositions, which can be liposome-encapsulated immunotransmitters) that can rapidly activate the human innate immune system to promote a broad-spectrum antiviral response against influenza and SARS-CoV-2 variants in mice and hamsters. In some cases, a single intranasal dose of NanoSTING can: (1) treat infection throughout the respiratory system and minimize symptoms, (2) reduce transmission by reducing infectious virus in the nasal passages, (3) prevent oseltamivir-resistant influenza and highly contagious SARS-CoV-2 strains (alpha and delta), and / or (4) provide long-lasting protection against reinfection by stimulating adaptive immunity. In some cases, administration of NanoSTING can upregulate interferon-stimulated pathways and / or antiviral pathways, for example in the nasal turbinates and / or lungs. In some embodiments, the NanoSTING composition can be a stable, low-cost, broad-spectrum antiviral therapeutic for prophylactic or early post-exposure treatment.

[0052] In some cases, the compositions and methods disclosed herein (e.g., nanoparticle formulations comprising NanoSTING, which may comprise cGAMP) can enable sustained release of cGAMP to the nasal compartment and lungs for 48 hours or longer after administration to a subject. The compositions disclosed herein (e.g., nanoparticle formulations comprising NanoSTING) can comprise lipids and a STING activator (e.g., cGAMP), but can also include one or more divalent metal ions (e.g., 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 promote type I interferon (IFN-I)-mediated responses. Quantitative models using human SARS-CoV-2 infection can be used to confirm that even small amounts of the compositions disclosed herein (e.g., NanoSTING compositions) can produce clinically observable benefits before or after exposure. The compositions disclosed herein (e.g., NanoSTING) can be used to prevent clinical disease (e.g., multiple variants of SARS-CoV-2 and / or influenza A, including oseltamivir-resistant influenza A), reduce viral load (e.g., by several orders of magnitude), reduce pathogen and / or disease transmission, and / or utilize adaptive immunity to provide lasting protection against reinfection. The stability and ease of administration and the comprehensive nature of the immune response elicited make the compositions disclosed herein (e.g., NanoSTING) provide an ideal non-invasive and broad-spectrum antiviral therapeutic composition for respiratory viruses.

[0053] Composition

[0054] A composition for preventing or treating a disease or pathological condition in a subject (e.g., a therapeutic composition) may include a delivery vehicle (e.g., a particle). The delivery vehicle of a composition for preventing or treating a disease or pathological condition in a subject may be a particle, such as a lipid-based particle. For example, a composition for preventing or treating a disease or pathological condition in a subject may include a lipid-based particle, such as a liposome (e.g., having a membrane with an outer surface and an inner space). In some embodiments, the composition includes one or more modulators (e.g., one or more types of modulators). In some embodiments, the modulator may include a small molecule, a protein or fragment thereof, and / or a polynucleotide or 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 because it can 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). A composition for preventing or treating a disease or pathological condition in a subject (or its spread to a second subject) may include one or more antigens. A composition for preventing or treating a disease or pathological condition in a subject (or its spread to a second subject) may include multiple antigens. In some embodiments, the antigens of the compositions described herein can elicit an immune response in a subject, which can be used to prevent or eliminate a disease (e.g., a respiratory disease) or a pathological condition (e.g., cancer) in a subject. In some embodiments, a composition for preventing or treating a disease or pathological condition in a subject can include a modulator (e.g., a STING agonist such as cGAMP) encapsulated in a particle (e.g., a liposome), wherein the antigen is associated with the liposome or a portion thereof (e.g., associated by incorporation into the liposome membrane).

[0055] In some embodiments, the compositions described herein can be used in methods of treating or preventing a disease or pathological condition in a subject (e.g., a mammal such as 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), for example, to prevent the subject from acquiring a disease or condition associated with exposure to (e.g., infection with) a pathogen (e.g., Figure 2A In some embodiments, the compositions disclosed herein can be administered in the form of a vaccine. In some embodiments, after exposure to a pathogen (e.g., a pathogen that causes respiratory disease or cancer), for example, after a subject acquires a disease or condition associated with the pathogen as a result of exposure to the pathogen (e.g., Figure 2B As shown, the compositions described herein can be administered to a subject to treat (e.g., in some embodiments, to ameliorate or cure) a disease or condition associated with exposure to (e.g., infection with) a pathogen infection.

[0056] Particles

[0057] The compositions of the present disclosure may include delivery vehicles, such as particles. The particles may include means for transporting a payload (e.g., a modulator payload) and / or one or more antigens (e.g., an antigen associated with a portion of the particle (e.g., a membrane or outer surface)). In some embodiments, the particle may include a membrane or wall. In some embodiments, the membrane or wall of the particle may define an internal space. In some embodiments, the internal space of the particle may include one or more modulators. In some embodiments, one or more antigens may be associated with a membrane or wall of the particle (e.g., the outer surface of a membrane or wall of the particle). The particle may include lipid-based particles, carbon-based particles, metal-based particles, or a combination thereof.

[0058] 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 (e.g., lipid-based particles) of the compositions disclosed herein may include multiple molecules, for example, assembled into a membrane. In some embodiments, the particles (e.g., lipid-based particles) or portions 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., NanoSTING compositions comprising lipid-based nanoparticles) may not include cationic lipids. In some embodiments, the particles (e.g., lipid-based particles) or portions thereof may be cationic. In some cases, the particles (e.g., lipid-based particles) may include cationic (e.g., positively charged) lipids. For example, particles (e.g., lipid-based particles) can include lipids selected from 1,2-dipalmitoyl-3-trimethylammonium-propane chloride (DPTAP) or 1,2-dioleoyl-3-trimethylammonium-propane hydrochloride (DOTAP). In some embodiments, particles (e.g., lipid-based particles) can be zwitterionic. In some embodiments, particles (e.g., lipid-based particles) or portions thereof can have a net zero charge. In some embodiments, particles (e.g., lipid-based particles) or portions thereof can be uncharged. In some embodiments, particles (e.g., liposomes) of compositions described herein can include dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, 1,2-bis(diphenylphosphine)ethane (DPPE), cholesterol, or a combination thereof. In some embodiments, particles (e.g., liposomes) of compositions described herein can include poly(ethylene glycol)-lipids (e.g., PEG lipids). In some embodiments, the particles (e.g., liposomes) of the composition may include DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-racemic-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 hydrochloride (DOTAP), or a combination thereof. In some embodiments, the particles may include a combination of DPPC and DPPG, for example, at a molar ratio of about 10:1. In some embodiments, the particles may include a combination of DPPC and cholesterol, for example, at a molar ratio of about 10:1.In some embodiments, the particles may include a combination of DPPC and DPPE-2000, for example, at a molar ratio of about 10:1. In some embodiments, the particles may include a combination of DPPG and cholesterol, for example, at a molar ratio of about 1:1. In some embodiments, the particles may include a combination of cholesterol and DPPE-PEG2000, for example, at a molar ratio of about 1:1. In some embodiments, the particles may include a combination of DPPG and DPPE-PEG2000, for example, at 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 in a molar ratio of 10:1:1:1, respectively. 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 composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 5:1:1:1, respectively. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:2:1:1, respectively. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:1:2:1, respectively. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:1:1:2, respectively. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:2:2:1, respectively. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:1:2:2, respectively. In some embodiments, the particles may be composed of DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a molar ratio of 10:2:1:2, respectively. In some embodiments, the molar ratio of the first molecule comprising the particle (e.g., lipid-based particle) to the second molecule comprising the particle can 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 higher than 1:100. In some embodiments, the molar ratio of the first molecule comprising the particle (e.g., lipid-based particle) of the present disclosure to the second molecule comprising 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 comprising the particle (e.g., lipid-based particle) may be 1:5. In some embodiments, the molar ratio of the first molecule comprising the particle (e.g., lipid-based particle) may be 1:10. In some embodiments, the molar ratio of the first molecule comprising the particle (e.g., lipid-based particle) may be 1:20. In some embodiments, the molar ratio of the first molecule comprising the particle (e.g., lipid-based particle) may be 1:50. In some embodiments, the first molecule comprising the particle (e.g., lipid-based particle) may be 1:100. In some embodiments, the first molecule comprising the particle (e.g., lipid-based particle) of the composition described herein may be dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, 1,2-bis(diphenylphosphino)ethane (DPPE), cholesterol, or a combination thereof. In some embodiments, particles comprising a composition described herein (e.g., lipid-based particles) can be DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-racemic-glycerol)), DPPE-PEG2000 (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), or cholesterol.

[0059] The particles of compositions described herein can be nanoparticles. Keeping a small particle size (e.g., an average hydrodynamic particle size of less than 300 nanometers (nm), less than 200nm, less than 150nm, less than 120nm, less than 115nm, less than 111nm, less than 110nm, less than 105nm, less than 100nm, less than 95nm, less than 90nm, less than 85nm, or less than 80nm) and / or low polydispersity (e.g., polydispersity index 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 particle stability and / or delivery efficiency. In some embodiments, the outer diameter of the particle can be 1 nanometer to 500 nanometers, 1 nanometer to 750 nanometers, or 1 nanometer to 1000 nanometers.In some embodiments, the outer diameter of the particle can be 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 100 nm, 10 nm to 200 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 100 nm. to 75 nm, 10 nm to 100 nm, 10 nm to 150 nm, 10 nm to 200 nm, 10 nm to 250 nm, 10 nm to 300 nm, 15 nm to 20 nm, 15 nm to 30 nm, 15 nm to 50 nm, 15 nm to 75 nm, 15 nm to 100 nm, 15 nm to 150 nm, 15 nm to 200 nm, 15 nm to 250 nm, 15 nm to 300 nm, 20 nm to 30 nm, 20 nm to 50 nm, 20 nm to 75 nm, 20 nm to 100 nm, 20 nm to 150 nm, 20 nm to 200 nm, 20 nm to 250 nm, 20 nm to 300 nm, 30 nm to 50 nm, 30 nm to 75 nm, 30 nm to 100 nm, 30 nm to 150 nm, 30 nm to 200 nm, 30 nm to 250 nm, 30 nm to 300 nm, 50 nm to 75 nm, 50 nm to 100 nm, 50 nm to 150 nm, 50 nm to 200 nm, 50 nm to 250 nm, 50 nm to 500 nm, In some embodiments, the outer diameter of the particle can be 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 outer diameter of the particle can be 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 outer diameter of the particle can be 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.

[0060] In some embodiments, the particle population may have an average (e.g., outer) diameter of 1 nm to 1,000 nm, for example, at room temperature of 25 degrees Celsius (° C.) 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 particle population may have an average (e.g., outer) diameter of 1 nm to 100 nm, for example, at room temperature of 25 degrees Celsius (° C.) 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 particle population may have an average (e.g., outer) diameter of 100 nanometers to up to 200 nanometers, for example, at 25 degrees Celsius (° C.) 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 particle population may have an average (e.g., outer) diameter of 200 nanometers to up to 300 nanometers, for example, at 25 degrees Celsius (° C.) 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 particle population can have a diameter of 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 125 nm, 1 nm to 150 nm, 1 nm to 175 nm, 1 nm to 200 nm, 1 nm to 300 nm, 1 nm to 1,000 nm, 20 nm to 30 nm, 20 nm to 50 nm, 20 nm to 75 nm, 20 nm to 100 nm, 20 nm to 125 nm, 1 nm to 150 nm, 1 nm to 175 nm, 1 nm to 200 nm, 1 nm to 300 nm, 1 nm to 1,000 nm, 20 nm to 30 nm, 20 nm to 50 nm, 20 nm to 75 nm, 20 nm to 100 nm, 20 nm to 125 nm, 20 nm to 150 nm, 20 nm to 175 nm, 2 0 nm to 200 nm, 20 nm to 300 nm, 20 nm to 1,000 nm, 30 nm to 50 nm, 30 nm to 75 nm, 30 nm to 100 nm, 30 nm to 125 nm, 30 nm to 150 nm, 30 nm to 175 nm, 30 nm to 200 nm, 30 nm to 300 nm, 30 nm to 1,000 nm, 50 nm to 75 nm, 50 nm to 100 nm, 50 nm to 125 nm, 50 nm to 150 nm, 50 nm to 175 nm, 50 nm to 200 nm, 5 0 nm to 300 nm, 50 nm to 1,000 nm, 75 nm to 100 nm, 75 nm to 125 nm, 75 nm to 150 nm, 75 nm to 175 nm, 75 nm to 200 nm, 75 nm to 300 nm, 75 nm to 1,000 nm, 100 nm to 125 nm, 100 nm to 150 nm, 100 nm to 175 nm, 100 nm to 200 nm, 100 nm to 300 nm, 100 nm to 1,000 nm, 125 nm to 150 nm, 125 nm to 175 nm , 125 nm to 200 nm, 125 nm to 300 nm, 125 nm to 1,000 nm, 150 nm to 175 nm, 150 nm to 200 nm, 150 nm to 300 nm, 150 nm to 1,000 nm, 175 nm to 200 nm, 175 nm to 300 nm, 175 nm to 1,000 nm, 200 nm to 300 nm, 200 nm to 1,000 nm, or 300 nm to 1,000 nm, at 25 degrees Celsius (° C.), for example, for up to 30 days. In some embodiments, a population of particles can have an average (e.g., outer) 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 1,000 nm, for example, at 25 degrees Celsius (° C.) for up to 30 days.In some embodiments, the particle population may have an average (e.g., outer) 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 1,000 nm, for example, at 25 degrees Celsius (° C.) for up to 30 days. In some embodiments, the particle population may have an average (e.g., outer) 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 1,000 nm, for example, at 25 degrees Celsius (° C.) for up to 30 days. In some cases, the average diameter may be an average hydrodynamic particle size.

[0061] In some embodiments, the particle population may have an average (e.g., outer) diameter of 1 nm to 1,000 nm and is maintained at a body temperature of 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 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days. In some embodiments, the particle population may have an average (e.g., outer) diameter of 1 nm to 100 nm and is maintained at a body temperature of 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 to 7 days, more than 7 days, 7 to 30 days, or more than 30 days. In some embodiments, the particle population may have an average (e.g., outer) diameter of 100 nm to up to 200 nm, for example, at 37 degrees Celsius (° C.) 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 particle population may have an average (e.g., outer) diameter of 200 nm to up to 300 nm, for example, at 37 degrees Celsius (° C.) 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 particle population can have a diameter of 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 125 nm, 1 nm to 150 nm, 1 nm to 175 nm, 1 nm to 200 nm, 1 nm to 300 nm, 1 nm to 1,000 nm, 20 nm to 30 nm, 20 nm to 50 nm, 20 nm to 75 nm, 20 nm to 100 nm, 20 nm to 125 nm, 1 nm to 150 nm, 1 nm to 175 nm, 1 nm to 200 nm, 1 nm to 300 nm, 1 nm to 1,000 nm, 20 nm to 30 nm, 20 nm to 50 nm, 20 nm to 75 nm, 20 nm to 100 nm, 20 nm to 125 nm, 20 nm to 150 nm, 20 nm to 175 nm, 2 0 nm to 200 nm, 20 nm to 300 nm, 20 nm to 1,000 nm, 30 nm to 50 nm, 30 nm to 75 nm, 30 nm to 100 nm, 30 nm to 125 nm, 30 nm to 150 nm, 30 nm to 175 nm, 30 nm to 200 nm, 30 nm to 300 nm, 30 nm to 1,000 nm, 50 nm to 75 nm, 50 nm to 100 nm, 50 nm to 125 nm, 50 nm to 150 nm, 50 nm to 175 nm, 50 nm to 200 nm, 5 0 nm to 300 nm, 50 nm to 1,000 nm, 75 nm to 100 nm, 75 nm to 125 nm, 75 nm to 150 nm, 75 nm to 175 nm, 75 nm to 200 nm, 75 nm to 300 nm, 75 nm to 1,000 nm, 100 nm to 125 nm, 100 nm to 150 nm, 100 nm to 175 nm, 100 nm to 200 nm, 100 nm to 300 nm, 100 nm to 1,000 nm, 125 nm to 150 nm, 125 nm to 175 nm , 125 nm to 200 nm, 125 nm to 300 nm, 125 nm to 1,000 nm, 150 nm to 175 nm, 150 nm to 200 nm, 150 nm to 300 nm, 150 nm to 1,000 nm, 175 nm to 200 nm, 175 nm to 300 nm, 175 nm to 1,000 nm, 200 nm to 300 nm, 200 nm to 1,000 nm, or 300 nm to 1,000 nm, for example, at 37 degrees Celsius (° C.) for up to 30 days. In some embodiments, a population of particles can have an average (e.g., outer) 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 1,000 nm, for example, at 37 degrees Celsius (° C.) for up to 30 days.In some embodiments, the particle population may have an average (e.g., outer) 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 1,000 nm, for example, at 37 degrees Celsius (° C.) for up to 30 days. In some embodiments, the particle population may have an average (e.g., outer) 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 1,000 nm, for example, at 37 degrees Celsius (° C.) for up to 30 days. In some cases, the average diameter may be an average hydrodynamic particle size.

[0062] The modulators and / or antigens disclosed herein can be associated with the particles disclosed herein. For example, in some embodiments, the antigens disclosed herein and the STING modulators can be located in different regions of the particles disclosed herein. In some embodiments, the modulators disclosed herein (e.g., STING modulators) can be encapsulated in the particles (e.g., STING modulators 16 can be encapsulated in particles 12, e.g., Figure 1 In some embodiments, all or part of the disclosed antigens can be associated with (e.g., attached to, adhered to, adsorbed to, electrostatically interacted with, covalently bound to, non-covalently bound to, integrated into, or prepared into) the surface of a particle (e.g., antigen 14 on the surface of particle 12, such as Figure 1 In some embodiments, all or part of the antigen is encapsulated in a lipid-based particle. In some embodiments, association (e.g., adsorption) of the antigen to the surface of the particle can increase the stability of the particle and / or increase the delivery efficiency after administration (e.g., to a target tissue).

[0063] In some embodiments, one or more antigens of the present disclosure may be encapsulated in (e.g., lipid-based) particles of the present disclosure. In some embodiments, one or more regulators of the present disclosure (e.g., including STING regulators) may be associated with the outer surface of the particles. In some embodiments, one or more antigens of the present disclosure may be encapsulated in (e.g., lipid-based) particles, while one or more regulators of the present disclosure (e.g., including STING regulators) are associated with the outer surface of the particles. In some embodiments, one or more antigens of the present disclosure may be associated with the outer surface of (e.g., lipid-based) particles of the present disclosure. In some embodiments, one or more regulators of the present disclosure (e.g., including STING regulators) may be encapsulated in particles. In some embodiments, one or more antigens of the present disclosure may be associated with the outer surface of (e.g., lipid-based) particles of the present disclosure, while one or more regulators of the present disclosure (e.g., including STING regulators) are encapsulated in particles. In some embodiments, one or more antigens of the present disclosure and one or more regulators (e.g., including STING regulators) of the present disclosure may be encapsulated in (e.g., lipid-based) particles. In some embodiments, one or more antigens and one or more regulators (e.g., including STING regulators) of the present disclosure can be associated with the surface of the (e.g., lipid-based) particles of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be integrated into the membrane of the (e.g., lipid-based) particles of the present disclosure. In some embodiments, one or more regulators (e.g., including STING regulators) of the present disclosure can be integrated into the membrane of the (e.g., lipid-based) particles of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be encapsulated in the (e.g., lipid-based) particles of the present disclosure, and one or more regulators (e.g., including STING regulators) are integrated into the membrane of the (e.g., lipid-based) particles of the present disclosure. In some embodiments, one or more antigens of the present disclosure can be associated with the outer surface of the (e.g., lipid-based) particles, and one or more regulators (e.g., including STING regulators) are integrated into the membrane of the (e.g., lipid-based) particles of the present disclosure. In some embodiments, one or more regulators (e.g., including STING regulators) can be encapsulated in the (e.g., lipid-based) particles of the present disclosure, and one or more antigens of the present disclosure are integrated into the membrane of the particles. In some embodiments, one or more modulators (e.g., including STING modulators) can be associated with the outer surface of the (e.g., lipid-based) particles of the present disclosure, while one or more antigens of the present disclosure are integrated into the membrane of the particle. In some embodiments, incorporating antigens and modulators (e.g., STING modulators) of the present disclosure into (e.g., lipid-based) particles can facilitate coordinated cytoplasmic delivery.

[0064] 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 to, covalently coupled to, electrostatically interacted with, prepared on a membrane (e.g., a membrane surface) of a particle (e.g., a lipid-based nanoparticle) described herein. In some embodiments, the compositions may include divalent cations selected from Mn 2+ Mg 2+ , Ca 2+ and Zn 2+ For example, a composition may include 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+ .

[0065] antigen

[0066] Compositions for preventing or treating respiratory diseases or pathological conditions of subjects may include antigens. Antigens (or portions thereof) of the present disclosure may be associated with (e.g., attached to, adhered to, adsorbed to, electrostatically interacted with, covalently bound to, non-covalently bound to, integrated into or prepared into) the surface of particles (e.g., lipid-based particles, such as liposomes). In some embodiments, antigens may be suitable for developing immunity against diseases in subjects. For example, in some embodiments, one or more antigens in the compositions of the present disclosure are capable of inducing an immune response against diseases in subjects.

[0067] The antigens of the compositions described herein may include weakened or killed forms of disease-causing pathogens or portions thereof (e.g., one or more pathogens described herein). In some embodiments, the antigen may include a peptide or protein associated with a pathogen or portion thereof. In some embodiments, the antigen may include a surface protein (e.g., a receptor protein) or portion thereof of a pathogen. In some embodiments, the antigen may be in the form of a polynucleotide, for example, which may be used to express a second antigen (e.g., a plasmid DNA molecule expressing a second antigen).

[0068] The antigens of the compositions described herein may comprise a spike protein or a portion thereof. In some embodiments, the antigens of the compositions described herein may comprise at least one component of a coronavirus spike protein (S-protein). In some embodiments, the antigens of the compositions described herein may comprise a monomeric form of a coronavirus spike protein (S-protein). In some embodiments, the antigens of the compositions described herein may comprise a polymeric form of a coronavirus spike protein (S-protein).

[0069] In some embodiments, the antigens 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 trimer form of the SARS-CoV2 spike protein (S), a trimer form 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).

[0070] In some embodiments, the antigen may comprise a monomeric or multimeric form of the SARS-CoV2 spike protein (S) comprising a D614G mutation, an A222V mutation, an S477N mutation, a D80Y mutation, an S98F mutation, or a combination thereof.

[0071] In some embodiments, the antigen can be a mixture of SARS-CoV2 spike proteins with different mutations. In some embodiments, the antigen can include monomeric or polymeric forms of nucleocapsid proteins. For example, the antigens of the present disclosure can include monomeric or polymeric forms of SARS-CoV2 nucleocapsid (N) proteins. In some embodiments, the antigen can be a monomeric or polymeric form of a SARS-CoV2 nucleocapsid (N) protein comprising the A220V mutation.

[0072] In some embodiments, the antigen may include 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, and 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 the following antigens: 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), variants of SARS-CoV-2 (e.g., 20A.EU1 or spike mutant D614G) or a combination thereof. In some embodiments, the antigen can include a (e.g., coronavirus) alpha variant spike protein monomer, a (e.g., coronavirus) alpha variant spike protein trimer, a (e.g., coronavirus) beta variant spike protein monomer, a (e.g., coronavirus) beta variant spike protein trimer, a (e.g., coronavirus) gamma variant spike protein monomer, a (e.g., coronavirus) gamma variant spike protein trimer, a (e.g., coronavirus) delta variant spike protein monomer, a (e.g., coronavirus) delta variant spike protein trimer, a receptor binding domain (RBD) portion of the (e.g., coronavirus) alpha variant spike protein, an RBD portion of the (e.g., coronavirus) beta variant spike protein, an RBD portion of the (e.g., coronavirus) gamma variant spike protein, and / or an RBD portion of the (e.g., coronavirus) delta variant spike protein.

[0073] In some embodiments, an antigen of a composition or method described herein (e.g., an antigen comprising a nucleocapsid (N) protein) can have an amino acid sequence according to SEQ ID NO: 1. 1. In some embodiments, an antigen of a composition or method described herein (e.g., an antigen comprising a nucleocapsid protein) can 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) can be encoded by a nucleotide sequence according to SEQ ID NO: 2. In some embodiments, an antigen can be encoded by a nucleotide sequence that is 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% identical to SEQ ID NO: 2. In some embodiments, the antigen of the composition or method described herein (e.g., an antigen comprising a spike (S) protein) can have an amino acid sequence of SEQ ID NO: 3. In some embodiments, the antigen of the composition or method described herein (e.g., an antigen comprising a spike (S) protein) can 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: 3. In some embodiments, the antigen (e.g., an antigen comprising a spike (S) protein) can be encoded by a nucleotide sequence according to SEQ ID NO: 4. In some embodiments, the antigen (e.g., an antigen comprising a spike (S) protein) can be encoded by a nucleotide sequence that is 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% identical to SEQ ID NO: 4.

[0074] In some embodiments, it may be advantageous for a composition described herein (e.g., where the composition is used in a method described herein) to include both an N-protein and an S-protein. For example, a composition 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, a composition 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, a composition described herein can 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.

[0075] In some embodiments, it may be beneficial for a composition as described herein to include equal amounts of N-protein and S-protein. In some embodiments, a composition or method as described herein may benefit from adjusting the ratio of N-protein to S-protein so that the composition includes unequal amounts of N-protein and S-protein. For example, a composition as described herein may include more N-protein than S-protein. In some embodiments, it may be beneficial for a composition as described herein to include more S-protein than N-protein. In some embodiments, the compositions described herein may comprise a mass ratio of N-protein to S-protein (e.g., mass ratio of N-protein:S-protein) of 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: 1, 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, 4:3 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 the spike protein of the composition described herein to the nucleocapsid protein of the composition is 1:2. In some embodiments, the compositions described herein may comprise a molar ratio of N-protein to S-protein (e.g., a molar ratio of N-protein:S-protein) of 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: 1 , 2:1 to 3:4, 3:4 to 4:5, 4:5 to 1 :1, 1 :1 to 5:4, 5:4 to 4:3, 4:3 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.

[0076] In some embodiments, the compositions or methods described herein may include an N-protein associated with (e.g., embedded in, covalently coupled to, or non-covalently coupled to) the membrane of a (e.g., lipid-based) nanoparticle and an S-protein that is not associated with (e.g., contained within or external to the nanoparticle and not coupled to (e.g., in a co-processing)) the membrane of the nanoparticle. In some embodiments, the compositions or methods described herein may include an S-protein associated with (e.g., embedded in, covalently coupled to, or non-covalently coupled to) the membrane of a (e.g., lipid-based) nanoparticle and an N-protein that is not associated with (e.g., contained within or external to the nanoparticle and not coupled to (e.g., in a co-processing)) the membrane of the nanoparticle. In some embodiments, the compositions or methods described herein may include an N-protein associated with (e.g., embedded in, covalently coupled to, or non-covalently coupled to) the membrane of a (e.g., lipid-based) nanoparticle and an S-protein associated with the membrane of the nanoparticle. In some embodiments, a composition or method 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 external to the nanoparticle and not coupled to the nanoparticle (e.g., in a co-processing)) and an S-protein that is not associated with the membrane of the nanoparticle.

[0077] In some embodiments, the antigen can be a mixture of SARS-CoV2 spike protein and nucleocapsid protein. In some embodiments, the antigen can be a mixture of SARS-CoV2 spike protein and nucleocapsid protein with different mutations.

[0078] In some embodiments, the antigen can be 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, and 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.

[0079] In some embodiments, the compositions of the present disclosure are suitable for developing immunity against cancer in a subject. In some embodiments, the antigens of the present disclosure are suitable for developing immunity against cancer in a subject. For example, in some embodiments, the antigens in the therapeutic compositions of the present disclosure can induce an immune response against cancer (e.g., the aforementioned cancer) in a subject.

[0080] In some embodiments, the antigen of the composition of the present disclosure can be a weakened or killed form of a tumor cell (or part thereof) associated with cancer. In some embodiments, the antigen can include a peptide or protein associated with a cancer cell. In some embodiments, the antigen can include a surface protein (e.g., a receptor protein) of a cancer cell.

[0081] In some embodiments, the antigen may comprise a mutant protein of a cancer cell. In some embodiments, the antigen may be a synthetic long peptide that targets a cancer cell mutation.

[0082] The antigen of the composition disclosed herein can be in various forms. For example, in some embodiments, the antigen can be a recombinant peptide or protein, or a peptide epitope recognized by a T cell. In some embodiments, the antigen of the composition disclosed herein can include a tandem minigene. In some embodiments, the antigen can be in the form of a nucleotide expressing the antigen (e.g., a plasmid DNA molecule expressing the antigen).

[0083] Conditioner

[0084] The compositions described herein for preventing or treating a disease in a subject (e.g., comprising particles, such as lipid-based particles, and optionally, antigens) may include a modulator. The modulator of the composition 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 composition described herein may be a pattern recognition receptor agonist. In some embodiments, the modulator of the composition described herein may be a pattern recognition receptor antagonist. The compositions disclosed herein may include multiple 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 composition described herein may activate the STING pathway in a subject (e.g., a subject to which the composition is administered). In some embodiments, the modulator of the composition described herein may inhibit the STING pathway in a subject (e.g., a subject to which the composition is administered).

[0085] The STING modulator of the composition described herein may include bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), aminobenzimidazole, a derivative of aminobenzimidazole, a nucleotide modulator, a plasmid DNA modulator, a nucleic acid modulator, CF501, SHR1032, or a combination thereof.

[0086] In some embodiments, a STING modulator can be an antagonist of the STING pathway. In some embodiments, STING antagonists may be particularly effective in treating and / or preventing disease, for example, in cases where disease-causing pathogens (e.g., RNA viruses, such as rhinoviruses) utilize the STING pathway to promote viral replication. In some embodiments, STING antagonists can be used to inhibit viral replication by reducing viral accessibility to the STING pathway. In some embodiments, STING antagonists include, but are not limited to, C-178, H-151, and combinations thereof.

[0087] In some embodiments, the regulator of the composition described herein can be arranged in the internal space of the particle of the composition. For example, the regulator can be encapsulated in the particle (e.g., lipid-based particle, such as liposome) of the composition described herein. In some embodiments, the regulator of the composition described herein can be associated with the surface of the particle of the composition (e.g., the outer surface of the film). In some embodiments, the regulator associated with the particle surface is bound to the particle surface by covalent or non-covalent means and associates. In some embodiments, the regulator associated with the particle surface is associated by being integrated into the particle surface (e.g., film).

[0088] 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 can have at least 85%, at least 90%, at least 95% or 100% identity. In some embodiments, the portion of the nucleic acid sequence of the antigen of the composition described herein can have at least 85%, at least 90%, at least 95% or 100% identity with the nucleic acid sequence of the modulator of the composition. In some embodiments, an antigen comprising a nucleic acid sequence having at least 85%, at least 90%, at least 95% or 100% identity with the nucleic acid sequence of the modulator described herein can be encapsulated in the particles (e.g., lipid-based particles) of the composition described herein (e.g., wherein the modulator is also encapsulated in the lipid-based particles). In some embodiments, the antigens and modulators described herein (e.g., wherein the antigens and modulators comprise nucleic acid sequences having at least 85%, at least 90%, at least 95% or 100% identity) are all encapsulated in the lipid-based particles described herein. In some embodiments, a composition comprising an antigen having sequences at least 85%, at least 90%, at least 95%, or 100% identical and a modulator may be delivered intranasally.

[0089] In some cases, it may be advantageous to formulate the compositions described herein within a desired pH range. For example, the formulation of the compositions described herein (e.g., for intranasal delivery) between pH 4.0 and pH 7.5 (e.g., pH 4.5 to pH 6.5 or pH 5.5 to pH 6.5) can avoid irritation and / or histological damage to intranasal tissues, which may occur if extremely acidic or extremely alkaline formulations are used for intranasal delivery (e.g., because the pH of the nasal cavity can be between 5.5 and 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 can have a pH of 4.0 to 4.5, 4.0 to 4.8, 4.0 to 5.0, 4.0 to 5.2, 4.0 to 5.5, 4.0 to 5.8, 4.0 to 6, 4.0 to 6.2, 4.0 to 6.5, 4.0 to 7, 4.0 to 7.5, 4.5 to 4.8, 4.5 to 5.0, 4.5 to 5.2, 4.5 to 5.5, 4.5 to 5.8, 4.5 to 6.0, 4.5 to 6.2, 4.5 to 6.5, 4.5 to 7.0, 4.5 to 7.5, 4.8 to 5.0, 4.8 to 5.2, 4.8 to 5.5, 4.8 to 5.8, 4.8 to 6.0, 4.8 to 6.2, 4.8 to 6.5, 4.8 to 7.0, 4.8 to 7.5, 5.0 to 5.2, 5.0 to 5.5, 5.0 to 5.8, 5.0 to 6.0, 5.0 to 6.2, 5.0 to 6.5, 5.0 to 7.0, 5 to 7.5, 5.2 to 5.5, 5.2 to 5.8, 5.2 to 6.0, 5.2 to 6.2, 5.2 to 6.5, 5.2 to 7.0, 5.2 to 7.5, 5.5 to 5.8, 5.5 to 6.0, 5.5 to 6.2, 5.5 to 6 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 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.

[0090] In some cases, it may be advantageous to formulate the compositions described herein within a desired osmotic pressure range. For example, a formulation of the compositions described herein (e.g., for intranasal delivery) with an osmotic pressure of 50 to 900 mOsm / kg can improve absorption while avoiding potential epithelial damage (e.g., which may occur when the composition osmotic pressure is very low) and avoiding increased mucosal secretion (e.g., which may occur when the composition osmotic pressure is very high) (e.g., because the nasal osmotic pressure can be about 280 mOsm / kg). In some cases, the compositions described herein may have an osmotic pressure of 50 mOsm / kg to 900 mOsm / kg. In some cases, the compositions described herein can have a viscosity of 50 mOsm / kg to 150 mOsm / kg, 50 mOsm / kg to 250 mOsm / kg, 50 mOsm / kg to 300 mOsm / kg, 50 mOsm / kg to 350 mOsm / kg, 50 mOsm / kg to 450 mOsm / kg, 50 mOsm / kg to 550 mOsm / kg, 50 mOsm / kg to 650 mOsm / kg, 50 mOsm / kg to 750 mOsm / kg, 50 mOsm / kg to 800 mOsm / kg, 50 mOsm / kg to 900 mOsm / kg, 100 mOsm / kg to 1100 mOsm / kg, 100 mOsm / kg to 1200 mOsm / kg, 100 mOsm / kg to 1300 mOsm / kg, 100 mOsm / kg to 1400 mOsm / kg, 100 mOsm / kg to 1500 mOsm / kg, mOsm / kg to 850mOsm / kg, 50mOsm / kg to 900mOsm / kg, 150mOsm / kg to 250mOsm / kg, 150mOsm / kg to 300mOsm / kg, 150mOsm / kg to 350mOsm / kg, 150mOsm / kg to 450mOsm / kg, 150mOsm / kg to 550mOsm / kg, 150mOsm / kg to 650mOsm / kg, 150mOsm / kg to 750mOsm / kg, 150mOsm / kg to / kg to 850mOsm / kg, 150mOsm / kg to 900mOsm / kg, 250mOsm / kg to 300mOsm / kg, 250mOsm / kg to 350mOsm / kg, 250mOsm / kg to 450mOsm / kg, 250mOsm / kg to 550mOsm / kg, 250mOsm / kg to 650mOsm / kg, 250mOsm / kg to 750mOsm / kg, 250mOsm / kg to 850mOsm / kg, 250 mOsm / kg to 900mOsm / kg, 300mOsm / kg to 350mOsm / kg, 300mOsm / kg to 450mOsm / kg, 300mOsm / kg to 550mOsm / kg, 300mOsm / kg to 650mOsm / kg, 300mOsm / kg to 750mOsm / kg, 300mOsm / kg to 850mOsm / kg, 300mOsm / kg to 900mOsm / kg, 350mOsm / kg to 450mOsm / kg,350mOsm / kg to 550mOsm / kg, 350mOsm / kg to 650mOsm / kg, 350mOsm / kg to 750mOsm / kg, 350mOsm / kg to 850mOsm / kg, 350mOsm / kg to 900mOsm / kg, 450mOsm / kg to 550mOsm / kg, 450mOsm / kg to 650mOsm / kg, 450mOsm / kg to 750mOsm / kg, 450mOsm / kg to 850mOsm / kg, 450mOsm / kg to 900mOsm / kg, 55 or 850 mOsm / kg to 900 mOsm / kg. In some cases, the compositions described herein can have an osmotic pressure of 50mOsm / kg, 150mOsm / kg, 250mOsm / kg, 300mOsm / kg, 350mOsm / kg, 450mOsm / kg, 550mOsm / kg, 650mOsm / kg, 750mOsm / kg, 850mOsm / kg, or 900mOsm / kg. In some cases, the compositions described herein can have an osmotic pressure of at least 50mOsm / kg, 150mOsm / kg, 250mOsm / kg, 300mOsm / kg, 350mOsm / kg, 450mOsm / kg, 550mOsm / kg, 650mOsm / kg, 750mOsm / kg, 850mOsm / kg, or 900mOsm / kg. In some cases, a composition described herein may have an osmotic pressure of at most 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.

[0091] In some cases, it may be advantageous to formulate the compositions described herein within a desired viscosity range. For example, a formulation of the compositions described herein (e.g., for intranasal delivery) having a viscosity of, for example, 1.1 cP (centipoise) to 50 cP (e.g., 1.5 cP at least 50 cP) can increase the residence time in the nasal cavity while adversely affecting the droplet size (e.g., affecting 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 can have a molecular weight 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 to 7cP, 1.1cP to 10cP, 1.1cP to 15cP, 1.1cP to 25cP, 1.1cP to 50cP, 1.1cP to 100cP, 1.5cP to 2cP, 1.5cP to 5cP, 1.5cP to 7cP, 1.5cP to 10cP, 1.5cP to 15cP, 1.5cP to 25cP, 1.5cP to 50cP, 1.5cP to 100cP, 2cP to 5cP, 2cP to 7cP, 2cP to 10cP, 2cP to 15cP, 2cP to 25cP, 2cP to 50cP, 2cP to 100cP, 5cP to 7cP, 5cP to 10cP, 5cP to 15cP, 5cP to 25cP, 5cP to 50cP, 5cP to 100cP, 7cP to 10cP, 7cP to 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 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 can 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 can 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.

[0092] How to use

[0093] In additional embodiments, the present disclosure relates to methods of treating or preventing a disease in a subject by administering to the subject a therapeutic composition of the present disclosure. Figure 2A In the more specific embodiment shown, the methods of the present disclosure include the step of administering a therapeutic composition to a subject (step 20) to treat or prevent a disease in the subject (step 22).

[0094] As described in more detail herein, the therapeutic compositions and methods of the present disclosure can have a variety of embodiments. For example, the therapeutic compositions of the present disclosure can include multiple types of STING modulators and antigens. In addition, the methods of the present disclosure can be used to administer the therapeutic compositions of the present disclosure to many subjects to treat or prevent a variety of diseases in a variety of ways.

[0095] application

[0096] In some embodiments, the compositions and / or methods described herein can be used to prevent or treat respiratory diseases or conditions, such as respiratory diseases or conditions associated with influenza, coronavirus, respiratory syncytial virus, rhinovirus. In some embodiments, the compositions and / or methods described herein can be used to prevent or treat 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 can be used to prevent and / or treat cancer, such as lung cancer.

[0097] There is an urgent need for safe and effective vaccines to prevent respiratory diseases and cancer. In some embodiments, the compositions described herein may include vaccines or therapeutic treatments (e.g., intranasal delivery) containing liposomes containing agonists of the stimulator of interferon genes (STING) pathway to achieve humoral immunity, T cell immunity, systemic immunity, and / or mucosal immunity.

[0098] Innate immunity is the first line of defense against invading pathogens. Innate immunity (unlike adaptive immunity, which can be tailored to each pathogen) can be triggered by pattern recognition receptors (PRRs) on host cells that recognize conserved pathogen-associated molecular patterns (PAMPs) 1. This recognition of patterns ensures an immediate response that does not require customization.

[0099] In the case of RNA viruses, recognition and activation of virus-specific RNA molecules can lead to the activation of interferon regulatory factors (IRFs) and nuclear factor κB (NF-κB). Together, these transcriptional regulators initiate a broad antiviral program, including the synthesis and secretion of type I and type III interferons and the subsequent upregulation of IFN-stimulated genes (ISGs).

[0100] This comprehensive antiviral program can exert a strong selective pressure on viral replication. Viruses have evolved sophisticated countermeasures to interfere with interferon signaling. The interaction between interferon-mediated responses and viral countermeasures is heterogeneous and may be an explanation for the heterogeneity of morbidity and mortality in humans.

[0101] Type 1 interferon responses can be suppressed in pathogen infection (e.g., coronavirus or other respiratory pathogens), and the balance between ISG and NF-κB-mediated proinflammatory responses can subsequently become dysregulated. Thus, patients with advanced disease following pathogen infection (e.g., infection with respiratory pathogens) may have low interferon signaling but excessive tumor necrosis factor (TNF) and interleukin 6 (IL-6) secretion. Indeed, people with anti-interferon autoantibodies that neutralize interferon function may be at high risk for developing advanced disease.

[0102] Similarly, people with inborn errors of type I IFN immunity may develop life-threatening COVID-19 at higher rates than people without such errors. In some embodiments, pre-treatment of cell lines with type 1 interferon can inhibit viral replication in otherwise susceptible cells. Without being limited by theory, these considerations may help explain the lack of a robust type I IFN response as the basis for late-stage COVID-19.

[0103] The stimulator of interferon genes (STING) pathway is a PRR that senses circular DNA dinucleotides and activates IRF3 and NF-κB, leading to the synthesis of ISGs. Although originally thought to be important for sensing bacteria and DNA viruses, the role of STING in RNA virus-mediated type I interferon and cytokine production requires more detailed investigation. As shown in this article, the role of STING in RNA virus-mediated type I interferon and cytokine production may be specific to the virus and cell type.

[0104] Double-stranded RNA from viruses such as coronaviruses in the cytoplasm of human cells can be sensed by RIG-I-like receptors, including retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation gene 5 (MDA5). In some embodiments, the sensing pathways downstream of these receptors lead to the activation of two IKK-related kinases, namely TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi). In some embodiments, these kinases are also involved in the activated STING pathway, indicating that although PRRs have evolved to sense different molecules, their downstream responses may be converging.

[0105] The use of STING agonists as therapeutics could exploit such conserved downstream effector responses to promote balanced activation of type I IFNs, even in the context of SARS-CoV2 infection. Release of type I IFNs using STING agonists independent of RIG-1-like receptors raises the possibility that they will not be affected by virus-evolved responses.

[0106] The release of interferons may enable a broad antiviral program to limit viral replication and thus reduce disease severity. On the other hand, RNA viruses (such as rhinoviruses) can hijack the STING pathway to promote viral replication. In this case, it would be useful to inhibit viral replication by suppressing inflammation using STING antagonists.

[0107] Without being limited by theory, the therapeutic compositions of the present disclosure may be used to treat or prevent disease through a variety of mechanisms of action. For example, in some embodiments, the therapeutic compositions of the present disclosure may be used to treat a disease in a subject. In some embodiments, the therapeutic compositions of the present disclosure may be used to prevent a disease in a subject. In some embodiments, the therapeutic compositions of the present disclosure may be used to treat and prevent a disease in a subject. In some embodiments, the therapeutic compositions of the present disclosure may be used to treat or prevent a disease in a subject by developing immunity to the disease in the subject.

[0108] For example, in some embodiments, the therapeutic compositions of the present disclosure can elicit an immune response against a disease in a subject. In some embodiments, the therapeutic compositions of the present disclosure can elicit such immunity by at least one of innate immunity, mucosal immunity, systemic immunity, cellular immunity, humoral immunity, T cell immunity, systemic neutralizing antibody production, induction of IgG responses, induction of IgA responses, induction of IgM responses, induction of T cell responses, induction of mucosal IgA responses in the lungs and nasal compartments, induction of Th1 T cell responses, induction of CD8+T cell responses, induction of CD4+T cell responses, induction of NK cell responses, activation or inhibition of the interferon gene stimulator (STING) pathway, or a combination thereof.

[0109] In some embodiments, the compositions disclosed herein can elicit an average dilution titer of at least 1:25, greater than 1:25 (e.g., an average dilution titer of IgG or IgA specific for a pathogen antigen or a portion thereof) in the serum of a subject. In some embodiments, the compositions disclosed herein can elicit an average dilution titer 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 (e.g., an average dilution titer of IgG or IgA specific for a pathogen antigen or a portion thereof) in the bronchoalveolar lavage fluid (BALF) of a subject.

[0110] In some embodiments, the compositions of the present disclosure can cause or contribute to the development of immunity to a disease in a subject, for example, by inducing an innate immune response to a disease in a subject. In some embodiments, the innate immune response elicited can result in activation of interferon regulatory factors (IRFs), nuclear factor κB (NF-κB), or activation of a combination thereof. In some embodiments, inducing an innate immune response can result in the synthesis and secretion of type I and type III interferons (IFNs) and the subsequent upregulation of IFN-stimulated genes (ISGs).

[0111] In some embodiments, the therapeutic compositions of the present disclosure can be used to develop immunity to the disease in the subject, for example, through mucosal immunity and / or systemic immunity. In some embodiments, the therapeutic compositions of the present disclosure can help develop immunity of the subject through mucosal immunity, systemic immunity and cellular immunity. In some embodiments, systemic immunity can be developed by the production of neutralizing antibodies against antigens. In some embodiments, cellular immunity can be developed in spleen cells and / or lung cells. In some embodiments, mucosal immunity can be developed by the production of IgA in the nasal compartment and lungs and the production of IgA secreting cells in the spleen.

[0112] The methods and therapeutic compositions of the present disclosure can provide numerous 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.

[0113] Furthermore, in some embodiments, the therapeutic compositions of the present disclosure can prevent the establishment of an initial viral reservoir (eg, in the nasal compartment) and can help control viral transmission between individuals and / or transmission within individuals.

[0114] In some embodiments, the therapeutic compositions of the present disclosure can correct defects in interferon activation, which may be one of the main mechanisms of respiratory virus-mediated escape. In addition, the therapeutic compositions of the present disclosure can provide a universal mucosal adjuvant for the development of broad-spectrum therapeutic compositions against many pathogens, such as coronaviruses or other respiratory pathogens (including other viral respiratory pathogens), for example, because the therapeutic compositions of the present disclosure can be used in some embodiments to elicit one or more of IgM, IgG, IgA, and T cell responses in a subject.

[0115] In some embodiments, the methods and therapeutic compositions of the present disclosure may have veterinary applications. In some embodiments, veterinary applications include the treatment or prevention of disease in different types of animals and other similar applications.

[0116] Preparation and administration

[0117] Compositions disclosed herein can have various forms. In some embodiments, compositions described herein can be delivered as soluble liquids. In some embodiments, therapeutic compositions disclosed herein are suitable for intranasal and / or inhalation administration to a subject. In some embodiments, intranasal delivery of compositions disclosed herein can be used for targeting intranasal compartment tissue. In some embodiments, inhalation administration of compositions disclosed herein can be used for targeting lung compartment tissue.

[0118] In some embodiments, a syringe or liquid dropper can be used to administer a composition described herein intranasally to a subject. In some embodiments, administering a composition described herein to a subject can include the use of a nozzle, a sprayer, and / or an atomizer. In some embodiments, the therapeutic compositions of the present disclosure can be self-administered.

[0119] In some embodiments, the compositions of the present disclosure also include one or more stabilizers. In some embodiments, stabilizers include, but are not limited to, antioxidants, sequestrants, UV stabilizers, or combinations thereof.

[0120] In some embodiments, the compositions of the present disclosure also include 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.

[0121] In some embodiments, the compositions of the present disclosure also include one or more excipients. In some embodiments, excipients include but are not limited to lactose, sucrose, starch powder, cellulose alkanoate, trehalose, cellulose alkyl esters, talcum powder, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gelatin, gum arabic, trehalose, sodium alginate, polyvinyl pyrrolidone, polyvinyl alcohol or a combination thereof.

[0122] 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 its components) may increase the storage stability (e.g., shelf stability) 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 its components) may make the compositions easy to prepare, for example, allowing for use (e.g., administration to subjects) in areas where material preparation facilities are not readily available. For example, the regulators, lipid-based particles, and / or antigens described herein may be lyophilized and then rehydrated and mixed (e.g., as described herein) to prepare the compositions described herein at a subject administration site away from permanent medical or pharmaceutical facilities. In some embodiments, the lyophilized compositions described herein (or portions thereof, such as lyophilized particles or lyophilized antigens or lyophilized regulators) may be stored at 4°C. In some embodiments, a lyophilized composition described herein (or a portion thereof) can be stored at 4° C. and used for up to 1 week, up to 2 weeks, up to 3 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, up to 6 months, up to 7 months, up to 8 months, up to 9 months, up to 10 months, or up to 12 months.

[0123] 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.

[0124] The compositions of the present disclosure can be administered by a variety of methods. For example, in some embodiments, administration is performed by methods including but not limited to intravenous administration, intramuscular administration, intradermal administration, intraperitoneal administration, subcutaneous administration, spray-based administration, aerosol-based administration, intraocular administration, oral administration, intraocular administration, intratracheal administration, intranasal administration, inhalation administration, or a combination thereof.

[0125] In some embodiments, the compositions of the present disclosure can be administered as a single dose (e.g., by intranasal administration). In some embodiments, the methods of the present disclosure may include administering a dose of the compositions described herein to an object (e.g., an animal object) (e.g., by intranasal administration) for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, or at least 28 days after exposure (or suspected exposure) to a pathogen that can cause a disease (such as, a respiratory disease described herein). For example, a method for treating a subject having, at risk of having, or suspected of having been exposed to a pathogen that can cause a disease (such as, a respiratory disease) can comprise administering to a subject (e.g., an animal subject) one or more doses of a composition described herein (e.g., by intranasal administration) for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, or at least 28 days after exposure (or suspected exposure) to the pathogen. In some cases, a method for treating a subject having an infectious disease (such as a respiratory disease) from a pathogen (e.g., selected from the pathogens described herein) can include administering to a subject (e.g., an animal subject) one or more doses of a composition described herein (e.g., by intranasal administration) for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, or at least 28 days after exposure (or suspected exposure) to the pathogen.In some cases, a method for treating a subject having an infectious disease (such as a respiratory disease) caused by a pathogen (e.g., a pathogen selected from the ones described herein) can include administering to a subject (e.g., an animal subject) one or more doses of a composition described herein (e.g., by intranasal administration) for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, or at least 28 days after exposure (or suspected exposure) to the pathogen.

[0126] In some cases, a method for slowing or stopping the rate of disease progression caused by a pathogen (e.g., selected from the pathogens described herein) in a subject can include administering to a subject (e.g., an animal subject) one or more doses of a composition described herein (e.g., by intranasal administration) for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 14 days, or at least 28 days after exposure (or suspected exposure) to the pathogen. In some cases, a method for slowing or stopping the rate of progression of a disease caused by a pathogen (e.g., selected from the pathogens described herein) in a subject can comprise administering to a subject (e.g., an animal subject) one or more doses of a composition described herein (e.g., by intranasal administration) for 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 subject's disease (or its stage, severity, or a change in its stage or severity).

[0127] In some cases, a method for reducing the risk of or preventing transmission of a pathogen (or a disease state caused by a pathogen) from a first subject to a second subject can include administering one or more doses of a composition described herein (e.g., by intranasal administration) to a first subject (e.g., an animal subject) for 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 the first subject has been exposed (or is suspected of being exposed) to the pathogen. In some cases, a method for reducing the risk of or preventing transmission of a pathogen (or a disease state caused by a pathogen) from a first subject to a second subject may comprise administering one or more doses of a composition described herein (e.g., by intranasal administration) to a second subject (e.g., an animal subject) for 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 its stage, severity, or change in its stage or severity) in the first subject. In some cases, a method for reducing the risk of or preventing transmission of a pathogen (or a disease state caused by a pathogen) from a first subject to a second subject can comprise administering one or more doses of a composition described herein (e.g., by intranasal administration) to a second subject (e.g., an animal subject) for 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 the risk of or preventing transmission of a pathogen (or a disease state caused by a pathogen) from a first subject to a second subject may comprise administering to a first subject (e.g., an animal subject) one or more doses of a composition described herein (e.g., by intranasal administration) for 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 a clinical diagnosis of the disease (or its stage, severity, or a change in its stage or severity) in the first subject.

[0128] In some cases, a method for reducing the risk of infection with a pathogen (or a disease state caused by a pathogen) or preventing a subject from infection with a pathogen (or a disease state caused by a pathogen) can include administering one or more doses of a composition described herein (e.g., by intranasal administration) 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.

[0129] In some embodiments, the therapeutic composition of the present invention can be given in multiple doses. In some embodiments, the method for treating or preventing a subject (e.g., an animal subject) disease can include giving 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 15 to 20, 20 to 25, 25 to 30, 30 to 40, 40 to 50, or more than 50 doses. In some embodiments, the composition of the present invention can be given to an object (e.g., an animal subject) at multiple time points (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 time points). In some embodiments, the method of the present invention can include giving to an object a first dose comprising a composition of the present invention and a second dose comprising a composition of the present invention. 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 methods of the present disclosure may include administering a first dose and a second dose to an object (e.g., an animal object) at intervals of 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. In some embodiments, the methods of the present disclosure may include administering a first dose and a second dose to an object (e.g., an animal object) at intervals of 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. In some embodiments, the methods of the present disclosure may include administering a first dose and a second dose to an object (e.g., an animal object) at intervals of 6 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 hours, 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 (e.g., wherein each dose in the multiple doses comprises one or more compositions of the present disclosure) to a subject (e.g., an animal subject) after administering the first dose in a plurality of doses, 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 administering the predose. In some embodiments, the methods of the present disclosure may include administering each dose (e.g., wherein each dose in the multiple doses comprises one or more compositions of the present disclosure) 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 administering the predose.In some embodiments, the method of the present disclosure may include administering each dose (e.g., wherein each dose in the multiple doses comprises one or more compositions of the present disclosure) to an object (e.g., an animal object) after 6 hours, 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, up to 5 days, 6 days, 7 days, 14 days, or 28 days after the first dose in the multiple doses. In some embodiments, the composition is administered to the object in two, three, four, five, or more than five doses. In some embodiments, the second, third, and fourth doses are administered (e.g., four doses) 12 to 36 hours after the predose. In some embodiments, the second, third, and fourth doses are administered (e.g., four doses) 1, 2, 4, 6, 8, 10, 12, 18, 24, 30, or 36 hours after the predose.

[0130] In some embodiments, a composition described herein (e.g., a "NanoSTING" composition comprising a lipid-based particle, a modulator, and an antigen) can be formulated and / or administered as a monotherapy (e.g., for treating a disease or condition in a subject, e.g., a disease or condition resulting from exposure to a pathogen).

[0131] The compositions or methods described herein (e.g., for treating a disease or condition of a subject, for preventing a disease or condition of a subject, or for preventing a disease or condition from spreading from a first subject to a second subject) may include nanoparticles (e.g., lipid-based nanoparticles) and modulators (e.g., pattern recognition receptor agonist proteins, such as STING agonists). In some embodiments, the composition or method comprising nanoparticles (e.g., lipid-based nanoparticles) and modulators may lack antigens. In some embodiments, the modulator of the composition or method comprising nanoparticles and modulators but lacking antigens may be encapsulated within the nanoparticles. In some embodiments, the composition comprising nanoparticles and modulators but lacking antigens may be administered to a subject (e.g., a human subject or a non-human mammalian subject) at a dose, dosage form, and or treatment as described herein or by vaccination on a schedule. For example, before a subject (e.g., a human subject or a non-human mammalian subject) is exposed to an infectious agent (e.g., a virus, such as a respiratory virus such as a coronavirus, influenza virus, parainfluenza virus, or rhinovirus), a composition comprising nanoparticles and modulators but lacking antigens may be administered to the subject. In some embodiments, before the object is infected and / or respiratory diseases or conditions occur (e.g., associated with respiratory viruses) or cancer (such as, lung cancer) occurs in some embodiments, a composition comprising nanoparticles and modulators but lacking antigens can be administered to the object (e.g., human object or non-human mammal object). In some embodiments, after the object (e.g., human object or non-human mammal object) is exposed to infectious substances (e.g., viruses, such as respiratory viruses such as coronavirus, influenza virus, parainfluenza virus or rhinovirus), a composition comprising nanoparticles and modulators but lacking antigens can be administered to the object. In some embodiments, after the object is infected and / or respiratory diseases or conditions occur (e.g., associated with respiratory viruses) or cancer (such as, lung cancer) occurs in some embodiments, a composition comprising nanoparticles and modulators but lacking antigens can be administered to the object (e.g., human object or non-human mammal object). In some embodiments, a composition comprising nanoparticles and modulators but lacking antigens can be administered to the object intranasally. In some embodiments, compositions and methods comprising nanoparticles and modulators but lacking antigens can be relatively cheap or logistically uncomplicated to manufacture, store and / or prepare. In some embodiments, a composition comprising nanoparticles and a modulator but lacking an antigen may be administered to a subject in combination with a composition comprising nanoparticles, a modulator, and an antigen, e.g., simultaneously, sequentially, or separately as part of a single treatment or vaccination regimen.

[0132] The compositions or methods described herein (e.g., for treating a disease or condition of a subject, for preventing a disease or condition of a subject, or for preventing a disease or condition from spreading from a first subject to a second subject) may include a modulator as described herein (e.g., a pattern recognition receptor agonist protein, such as a STING agonist). In some embodiments, a composition or method comprising a modulator may lack an antigen and a nanoparticle (e.g., a liposome). In some embodiments, a modulator may 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 of the subject (e.g., cancer or a disease or condition associated with a respiratory virus or respiratory disease). In some embodiments, a composition comprising a modulator but lacking an antigen or nanoparticle may be administered to a subject (e.g., a human subject or a non-human mammalian subject) at a dose, dosage form, and / or treatment or vaccination time course as described herein. For example, a composition comprising a modulator but lacking an antigen or nanoparticle may be administered to a subject before the subject (e.g., a human subject or a non-human mammalian subject) is exposed to an infectious agent (e.g., a virus, such as a respiratory virus such as a coronavirus, influenza virus, parainfluenza virus, or rhinovirus). In some embodiments, before the subject is infected and / or a respiratory disease or condition (e.g., associated with a respiratory virus) or cancer (e.g., lung cancer) occurs in some embodiments, a composition comprising a regulator but lacking an antigen or nanoparticle can be administered to the subject (e.g., a human subject or a non-human mammal subject). In some embodiments, after the subject (e.g., a human subject or a non-human mammal subject) is exposed to an infectious agent (e.g., a virus, such as a respiratory virus such as a coronavirus, influenza virus, parainfluenza virus, or rhinovirus), a composition comprising a regulator but lacking an antigen or nanoparticle can be administered to the subject. In some embodiments, after the subject is infected and / or a respiratory disease or condition (e.g., associated with a respiratory virus) occurs or cancer (e.g., lung cancer) occurs in some embodiments, a composition comprising a regulator but lacking an antigen or nanoparticle can be administered to the subject (e.g., a human subject or a non-human mammal subject). In some embodiments, a composition comprising a regulator but lacking an antigen or nanoparticle can be administered to the subject intranasally. In some embodiments, compositions and methods comprising a regulator but lacking an antigen or nanoparticle can be relatively cheap or logistically uncomplicated to manufacture, store, and / or prepare. In some embodiments, a composition comprising nanoparticles and a modulator but lacking an antigen may be administered to a subject in combination with a composition comprising nanoparticles, a modulator, and an antigen, e.g., simultaneously, sequentially, or separately as part of a single treatment or vaccination regimen.

[0133] 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 preventing a subject from contracting the disease or condition or transmitting the disease or condition to a second subject) can include 0.1 micrograms to 200 micrograms 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 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 2 0μg, 1μg to 30μg, 1μg to 50μg, 1μg to 100μg, 1μg to 120μg, 1μg to 200μg, 2μg to 3μg, 2μg to 5μg, 2μg to 10μg, 2μg to 20μg, 2μg to 30μg, 2μg to 50μg, 2μg to 100μg, 2μg to 120μg, 2μg to 200μg, 3μg to 5μg, 3μg to 10μg, 3μg to 20μg, 3μg to 30μg, 3μg to 50μg, 3μg to 100μg, 3μg to 120μg, 3μg to 200μg, 5μg to 10μg, 5μg to 20μg, 5μg to 30μg, 5μg to 50μg, 5μg to 100μg, 5μg to 120μg, 5μg to 200μg, 10μg to 20μg, 10μg to 30μg, 10μg to 50μg, 10μg to 100μg, 10μg to 120μg, 10μg to 200μg, 20μg to 30μg, 20μg to 50μg , 20 micrograms to 100 micrograms, 20 micrograms to 120 micrograms, 20 micrograms to 200 micrograms, 30 micrograms to 50 micrograms, 30 micrograms to 100 micrograms, 30 micrograms to 120 micrograms, 30 micrograms to 200 micrograms, 50 micrograms to 100 micrograms, 50 micrograms to 120 micrograms, 50 micrograms to 200 micrograms, 100 micrograms to 120 micrograms, 100 micrograms to 200 micrograms, or 120 micrograms to 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, a composition of the present disclosure may comprise 0.1 microgram, 1 microgram, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).In some embodiments, the compositions of the present disclosure may include at least 0.1 micrograms, 1 microgram, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, the compositions of the present disclosure may include at most 0.1 micrograms, 1 micrograms, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).

[0134] 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 preventing a subject from contracting the disease or condition or transmitting the disease or condition to a second subject) can contain 0.1 micrograms to 200 micrograms of particles (e.g., lipid-based particles (liposomes), such as lipid-based nanoparticles). In some embodiments, the compositions of the present disclosure may comprise 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 2 0μg, 1μg to 30μg, 1μg to 50μg, 1μg to 100μg, 1μg to 120μg, 1μg to 200μg, 2μg to 3μg, 2μg to 5μg, 2μg to 10μg, 2μg to 20μg, 2μg to 30μg, 2μg to 50μg, 2μg to 100μg, 2μg to 120μg, 2μg to 200μg, 3μg to 5μg, 3μg to 10μg, 3μg to 20μg, 3μg to 30μg, 3μg to 50μg , 3 μg to 100 μg, 3 μg to 120 μg, 3 μg to 200 μg, 5 μg to 10 μg, 5 μg to 20 μg, 5 μg to 30 μg, 5 μg to 50 μg, 5 μg to 100 μg, 5 μg to 120 μg, 5 μg to 200 μg, 10 μg to 20 μg, 10 μg to 30 μg, 10 μg to 50 μg, 10 μg to 100 μg, 10 μg to 120 μg, 10 μg to 200 μg, 20 μg to 30 μg, 20 μg to 50 In some embodiments, the present invention relates to particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) of 20 to 100 μg, 20 to 120 μg, 20 to 200 μg, 30 to 50 μg, 30 to 100 μg, 30 to 120 μg, 30 to 200 μg, 50 to 100 μg, 50 to 120 μg, 50 to 200 μg, 100 to 120 μg, 100 to 200 μg, or 120 to 200 μg of a drug. 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), such as 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), such as lipid-based nanoparticles). In some embodiments, the compositions of the present disclosure may comprise at most 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), such as lipid-based nanoparticles).

[0135] In some embodiments, a composition of the present disclosure (e.g., for treating a subject having a disease or condition (e.g., a respiratory disease), or preventing a subject from contracting the disease or condition or transmitting the disease or condition to a second subject) comprises 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles), which may comprise 0.1 micrograms to 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, the compositions of the present disclosure comprise 20 micrograms of particles (e.g., lipid-based particles (liposomes) such as lipid-based nanoparticles), which may comprise 0.1 microgram to 1 microgram, 0.1 microgram to 2 micrograms, 0.1 microgram to 3 micrograms, 0.1 microgram to 5 micrograms, 0.1 microgram to 10 micrograms, 0.1 microgram to 20 micrograms, 0.1 microgram to 30 micrograms, 0.1 microgram to 50 micrograms, 0.1 microgram to 100 micrograms, 0.1 microgram to 120 micrograms, 0.1 microgram to 200 micrograms, 1 microgram to 2 micrograms, 1 microgram to 1 ug to 3ug, 1ug ​​to 5ug, 1ug ​​to 10ug, 1ug ​​to 20ug, 1ug ​​to 30ug, 1ug ​​to 50ug, 1ug ​​to 100ug, 1ug ​​to 120ug, 1ug ​​to 200ug, 2ug to 3ug, 2ug to 5ug, 2ug to 10ug, 2ug to 20ug, 2ug to 30ug, 2ug to 50ug, 2ug to 100ug, 2ug to 120ug, 2ug to 200ug, 3ug to 5ug, 3ug to 10ug, 3ug to 20ug. ug, 3ug to 30ug, 3ug to 50ug, 3ug to 100ug, 3ug to 120ug, 3ug to 200ug, 5ug to 10ug, 5ug to 20ug, 5ug to 30ug, 5ug to 50ug, 5ug to 100ug, 5ug to 120ug, 5ug to 200ug, 10ug to 20ug, 10ug to 30ug, 10ug to 50ug, 10ug to 100ug, 10ug to 120ug, 10ug to 200ug, 20ug to 3 In some embodiments, the present invention relates to an agent comprising: a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising: a STING pathway activator such as cGAMP, a stimulant comprising:In some embodiments, a composition of the present disclosure comprising 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) may contain 0.1 micrograms, 1 micrograms, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., cGAMP)). In some embodiments, a composition of the present disclosure comprising 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) may contain at least 0.1 micrograms, 1 micrograms, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., cGAMP)). In some embodiments, a composition of the present disclosure comprising 20 micrograms of a particle (e.g., a lipid-based particle (e.g., a liposome), such as a lipid-based nanoparticle) may contain at most 0.1 micrograms, 1 microgram, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (such as, cGAMP)).

[0136] In some embodiments, the compositions of the present disclosure may include a variety of 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 types of antigens. For example, the compositions of the present disclosure may include monomeric antigens (e.g., monomeric S-proteins), trimeric antigens (e.g., trimeric S-proteins), chimeric antigens (e.g., chimeric S-proteins) and / or variants of antigens (e.g., variant S-proteins).

[0137] 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 preventing a subject from contracting the disease or condition or transmitting the disease or condition to a second subject) may comprise 0.1 micrograms to 200 micrograms of an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or an RSV antigen protein). In some embodiments, the compositions of the present disclosure may comprise 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, 1 μg to 30 μg, 1 μg to 50 μg, 1 μg to 100 μg, 1 μg to 120 μg, 1 μg to 200 μg, 2 μg to 3 μg, 2 μg to 5 μg, 2 μg to 10 μg, 2 μg to 20 μg, 2 μg to 30 μg, 2 μg to 50 μg, 2 μg to 100 μg, 2 μg to 120 μg, 2 μg to 200 μg, 3 μg to 5 μg, 3 μg to 10 μg, 3 μg to 20 μg, 3 μg to 30 μg, 3 μg to 50 μg, 3 μg to 100 μg, 3 μg to 120 μg, 3 μg to 200 μg, 5 μg to 10 μg, 5 μg to 20 μg, 5 μg to 30 μg, 5 μg to 50 μg, 5 μg to 100 μg, 5 μg to 120 μg, 5 μg to 200 μg, 10 μg to 20 μg, 10 μg to 30 μg, 10 μg to 50 μg, 10 μg to 100 μg, 10 μg to 120 μg, 10 μg to 200 μg, 20 μg to 30 μg, 20 μg to 50 μg, 20 μg to ug 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 an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or an RSV antigen protein). In some embodiments, the compositions of the present disclosure may contain 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 an RSV antigen protein).In some embodiments, the compositions of the present disclosure may contain 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 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 at most 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, a composition of the present disclosure (e.g., for treating a subject having a disease or condition (such as a respiratory disease), or preventing a subject from contracting the disease or condition or transmitting the disease or condition to a second subject) comprises 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles), which may comprise 0.1 micrograms to 200 micrograms of an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or an RSV antigen protein).In some embodiments, the compositions of the present disclosure comprise 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles), which may comprise 0.1 microgram to 1 microgram, 0.1 microgram to 2 micrograms, 0.1 microgram to 3 micrograms, 0.1 microgram to 5 micrograms, 0.1 microgram to 10 micrograms, 0.1 microgram to 20 micrograms, 0.1 microgram to 30 micrograms, 0.1 microgram to 50 micrograms, 0.1 microgram to 100 micrograms, 0.1 microgram to 120 micrograms, 0.1 microgram to 200 micrograms, 1 microgram to 2 micrograms, 1 microgram to 50 micrograms, 0.1 microgram to 100 micrograms, 0.1 microgram to 120 micrograms, 0.1 microgram to 200 micrograms, 1 microgram to 2 micrograms, 1 microgram to 50 micrograms, 3 μg, 1 μg to 5 μg, 1 μg to 10 μg, 1 μg to 20 μg, 1 μg to 30 μg, 1 μg to 50 μg, 1 μg to 100 μg, 1 μg to 120 μg, 1 μg to 200 μg, 2 μg to 3 μg, 2 μg to 5 μg, 2 μg to 10 μg, 2 μg to 20 μg, 2 μg to 30 μg, 2 μg to 50 μg, 2 μg to 100 μg, 2 μg to 120 μg, 2 μg to 200 μg, 3 μg to 5 μg, 3 μg to 10 μg, 3 μg to 20 μg, 3 μg to 3 μg to 30 μg, 3 μg to 50 μg, 3 μg to 100 μg, 3 μg to 120 μg, 3 μg to 200 μg, 5 μg to 10 μg, 5 μg to 20 μg, 5 μg to 30 μg, 5 μg to 50 μg, 5 μg to 100 μg, 5 μg to 120 μg, 5 μg to 200 μg, 10 μg to 20 μg, 10 μg to 30 μg, 10 μg to 50 μg, 10 μg to 100 μg, 10 μg to 120 μg, 10 μg to 200 μg, 20 μg to 30 μg, 20 μg to 30 μg, 100 μg, 100 μg, 100 μg, 20 μg, 20 μg, 20 μg, 200 μg, 30 μg, 30 μg, 100 μg, 20 μg, 200 μg, 30 μg, 30 μg, 30 μg, 30 μg, 50 μg, 100 μg, 100 μg, 200 μg, 120 μg, 100 μg, 200 μg, or 120 μg of an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or an RSV antigen protein). In some embodiments, a composition of the present disclosure comprises 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) may contain 0.1 micrograms, 1 microgram, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of an antigen (e.g., a nucleocapsid protein (N-protein), a spike protein (S-protein), or an RSV antigen protein).In some embodiments, the compositions of the present disclosure contain 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) that may contain at least 0.1 micrograms, 1 microgram, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms 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 contain 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) that may contain at most 0.1 micrograms, 1 micrograms, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of antigen (e.g., nucleocapsid protein (N-protein), spike protein (S-protein), or RSV antigen protein).

[0138] In some cases, a composition of the present disclosure (e.g., for treating a subject having a disease or condition (e.g., a respiratory disease), or preventing a subject from contracting the disease or condition or transmitting the disease or condition to a second subject) comprises at least 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) and at least 4 micrograms of an antigen, and may comprise 0.1 micrograms to 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, the compositions of the present disclosure comprise at least 20 μg of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) and at least 4 μg of antigen, and may comprise 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 2μg, 1μg to 3μg, 1μg to 5μg, 1μg to 10μg, 1μg to 20μg, 1μg to 30μg, 1μg to 50μg, 1μg to 100μg, 1μg to 120μg, 1μg to 200μg, 2μg to 3μg, 2μg to 5μg, 2μg to 10μg, 2μg to 20μg, 2μg to 30μg, 2μg to 50μg, 2μg to 100μg, 2μg to 120μg, 2μg to 200μg, 3μg to 5μg, 3μg to 10μg, 3μg to 20 μg, 3 μg to 30 μg, 3 μg to 50 μg, 3 μg to 100 μg, 3 μg to 120 μg, 3 μg to 200 μg, 5 μg to 10 μg, 5 μg to 20 μg, 5 μg to 30 μg, 5 μg to 50 μg, 5 μg to 100 μg, 5 μg to 120 μg, 5 μg to 200 μg, 10 μg to 20 μg, 10 μg to 30 μg, 10 μg to 50 μg, 10 μg to 100 μg, 10 μg to 120 μg, 10 μg to 200 μg, 20 μg to 30 , 20 to 50 μg, 20 to 100 μg, 20 to 120 μg, 20 to 200 μg, 30 to 50 μg, 30 to 100 μg, 30 to 120 μg, 30 to 200 μg, 50 to 100 μg, 50 to 120 μg, 50 to 200 μg, 100 to 120 μg, 100 to 200 μg, or 120 to 200 μg of a modulator (e.g., a SITNG pathway activator (e.g., a STING agonist)).In some embodiments, the compositions of the present disclosure comprise at least 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) and at least 4 micrograms of antigen, and may comprise 0.1 micrograms, 1 micrograms, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, the compositions of the present disclosure comprise at least 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) and at least 4 micrograms of antigen, and may comprise at least 0.1 micrograms, 1 micrograms, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)). In some embodiments, the compositions of the present disclosure comprise at least 20 micrograms of particles (e.g., lipid-based particles (e.g., liposomes), such as lipid-based nanoparticles) and at least 4 micrograms of antigen, and may comprise at most 0.1 micrograms, 1 microgram, 2 micrograms, 3 micrograms, 5 micrograms, 10 micrograms, 20 micrograms, 30 micrograms, 50 micrograms, 100 micrograms, 120 micrograms, or 200 micrograms of a modulator (e.g., a STING pathway activator (e.g., a STING agonist)).

[0139] In some embodiments, a dose administered to a subject according to 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 particles or comprising a modulator, particles, and an antigen). In some embodiments, a dose administered to a subject according to 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 to 1μg, 0.5μg to 2.5μg, 0.5μg to 5μg, 0.5μg to 7.5μg, 0.5μg to 9.3μg, 0.5μg to 10μg, 0.5μg to 15μg, 0.5μg to 20μg, 1μg to 2.5μg, 1μg to 5μg, 1μg to 7.5μg, 1μg to 9.3μg, 1μg to 10μg, 1 ug to 15ug, 1ug ​​to 20ug, 2.5ug to 5ug, 2.5ug to 7.5ug, 2.5ug to 9.3ug, 2.5ug to 10ug, 2.5ug to 15ug, 2.5ug to 20ug, 5ug to 7.5ug, 5ug to 9.3ug, 5ug to 10ug, 5ug to 15ug, 5ug to 20ug, 7.5ug In some embodiments, the dose administered to a subject according to the compositions or methods 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., comprising a modulator and particles or comprising a modulator, particles, and an antigen). In some embodiments, a dose administered to a subject according to a composition or method described herein may comprise at least 0.1 micrograms, 0.5 micrograms, 1.0 micrograms, 1.2 micrograms, 2.3 micrograms, 2.5 micrograms, 4.7 micrograms, 5.0 micrograms, 7.5 micrograms, 9.3 micrograms, 10 micrograms, 15 micrograms, or up to 20 micrograms of a composition described herein (e.g., comprising a modulator and particles or comprising a modulator, particles, and an antigen).In some embodiments, a dose administered to a subject according to a composition or method described herein may comprise up to 0.1 micrograms, 0.5 micrograms, 1.0 micrograms, 1.2 micrograms, 2.3 micrograms, 2.5 micrograms, 4.7 micrograms, 5.0 micrograms, 7.5 micrograms, 9.3 micrograms, 10 micrograms, 15 micrograms, or up to 20 micrograms of a composition described herein (e.g., comprising a modulator and particles or comprising a modulator, particles, and an antigen).

[0140] In some embodiments, the compositions of the present disclosure can be given in combination with other therapeutic treatments. In some embodiments, other therapeutic therapies but are not limited to immunotherapy, virotherapy, targeted inhibition, radiotherapy, chemotherapy or a combination thereof. In some embodiments, the compositions as described herein can include and / or be given together with 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)) in a treatment regimen (e.g., given simultaneously or non-simultaneously).

[0141] Object

[0142] The therapeutic composition of the present disclosure can be administered to an object in need. For example, in some embodiments, the object is a mammal (e.g., a human). In some embodiments, the object is a domestic animal. For example, the object can be a dog or a cat. In some embodiments, the object 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 object can be a human. In some embodiments, the object can be susceptible to or suffer from a respiratory infection. In some embodiments, the object is susceptible to respiratory viruses or suffers from a condition caused by respiratory viruses. For example, a composition or method can be administered to an object that has been exposed to or infected with a respiratory virus (e.g., 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 object can be susceptible to or suffer from cancer. In some embodiments, the subject may be selected for treatment due to the symptoms of one or more pathogen infection or disease infection (e.g., respiratory pathogen infection or respiratory disease infection). For example, the subject to be treated can be selected according to one or more symptoms, including persistent cough, elevated body temperature (e.g., greater than 100.4°C measured by forehead skin), body chills, joint pain, dyspnea or shortness of breath, pulmonary effusion, fatigue, headache, loss of taste or smell, or diagnostic tests (such as, PCR tests) are present. In some embodiments, based on population risk factors (such as obesity, advanced age (e.g., 65 years old or older), immune damage or pregnancy), the subject is selected for treatment with the composition disclosed herein. In some embodiments, the subject is selected for treatment based on the risk of pathogen infection or disease infection (e.g., respiratory pathogen infection or respiratory disease infection), for example, if the subject's occupation involves close interaction with customers, frequent interaction with high-risk groups, handling of biological samples, or close contact with potential infected individuals.

[0143] Treatment or prevention of disease

[0144] Pathogens and cancer have caused significant health and economic problems. For example, viral infections caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) have led to the COVID-19 pandemic, which is currently the most pressing health and economic crisis in the world. In addition, cancers (such as lung cancer) have a high mortality rate.

[0145] In addition, there are very limited therapeutic options to effectively treat and prevent cancer and infections caused by pathogens. For example, there are currently no drugs available to treat or prevent viral infections caused by SARS-CoV-2.

[0146] In addition, many vaccines aimed at preventing infection caused by pathogens are intramuscular vaccines, which are designed to induce systemic immunity without conferring innate 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 break through before spreading to the lungs.

[0147] Furthermore, the effectiveness of vaccine formulations is highly unpredictable. For vaccines that enter Phase I trials, only about one-third will receive final FDA approval. This success rate means that even with existing technologies to prime the immune system, the development of effective immunity to each pathogen requires the design and optimization of the right antigen and adjuvant.

[0148] Even with FDA approval, it does not mean the vaccine is effective. Developing a vaccine is a long process, and even in the context of the COVID19 pandemic, there are many challenges.

[0149] Although most platforms target the spike protein as an antigen, it is desirable to optimize the antigen design to ensure the effectiveness of the vaccine. The difficulty in selecting an appropriate vaccine design has been highlighted for HIV-1, hepatitis C, and malaria, where a vaccine has yet to be found despite decades of effort and multiple candidate vaccines.

[0150] Furthermore, in the case of respiratory pathogens, the nasal compartment is the first barrier that the pathogen needs to breach before spreading to the lungs. However, current intramuscular vaccines are designed to elicit systemic immunity and do not confer innate immunity, such as mucosal immunity.

[0151] Therefore, there is an urgent need for safe and durable therapeutic compositions to treat and prevent a variety of 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 to treat or prevent 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.

[0152] The therapeutic compositions and methods of the present disclosure can be used 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 can be an infection caused by a pathogen. In some embodiments, the disease can be a respiratory disease. In some embodiments, the pathogen can be a respiratory pathogen. In some embodiments, the respiratory pathogen can be a virus. In some embodiments, the virus can 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 variant thereof, or a combination thereof.

[0153] In some embodiments, the pathogen is a coronavirus. In some embodiments, coronaviruses include, but are 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), variants of SARS-CoV-2 (e.g., 20A.EU1 or spike mutant D614G) or combinations thereof. In some embodiments, coronaviruses are SARS-CoV-2 viruses, their alpha variants (e.g., B.1.1.7), their delta variants (e.g., B.1.617.2), their Omicron variants (e.g., B.1.1.529), or combinations thereof.

[0154] In some embodiments, the pathogen is an influenza virus. In some embodiments, the influenza virus comprises an oseltamivir-sensitive strain, a treatment-resistant strain, or a combination thereof.

[0155] In some embodiments of the compositions and methods of the invention, a subject (e.g., a mammalian subject, such as a human, mouse, hamster, dog, cat, or non-human primate) can be treated prophylactically to prevent viral infection (e.g., SARS-CoV-2 coronavirus) by intranasal delivery of the compositions described herein (including modulators, such as STING agonists and lipid-based nanoparticles) and antigens (e.g., spike proteins associated, embedded, or coupled to the outer surface of lipid-based nanoparticles) in a single dose or as part of a multi-dose regimen. In some embodiments, treatment with the composition can induce an immune response in the subject in one or more body cavities, including the blood, spleen, lungs, and / or nasal compartments. In some embodiments, the compositions and methods described herein can induce 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 lung tissue (e.g., BALF) or blood (e.g., serum) of the subject. In some embodiments, the compositions and methods described herein can increase the number of B cells secreting IgA and / or the number of antigen-specific T cells (e.g., spike protein-specific T cells), for example, in the spleen. In some embodiments, after administering the compositions described herein to a subject, an increase in antigen-specific T cells can be detected in the lungs. In some embodiments, after administering the compositions described herein to a subject, an increase in anti-antigen IgA and / or an increase in the presence and / or activity of germinal center (GC) B cells or follicular helper T (Tfh) cells can be detected in the nasal compartment.

[0156] In some embodiments, the disease to be treated or prevented in the subject is cancer. In some embodiments, the 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 a combination thereof. In some embodiments, the cancer can be lung cancer.

[0157] Example

[0158] Example 1: Preparation, characterization and stability of NanoSTING

[0159] This example describes the preparation, characterization, and stability of NanoSTING, a liposomal formulation containing the natural immunotransmitters, cyclic GMP-AMP and cGAMP ( Figure 3A ).

[0160] The nanoparticles promoted stability and delivery to alveolar macrophages, promoting responses in the upper airways and lungs. Dynamic light scattering (DLS) analysis showed that the average particle size of NanoSTING was 98 nm and the polydispersity index was 0.25 ( Figure 3E). The zeta potential of NanoSTING is -40 mV ( Figure 3F Administration of NanoSTING was able to induce an interferon response by using THP-1 monocytes modified to conditionally secrete luciferase downstream of an interferon regulatory factor (IRF)-responsive promoter ( Figure 3B ). THP-1 cells were inoculated with NanoSTING at doses ranging from 2.5-10.0 μg and kinetic measurements were performed over 24 hours by measuring luciferase activity in the supernatant. With 5 and 10 μg NanoSTING, low levels of luciferase activity were observed at 6 hours, with maximal secretion at 24 hours ( Figure 4 The longitudinal nanoparticle stability of NanoSTING 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 the charge changes of the nanoparticles. Although the hydrodynamic diameter of NanoSTING remained essentially unchanged over 30 days at 25°C ( Figure 3C ), but after 2 weeks at 37°C, the hydrodynamic diameter increased slightly (mean: 114 nm at 25°C, 154 nm at 37°C) ( Figure 3D ). The zeta potential did not change significantly at either temperature (-45 mV at both 25°C and 37°C). These results suggest that NanoSTING is immunologically active and that the nanoparticles remain stable even without refrigeration.

[0161] Example 2: Sustained secretion of interferon-β (IFNβ) in the nasal compartment after NanoSTING delivery

[0162] This example demonstrates the ability of NanoSTING to deliver cGAMP and induce secretion of effector cytokines in the nasal compartment of mice.

[0163] Although cGAMP is a potent natural activator of STING and thus serves as an immunotransmitter, its clinical utility is hampered by its lack of cellular permeability and rapid degradation by plasma ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), resulting in an in vivo half-life of only about 35 minutes. Different amounts of NanoSTING (10-40 μg) were delivered intranasally to groups of BALB / c mice. Next, nasal turbinates and lungs were harvested and cGAMP levels were measured using quantitative ELISA ( Figure 5A and 5B A dose-dependent increase in GAMP concentrations in the nasal turbinates was observed; at the low dose (10 μg), cGAMP concentrations were quantified for up to 12 hours and returned to baseline at 24 hours; whereas at the higher doses (20-40 μg), cGAMP concentrations were quantified for 24 hours and returned to baseline at 48 hours ( Figure 5BIn the lung, cGAMP was detectable only at higher concentrations (20 and 40 μg) ( Figure 5C In the same animals, serum was profiled and no cGAMP was detected in the circulation at any time point, even at the highest dose (40 μg) ( Fig.7D , 7E These data demonstrate that NanoSTING can transport cGAMP to nasal passage cells in a concentration- and time-dependent manner without systemic exposure.

[0164] The biological significance 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 included effector cytokines, C–X–C motif chemokine ligand 10 (Cxcl10), and interferon beta (Ifnb); interferon-stimulated genes (ISGs) including Isg15, interferon regulatory factor 7 (Irf7), myxovirus resistance proteins 1 and 2 (Mx1 and Mx2), and interferon-induced protein 1 with tetratricopeptide repeats (Ifit1); and nonspecific proinflammatory cytokines (Il6, Il10, and Tnf). BALB / c mice received varying doses of intranasal NanoSTING, and nasal turbinates were quantitatively analyzed by qRT-PCR (6-48 h) ( Figure 5A The effector cytokines Cxcl10 and Ifnb showed maximal induction that remained elevated at 48 hours (7,000 to 20,000-fold induction) ( Figure 5D and 5E Five ISGs showed strong induction from 6 h (300- to 1,000-fold) to 24 h, followed by decay at 24-48 h ( Fig. 5F , 5G , 5H, 5-I, 5J, and 5N). The inflammatory response of NanoSTING was associated with the IFN pathway, as the pro-inflammatory cytokine Il6 showed a transient induction (5,000-fold) at 6 hours, which was significantly attenuated at 24 hours and reached baseline at 48 hours ( Figure 5K In addition, Tnf and Il10 showed only weak induction (15- to 60-fold) ( Figure 5L and 5M These results suggest that NanoSTING elicits a rapid and sustained inflammatory response, triggering effector cytokines and ISGs but only minimally activating nonspecific proinflammatory cytokines.

[0165] Since the qRT-PCR data indicated robust induction of the effector cytokines Ifnb and Cxcl10, the concentrations of IFN-β and CXCL10 proteins in the nasal turbinates were quantified. Consistent with the transcriptional data, quantitative ELISA confirmed that IFNβ and CXCL10 were detectable in both the nasal turbinates and the lungs for up to 24 h ( Fig. 7A , 7B and 7C). No expression of IFN-β and CXL10 was observed in test sera from the same animals ( Fig.7D , 7E and 7F), suggesting that the stimulation of innate immunity by intranasal NanoSTING is local rather than systemic.

[0166] Example 3: Identification of a potent IFN-I signature in hamster lungs following NanoSTING administration

[0167] This example describes experimental confirmation of the induction of interferon-dependent and interferon-independent pathways following intranasal administration of NanoSTING in hamsters.

[0168] Hamsters are a good model for SARS-CoV-2 challenge that mimics severe disease in humans; the animals exhibit easily quantifiable clinical disease characterized by rapid weight loss, very high viral loads in the lungs, and extensive lung pathology. Furthermore, unlike the K18-hACE2 transgenic model, hamsters can recover from disease (like most humans), thus providing the opportunity to study the effects of treatments on the lungs (disease) and nasal passages (transmission).

[0169] Biodistribution was examined by varying the delivery volume of intranasally delivered NanoSTING. Intranasal administration of low and high volumes (40 μl and 120 μl) of Evans blue dye resulted in staining of the nasal turbinates, lungs, and stomach of hamsters ( Figure 8C , 8D and 8E). However, at both volumes, significant amounts of dye were delivered to the nasal turbinates and lungs (the intended target organs) ( Figure 8C ), and the normalized ratio of distribution to these tissues is independent of the volume administered ( Fig.8D and 8E ). These results indicate that the biodistribution after intranasal delivery of the liquid formulation is not affected by the inoculum size in the hamster model.

[0170] To evaluate the effects of intranasal NanoSTING on the lungs, groups of hamsters received daily doses of NanoSTING (60 μg) or PBS (control) for four consecutive days. The two groups of animals showed no differences in clinical signs, such as body temperature or body weight ( Fig. 8A and 8B). On day 5, lungs of hamsters were isolated and unbiased whole transcriptome profiling was performed using RNA sequencing (RNA-seq). A total of 2,922 differentially expressed genes (DEGs) were identified between the two groups at a false discovery rate (FDR q value < 0.25) ( Fig. 10A ). A type I IFN response was induced in NanoSTING-treated lungs, which contained classical ISGs including Mx1, Isg15, Uba7, Ifit2, Ifit3, Ifit35, Irf7, Adar, and Oas2 ( Fig. 10B ). The effector cytokines Cxcl9-11 and Ifnb were also induced in treated hamsters ( Fig. 10C ) and showed strong induction of direct antiviral proteins such as Ddx60 and Gadd45g ( Fig.11A Next, gene set enrichment analysis was performed to compare pathways that were differentially induced after NanoSTING treatment. Changes in these groups were examined against the Molecular Signature Database (Hallmark, C2, and C7 curated gene sets). Unique clustering of pathways associated with type I and type III interferons was observed in the lungs of NanoSTING-treated animals. The specificity of the response was confirmed by qRT-PCR analysis quantifying Mx1-2, Isg15, Irf7, Cxcl11, Ifnb, Il6, and Il10 ( Fig. 9A Since the gene signature of STING's interferon-independent activity is known, gene set enrichment analysis (GSEA) was performed and confirmed that NanoSTING activated interferon-independent pathways and related proteins ( Fig.11A and 11B ).

[0171] Collectively, these results suggest that NanoSTING effectively engages both IFN-dependent and IFN-independent antiviral pathways in the lung via cGAMP-mediated STING activation.

[0172] Example 4: Using quantitative models to predict the effects of early treatment of NanoSTING on viral replication

[0173] This example describes the results of modeling experiments performed to analyze the level of SARS-CoV-2 replication following administration of NanoSTING.

[0174] In vivo mechanistic experiments demonstrated that NanoSTING induces a broad antiviral program by engaging the innate immune system. A mathematical modeling approach combined with human viral load data was used to determine the therapeutic window and quantify the relative amount of type I IFN (or related pathways) required for NanoSTING to produce a therapeutic effect. To simplify the framework of the model, testing was performed under the assumption that cGAMP in vivo only stimulates interferon responses. Under this assumption, the range of relative interferon ratios (RIR, 0-1) required for elicitation by NanoSTING was modeled compared to the population-level peak interferon response observed during SARS-CoV-2 infection ( Fig. 11C and 11D ), and the effect on viral elimination was examined. Based on the model, an RIR of only 0.27 (27% of natural infection) was sufficient to achieve a 50% reduction in viral load (based on the area under the curve, AUC), and an RIR value of at least 0.67 was sufficient to achieve a 100% reduction in viral load ( Fig.11E Next, we modeled the window for initiating treatment, which showed that intervention was most effective when initiated within 2 days of infection ( Fig.11F ). However, if treatment is started after the peak of viral replication, even with an RIR of 1, improved outcomes are not easily achieved ( Fig.11F , 11G , 11H, 11-I, 11J and 11K).

[0175] Collectively, these results from the quantitative model predict that: (a) a single dose of NanoSTING is required to elicit only moderate amounts of IFN, and this is likely achievable as our data support a substantial induction of IFNβ ( Figure 5E , 5M and 11b) and natural infection with viruses (such as SARS-CoV-2 and influenza A) is known to suppress interferon production, and (b) the therapeutic window is optimized both for prophylactic treatment and for treatment shortly after infection.

[0176] Example 5: NanoSTING treatment protects against SARS-CoV-2 Delta strain and induces protection against SARS-CoV-2 reinfection

[0177] This example describes the level of protection against the SARS-CoV-2 Delta (B.1.617.2) strain following a single dose of NanoSTING in hamsters.

[0178] The Delta strain (B.1.617.2) was chosen because it causes both upper and lower respiratory tract illness and increased disease severity compared with previous variants (WH and Beta). Groups of 12 animals were treated with a single intranasal dose of 120 μg NanoSTING and then 24 hours later with 10 4 The delta variant was infected with half tissue culture infectious dose (TCID50) Fig. 12A Animals in the placebo-treated (PBS) group showed severe weight loss, with an average peak weight loss of 8.3%. In contrast, animals treated with NanoSTING showed little weight loss (average peak weight loss of only 2.0%) ( Fig. 12C This small amount of weight loss was similar to the results obtained with adenovirus vector vaccines challenged with either the WH strain or the Beta strain. Half of the animals were sacrificed on day 2 (peak of viral replication) to quantify infectious viral load. Even for the highly contagious Delta strain, NanoSTING reduced infectious viral load in the lungs by 300-fold compared to placebo-treated animals 2 days after infection ( Fig.12D The reduction in lung viral load was strongly associated with the prevention of weight loss in these animals. Quantitative analysis of viral load in the nasal cavity showed that NanoSTING treatment reduced infectious viral load in the nasal cavity by 1,000-fold at 2 days post-infection compared with placebo-treated animals ( Fig.12D ). The decrease in viral replication in the nasal compartment mimics the propensity for human transmission and confirms that NanoSTING treatment reduces the likelihood of transmission. To map the duration of efficacy of prophylactic NanoSTING treatment, hamsters were next given a single intranasal dose of NanoSTING and then 72 hours later with 10 4 TCID50 of Delta strain challenge ( Fig.13A Even when given 72 hours before exposure, NanoSTING showed modest protection against weight loss and significant reductions in infectious viral load ( Fig.13A , 13B To test the effect of NanoSTING after virus exposure, intranasal NanoSTING ( Fig.14A ). Infectious virus in the nasal passages and lungs was reduced by 340-fold and 13-fold, respectively ( Fig. 14B and 14C ).

[0179] These results demonstrate that a single-dose treatment with NanoSTING effectively reduced clinical symptoms, protected the lungs, and reduced infectious virus in the nasal passages.

[0180] One of the advantages of using the innate immune system to clear viral infections is that the process mimics natural infection while minimizing the risk of clinical symptoms. To test whether adaptive immunity plays a role, hamsters were given NanoSTING transdermally and 24 hours later, they were treated with 10 4 TCID50 of the delta variant challenged animals ( Fig. 12A Administration of NanoSTING reduced weight loss during the primary challenge. Unlike placebo-treated animals, animals treated with NanoSTING regained weight, and by day 4, their weight was not significantly different from that of unchallenged animals. The reduction in weight loss was attributed to a reduction in infectious viral load in lung and nasal tissues ( Fig. 12C , 12D and 12E). On day 28, animals were challenged again with the delta variant. NanoSTING-treated animals did not lose weight at all during the second challenge, and the animals weighed the same as the previously unchallenged animals ( Fig.12F ).

[0181] These results suggest that a single intranasal treatment of NanoSTING activates the antiviral program of innate immunity and protects against clinical disease during primary infection, while promoting adaptive immunity to provide long-lasting protection against reinfection.

[0182] Example 6: Effect of NanoSTING treatment on the IFN-escape SARS-CoV-2 alpha variant (B.1.1.7).

[0183] This example examines the effects of different doses of NanoSTING and different doses of treatment on the SARS-CoV-2 alpha strain.

[0184] The alpha (B.1.1.7) variant is known to be resistant to IFN-1 signaling in vitro and thus provides a challenging model to test the efficacy of NanoSTING. Hamsters were pretreated with two intranasal doses of NanoSTING (30 μg and 120 μg) and 24 hours later with 10 4 TCID50 alpha variants were used to attack ( Fig.16A Treatment with NanoSTING at either dose protected the hamsters from severe weight loss ( Fig. 16B A composite scoring scale (ranging from 1-12) was used to interpret pathology and disease to analyze lung tissue at day 6 after viral challenge. NanoSTING-treated animals had significantly reduced gross pathology scores, with minimal evidence of inflammatory cell invasion or alveolar damage ( Fig.17A and 17BIn addition, the viral load in the lung and nasal compartments was quantified, and a significant reduction in viral load was found in both compartments even on day 2 ( Fig. 17C and 17D ).

[0185] Thus, these results suggest that intranasal NanoSTING treatment reduces SARS-CoV-2 replication in vivo by several orders of magnitude and provides protection against IFN-I escape strains of SARS-CoV-2.

[0186] Example 7: Evaluation of the Transmission of SARS-CoV-2 Omicron Variants after NanoSTING Treatment

[0187] This example describes experiments demonstrating that NanoSTING treatment can block transmission and prevent infection with the more contagious SARS-CoV-2 Omicron variant.

[0188] The Omicron variant (B.1.1.529) is the most infectious of the known SARS-CoV-2 strains. Using the Omicron variant sets the highest standard for NanoSTING to prevent viral transmission. To assess whether prophylactic treatment of infected hamsters could prevent transmission to contact hamsters and whether postexposure treatment of contact hamsters could mitigate viral replication, we performed experiments in three groups of 16 hamsters. In each group, eight hamsters were infected intranasally with 10 copies of the Omicron variant of SARS-CoV-2. 4 TCID50, one day after infection, each hamster was paired with a sentinel hamster ( Fig.22A None of the infected animals showed any weight loss ( Fig.22D ). Sentinel hamsters were: (a) co-housed with NanoSTING (120 μg) treated index hamsters (Group 2) or (b) treated with NanoSTING after co-housed with infected but untreated hamsters (Group 3). As with other SARS-CoV-2 strains tested, pre-treatment of infected hamsters with NanoSTING prevented transmission in nearly all animals (7 / 8 treated animals were virus-free vs. 1 / 8 untreated animals). Notably, post-exposure treatment of sentinel hamsters was also effective in preventing infection (6 / 8 treated animals were virus-free), and all animals showed a reduction in viral load ( Fig. 22B and 22C ).

[0189] These results suggest that NanoSTING is highly effective in blocking transmission, even for the highly contagious Omicron strain.

[0190] Example 8: NanoSTING treatment has a similar effect on preventing influenza as oseltamivir

[0191] Multiple experiments performed in this example demonstrate that administration of NanoSTING is an effective treatment for influenza virus, as measured by weight loss, survival rate, and viral titer.

[0192] Influenza viruses have evolved multiple mechanisms to suppress the host's innate immunity, including the inhibitory effect of the NS1 protein on the interferon response. One of the main treatments for influenza is post-exposure prophylaxis with oseltamivir, which inhibits the influenza neuraminidase protein. Therefore, oseltamivir (Tamiflu) can be used as a benchmark to evaluate the efficacy of NanoSTING treatment.

[0193] A group of ten mice were treated with a single intranasal dose of NanoSTING and then challenged 24 hours later with 4xLD50 (median lethal dose) of influenza A / California / 04 / 2009 (H1N1) susceptible strain ( Fig.21A Animals were evaluated for 14 days, with weight loss and survival as primary endpoints. A group of mice was treated with a clinically relevant dose of oseltamivir (30 mg / kg / day) twice daily for five days as a positive control. Untreated animals began to lose significant weight on day 3 (peak of viral infection), and by day 11, only one animal was alive, having lost 23% of its body weight ( Fig.21B On day 3, the NanoSTING-treated mice showed no significant weight loss, similar to unchallenged animals and even significantly improved compared to oseltamivir-treated animals ( Fig.21B The effects of a single dose of NanoSTING (40 μg) were attenuated after day 3. However, even accounting for this reduction, mean peak weight loss and weight loss kinetics were not significantly different from those of oseltamivir-treated animals ( Fig.21A and 21B ). Taken together, these results suggest that NanoSTING provides influenza protection in animals equivalent to that of a clinically relevant dose of oseltamivir.

[0194] However, even taking this reduction into account, the mean peak weight loss, weight loss kinetics, and survival of treated animals were not significantly different from those of oseltamivir-treated animals ( Fig.18B Animals were pretreated with oseltamivir (30 mg / kg / day) or NanoSTING (40 μg) and then challenged with 4xLD50 (median lethal dose) influenza A / California / 04 / 2009 (H1N1) susceptible strain ( Fig.18AThe average peak weight loss rates of animals treated with NanoSTING, placebo, and oseltamivir were -14%, -27%, and -32%, respectively, which shows the protective effect of NanoSTING on animals ( Fig.18B ).

[0195] A single amino acid mutation (His275Tyr) in neuraminidase has resulted in oseltamivir-resistant influenza virus infection in humans. Since NanoSTING relies on the host's innate immune response, an experiment was performed to evaluate its efficacy against oseltamivir-resistant influenza A in mice. A group of ten mice were treated with a single intranasal dose of NanoSTING (40 μg) and then challenged 24 hours later with a 4xLD50 (half lethal dose) of a susceptible, resistant strain of influenza A virus ( Fig.19A The animals were evaluated for 14 days, with weight loss and survival as primary endpoints. A group of mice was treated with a clinically relevant dose of oseltamivir (30 mg / kg / day) twice daily for five days as a positive control. The oseltamivir-treated mice experienced a 32% decrease in mean peak body weight, while the NanoSTING-treated animals experienced an 8.2% decrease ( Fig.19B The survival data of animals treated with NanoSTING were significantly different from those treated with oseltamivir ( Fig.19C ). Overall, NanoSTING was protective compared with oseltamivir, as evidenced by weight loss and improved survival.

[0196] Next, an experiment was designed to test the effect of NanoSTING treatment on viral load in lung tissue. A group of ten mice were treated with a single intranasal dose of NanoSTING (40 μg) and then challenged 24 hours later with a 4xLD50 (half lethal dose) of a sensitive, resistant strain of influenza A virus ( Fig. 20A The animals were evaluated for 7 days, with changes in body weight and viral titers as primary endpoints. Oseltamivir treatment had no significant effect in this experiment, with 90% of the mice succumbing to the disease. A single dose of NanoSTING prevented the animals from losing weight (mean peak weight loss on day 7 was 5.2% vs. 32% in the placebo group) ( Fig. 20B Seven days after virus exposure, there were 500-fold fewer infectious particles in the lungs than in the placebo-treated group, suggesting NanoSTING has the ability to help prevent illness and death. Fig. 20C ).

[0197] Together, these results suggest that NanoSTING treatment is effective against oseltamivir-resistant strains of influenza A virus. These experiments confirm that NanoSTING can function as a broad-spectrum antiviral agent against influenza viruses, including resistant strains.

[0198] Example 9: Preparation of STING-loaded liposomes and vaccine formulations

[0199] 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, USA).

[0200] Liposomes can be composed of DPPC, DPPG, cholesterol (Chol) and DPPE-PEG2000 in a molar ratio of 10:1:1:1. To prepare liposomes, we mixed the lipids in CHCl3 and CH3OH and then evaporated the solution using a vacuum rotary evaporator at 45°C for about 80 minutes. The resulting lipid film was dried until all organic solvents evaporated. Next, the lipid film was hydrated by adding preheated cGAMP solution (pH 7.4, 0.3 mg / ml cGAMP in PBS buffer). We mixed the hydrated lipids at an elevated temperature of 65°C for another 30 minutes and then subjected them to freeze-thaw cycles. We then sonicated the mixture for 60 minutes using a Brandson sonicator (40 kHz). Next, we removed the uncaptured free cGAMP using an Amicon ultrafiltration device (MW cutoff of 10 kDa). Subsequently, we washed the cGAMP-liposomes three times with PBS buffer. We measured the cGAMP concentration in the filtrate using a Take3 Micro-Volume absorbance analyzer from Cytation5 (BioTek) against a cGAMP calibration curve at 260 nm. We calculated the final concentration of liposome-encapsulated cGAMP and the encapsulation efficiency by subtracting the concentration of free drug in the filtrate. We purchased SARS-CoV-2 (2019-nCoV) nucleocapsid recombinant protein from BEI Resources (VA, USA #NR-53797) and tested two different doses (10 μg and 20 μg) of the combined STING-liposome suspension. We kept the vaccine formulation at room temperature to allow protein adsorption to the liposomes. We stored the formulated vaccine at 4°C for up to 2 months. The mean particle size, polydispersity index, and zeta potential were characterized using a Litesizer 500 (Anton Paar) at room temperature.

[0201] THP-1DUAL cell line (Invivogen) was cultured in a humidified incubator at 37°C and 5% CO2 and grown in RPMI / 10% FCS (Corning, New York, USA). In addition, we supplemented THP-1 dual cell line cultures with the corresponding selection agents (100 μg / ml bleomycin + 10 μg / ml blasticidin) and the corresponding selection cell inhibitors from Invivogen.

[0202] Example 10: Preparation of STING-loaded liposomes and vaccine formulations

[0203] Cell stimulation experiments with luciferase reporter enzyme assays

[0204] We performed cell stimulation experiments according to the manufacturer's instructions (Invitrogen, CA, USA). First, we cultured cells at 1 × 105 10 cells / well were seeded in 180μl growth medium in a 96-well plate. Next, we serially diluted NanoSTING in growth medium. Then, we incubated the cells at 37°C for 24 hours. To detect IRF activity, 10μl culture supernatant / well was collected at 6 hours, 12 hours, and 24 hours and transferred to a white (opaque) 96-well plate. Next, 50μl QUANTI-Luc was added to each well. TM After adding the substrate solution (Invitrogen), we immediately performed luminescence measurements (expressed in relative light units (RLU)) using Cytation 7 (Cytation 7, Bio-Tek Instruments) and read the plate.

[0205] Example 11: Cytotoxicity Test Formulation

[0206] The cytotoxic activity of the NanoSTING composition 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 / 24 hours. Afterwards, the cells were treated with [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO). Four hours later, all the culture medium including 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, Bio-Tek Instruments). Fig.27A and Fig.27B The data are expressed as percentage viability compared to untreated cells (negative control - considered as 100% viability) and cells treated with Triton X-100 (positive control - considered as 100% cell death).

[0207] Example 12: DNA binding assay

[0208] DNA binding studies were performed to examine the suitability of the assay for detecting DNA condensation, and we used branched PEI as a positive control (Sigma Chemical Company, MO#408727). DiYO-1 (AAT Biorequest#17579) and plasmid (pMB57.6)-DNA complexes were mixed in equal volumes of DNA and DiYO-1 (in 20mM HEPES, 100mM NaCl, pH=7.4) to reach final concentrations of 400nM and 8nM, respectively. The solution was left at room temperature for 5 hours before use. Next, different concentrations of PEI (R=0, 1, 2, 5, where R is the molar ratio of PEI nitrogen to DNA phosphate) were added to the DNA-DiYO-1 solution, vortexed for 1 minute and left to stand for 2 hours to reach equilibrium. The fluorescence intensity of the solution was measured at excitation and emission wavelengths of 470nm and 510nm, respectively. The same operation was also performed using SARS-CoV2 N protein instead of PEI. N protein was added to the DNA-DiYO-1 solution at concentrations of 0.1 and 0.5 μM.

[0209] Example 13: Mice and Immunization

[0210] Female, 7-9 week old BALB / c mice were from Charles River Laboratories. Prior to compound administration, mice were anesthetized by intraperitoneal injection of ketamine and xylazine. Animals were given a composition with one of two different concentrations of nucleocapsid protein (10 μg and 20 μg) and 20 μg of liposome-STING adjuvant.

[0211] Example 14: Weight Monitoring and Sample Collection

[0212] The body weight of the animals was monitored every 7 days for four weeks after administration of the NanoSTING composition or control treatment. Serum was collected on days 7, 14, 21 and 27 after administration for detection of humoral immune responses. The blood was kept at room temperature (RT) for 10 minutes to promote coagulation and then centrifuged at 2000g for 5 minutes. Serum was collected and stored at -80°C and subsequently used for ELISA testing. On day 27 after intranasal administration, bronchoalveolar lavage fluid (BALF), lungs and spleen were collected. Serum and other biological fluids (containing protease inhibitors) were stored at -80°C for a long time. After dissociation, splenocytes and lung lymphocytes were frozen in FBS + 10% DMSO and stored in liquid nitrogen vapor phase until further use.

[0213] Example 15: ELISA test

[0214] Anti-N-protein antibody titers in serum or other biological fluids were determined using ELISA. Briefly, 1 μg / ml N protein (Sino Biological, Pennsylvania, USA) in PBS was coated on ELISA plates (Corning, New York, USA) overnight at 4°C or 2 hours at 37°C. The plates were blocked at room temperature with PBS+1% BSA (Fisher Scientific, Pennsylvania, USA)+0.1% polysorbate 20 (Sigma-Aldrich, Maryland, USA) for 2 hours. After cleaning, samples of different dilutions were added. Detection was by HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, 1:5,000; Pennsylvania, USA), anti-mouse IgA (Bethyl Laboratories, 1:10,000; Texas, USA) and detection antibody against mouse IgA (1:250) from mouse total IgA ELISA kit from Invitrogen (California, USA). Positive control (anti-N IgG) was obtained from Abeomics (California, USA).

[0215] Example 16: Spleen and lung processing for ELISPOT and flow cytometry

[0216] To isolate lung lymphocytes, the lung vasculature was perfused with Ca-free 2+ Mg 2+ Inject 5 ml of 1 mM EDTA solution in PBS into the right ventricle. Use a scalpel to cut each lung into 100-300 mm 2The chopped tissue was transferred to a tube containing 5 ml digestion buffer containing collagenase D (2 mg / ml, Roche #11088858001) and DNAse (0.125 mg / ml, Sigma #DN25) in 5 ml RPMI, in a 37 ° C water bath for 1 hour and 30 minutes, vortexed every 10 minutes. The remaining intact tissue was destroyed by passing through a 21-gauge needle (6-8 times). The sample was incubated at room temperature for 90 minutes, and 500 μl of ice-cold stop buffer (1x PBS, 0.1M EDTA) was added to terminate the reaction. Tissue debris and dead cells were removed with a 40 μm disposable cell strainer (Falcon), and cells were collected after centrifugation at 300 g. Red blood cells were lysed by resuspending the cell pellet in 3 ml ACK lysis buffer (Invitrogen), incubated at room temperature for 3 minutes, and then centrifuged at 300 g. The supernatant was discarded and the cell pellet was resuspended in 5 ml of complete RPMI medium (Corning, New York, USA). Then, the spleen was homogenized in RPMI medium and passed through a 40 μm cell strainer using the hard end of a syringe plunger. Afterwards, the splenocytes were incubated at room temperature for 3 minutes in 3 ml of ACK lysis buffer to remove red blood cells (RBC) and then passed through a 40 μm filter to obtain a single cell suspension. Pulmonary lymphocytes and splenocytes were counted using the trypan blue exclusion method.

[0217] Example 17: ELISPOT assay

[0218] IFN-γ and IL-4 ELISpot assays were performed using mouse IFN-γ ELISPOT basic kit (ALP) and mouse IL-4 ELISPOT basic kit and following the manufacturer's instructions (Mabtech, Virginia, USA). For cell activation control, cultures were treated with 10 ng / ml phorbol 12-myristate 13-acetate PMA (Sigma, St. Louis, Missouri, USA) and 1 μg / ml ionomycin (Sigma, St. Louis, Missouri, USA). Complete medium (RPMI with 10% FBS) was used as a negative control. Splenocytes and lung lymphocytes (3x10 5) were stimulated in vitro with a peptide pool consisting mainly of 15-mer sequences with 11 amino acid overlaps, covering the complete sequence of the nucleocapsid phosphoprotein ("N") of SARS-CoV-2, at a concentration of 1.5 μg / ml / peptide (Miltenyi Biotec; 130-126-699, Germany), at 37°C for 16-18 hours on 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 next day, the cells were washed, and the antibodies were detected using biotinylated R4-6A2 anti-IFNγ (Mabtech #3321-6-250) and BVD6-24G2 anti-IL-4 (Mabtech #3311-6-250) and prepared plates. The wells were washed and treated with Extravidin-ALP antibody (Sigma, St. Louis, MO, USA) diluted 1:30,000 at room temperature for 1 hour. After washing, the points were developed by adding 70 μL / well of BCIP / NBT-plus substrate (Mabtech #3650-10) to the wells. The plates were incubated for 20-30 minutes to develop color and then washed with water. The points were quantified using Cytation7 (Bio-Tek Instruments). Each point corresponds to a single cytokine secreting cell. The numerical values ​​were analyzed as the average of three repeated measurements minus the background.

[0219] Example 18: Cell surface staining and intracellular cytokine staining by flow cytometry

[0220] After stimulation for 18 hours at 37°C with a nucleocapsid protein-peptide pool (Miltenyi Biotec; 130-126-699, Germany) at a concentration of 1.5 μg / ml / peptide, spleen and lung lymphocytes from immunized and control animals were stimulated for 16-18 hours to detect nucleocapsid protein-specific CD8+ T cell responses, and then brefeldin A (5 μg / ml BD Biosciences #BD 555029) was added for the last 5 hours of incubation. 10 ng / ml PMA (Sigma, St. Louis, MO, USA) and 1 μg / ml ionomycin (Sigma, St. Louis, MO, USA) were used as positive controls. No peptide stimulation was used as background control. Cells were collected and stained with Live / Dead Aqua (Thermo Fisher #L34965) in PBS, followed by Fc receptor blocking with anti-CD16 / CD32 (Thermo Fisher #14-0161-85), and then stained with the following antibodies in flow cytometry staining buffer (FACS) for 30 minutes on ice: anti-CD4 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), anti-CD45 (clone 30-F11; BD; #564279). Cells were washed twice with FACS buffer and then fixed with 100 μl IC fixation buffer (eBioscience) at RT for 30 minutes. Cells were permeabilized with 200 μl permeabilization buffer (BD Cytofix solution kit) for 10 minutes. Intracellular staining was performed using antibodies Alexa Fluor 488 interferon (IFN)γ (clone XMG1.2; BD; #557735) and granzyme B (clone GB11; Biolegend; #515407) at 4°C overnight. Cells were then washed with FACS buffer and analyzed on an LSR-Fortessa flow cytometer (BD Biosciences) using FlowJo. TM Software version 10.8 (Tree Star, Ashland, OR, USA). Results were calculated as the total number of cytokine-positive cells minus the background. The amount of antibody used was optimized by titration.

[0221] Example 19: Preparation and Characterization and Composition

[0222] This example demonstrates that NanoSTING is a liposomal adjuvant that can achieve mucosal immunity. Liposomal nanoparticles improve stability and have been shown to promote delivery to alveolar macrophages, thereby promoting responses in the lungs. Dynamic light scattering (DLS) analysis showed that NanoSTING had an average particle size of 100nm and a polydispersity index of 0.21. The zeta potential of NanoSTING was -50mV. The ability of NanoSTING to induce an interferon (IRF) response was confirmed by using THP-1 monocytes, which stably express secreted luciferase downstream of an IRF-responsive promoter. THP-1 monocytic cells were stimulated with NanoSTING at concentrations ranging from 1.2-9.3μg, and kinetic measurements were performed for 24 hours by measuring luciferase activity in the supernatant. Although lower levels of luciferase secretion were observed at 6 hours, the secretion was maximal at 24 hours, and all concentrations tested at this time point showed similar responses. In summary, these results indicate that optimal activation of the STING pathway by these NanoSTING compositions was observed at 24 hours.

[0223] As an immunogen, a 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" method was used to adsorb the protein to the liposomes. Using a standard S-protein quantitative ELISA, 61.5% of the S-protein was confirmed to be adsorbed to the liposomes. The adsorbed trimeric S protein (NanoSTING-S) had an average particle size of 104nm, an average zeta potential of 133mV, and a polydispersity index of 0.24. Compared to NanoSTING (81nm, -35mV), NanoSTING-S is expected to be more negatively charged based on the protein isoelectric point (pI). Unlike the trimeric S protein, which is known to aggregate in solution, NanoSTING-S was tested after six months of storage at 4°C and confirmed no evidence of aggregation or zeta potential changes.

[0224] Example 20: Single-dose immunization of mice with NanoSTING-S vaccine produces cross-reactive humoral and cellular immunity

[0225] Mice were immunized with a single intranasal dose of NanoSTING-S, and no clinical symptoms, including weight loss, were observed throughout the observation period. 100% seroconversion was observed on day 7, with peak responses on day 21. ELISA was performed on day 28 to quantify the binding of the variant full-length protein and the receptor binding domain (RBD) protein as a surrogate for neutralization. High serum IgG titers were observed not only against beta, but also against alpha (B.1.1.7), gamma (P.1), and delta (B.1.617.2) full-length proteins. High serum IgG titers were also observed against the RBD of both beta and alpha variants. SARS-CoV-2-specific antibody responses were assessed in bronchoalveolar lavage fluid (BALF) and robust IgG titers against full-length beta were confirmed. IgA-mediated protection is an important component of mucosal immunity against respiratory pathogens. To confirm the role of intranasal NanoSTING as a mucosal adjuvant, IgA responses were tested in serum. Strong IgA responses were detected against all three variants tested. Overall, these results demonstrate that a single-dose immunization with NanoSTING-S generates robust IgG and IgA responses that cross-react with different spike mutants, including the delta variant.

[0226] To assess vaccine-induced S-specific T cell responses, splenocytes and lung lymphocytes were harvested 28 days after immunization. T cells from spleen and lung were stimulated with overlapping 15-mer peptide pools, and antigen-specific T cells were quantified using IFN-γ (Th1 / Tc1 response) and IL-4 (Th2 response) ELISPOT assays. NanoSTING-S immunized mice exhibited robust and significant lung and spleen T cell responses, with an average of 500 and 176 IFN-γ spots / 10 6 cells. Lung and spleen cells were also stimulated with a pool of peptides containing S protein mutations distinct from the WH S protein. Significant Th1 responses were observed against these mutation-specific S peptides, 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 following immunization with NanoSTING-S. Together, these results suggest that intranasal vaccination elicits a strong cross-reactive Th1 response, but with no evidence of a Th2 response.

[0227] Example 21: NanoSTING-S-induced immune response provides protection against highly infectious delta strains

[0228] To test the protective efficacy of NanoSTING-S, a golden hamster challenge model was used. This animal model replicates more severe disease in humans, with animals exhibiting easily quantifiable clinical disease characterized by rapid weight loss, very high lung viral loads, and extensive lung lesions. Additionally, unlike the K18-hACE2 model, hamsters can recover from disease (as do humans), thus providing the opportunity to study the effects of treatment on both the lungs (disease) and the nasal passages (transmission). The animals were challenged with the Delta strain for two reasons: (1) the Delta strain is highly contagious, causes severe lung damage, and has become the dominant strain in humans; and (2) Delta-specific S mutations within the RBD (including L452R and T478K) are not present in the immunogen, thus, challenge with Delta provided the opportunity to assess cross-protection.

[0229] Based on the results of vaccines against other respiratory pathogens, 10 hamsters were immunized with two doses of NanoSTING-S intranasally. 4 Hamsters vaccinated with the delta variant were challenged with TCID50. Animals in the sham-vaccinated group experienced severe weight loss, with an average peak weight loss of 8.3%. In contrast, animals vaccinated with NanoSTING-S were largely protected from weight loss (average peak weight loss of 2.3%), similar to results obtained with adenovirus vector vaccines challenged with the WH strain or the beta strain. Half of the animals were sacrificed 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 on day 2 compared with sham-vaccinated animals; by day 5, infectious virus was undetectable in all animals. Viral replication in the animals' lungs mimics human clinical disease and death, while viral replication in the nasal cavity mimics human transmission. NanoSTING-S reduced infectious viral load in the nasal cavity by 380-fold on day 2 compared with non-immunized animals. Vaccinated animals showed a further significant reduction in the amount of infectious virus by day 5. The reduction in viral loads achieved intranasally was superior to that achieved with intramuscular adenovirus vaccines, indicating the advantage of mucosal vaccination.

[0230] Lung tissue collected on day 6 post-challenge was analyzed using a composite scoring scale (ranging from 1-12) to illustrate pathology and disease severity. Prominent immune cell infiltration and extensive viral pneumonia were observed in the lungs of mock-vaccinated hamsters. It was observed that the overall pathology scores were significantly reduced in both vaccinated and challenged animals, with minimal evidence of inflammatory cell invasion or alveolar damage. Overall, the results from NanoSTING-S-immunized hamsters challenged with the Delta strain coronavirus suggest that the NanoSTING composition has a cross-protective effect, effectively protecting the lungs and minimizing infectious virus in the nasal passages.

[0231] Example 22: Single-dose immunization of mice with NanoSTING-N vaccine produces long-lasting humoral and cellular immunity

[0232] Since the N protein is largely conserved among different SARS-CoV-2 variants, vaccines based on the N protein have been investigated. Independent studies using N protein-based viral vectors to immunize K18-hACE2 mice and challenge early lineage variants (WH and alpha) showed mixed results with partial protection or complete lack of protection.

[0233] The SARS-CoV-2 N protein is predicted to contain a nucleic acid (RNA) binding domain, a C-terminal dimerization domain, and three intrinsically disordered domains that promote phase separation with nucleic acids. Insect-cell-derived recombinant N protein was used as an immunogen, and SDS-PAGE confirmed a dominant band of 47 kD in size. To confirm the functional ability of the recombinant N protein, the interaction of the N protein with plasmid DNA was investigated. The DNA condensation probe DiYO-1, a dual intercalating fluorophore whose quantum yield increases by several orders of magnitude upon binding to double-stranded 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 the known synthetic polycationic DNA condensing agent polyethyleneimine (PEI) R=5. These results confirm that the recombinant N-protein is an effective binder for dsDNA. To formulate the vaccine NanoSTING-N, the recombinant N protein was gently mixed with NanoSTING to allow the protein to adsorb to the liposomes. Although the N protein showed a strong tendency to aggregate when stored at 4°C, NanoSTING-N was stable with no changes in size or zeta potential. These results suggest that the NanoSTING-N vaccine exists in the form of a stable nanoparticle colloid.

[0234] Two groups of mice were immunized by intranasal administration of liposomes containing cGAMP and 10 μg or 20 μg of WH 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 obvious abnormalities over 28 days. Fourteen days after immunization (d14), 100% of mice receiving NanoSTING-N were detected to have seroconverted and strong anti-N IgG levels were detected, with an average dilution titer of 1:640, and the response increased to an average dilution titer of d27. By day 15 (d15), serum concentrations of anti-S IgG antibodies increased and an average dilution titer of 1:4,400 was detected. IgG responses were similar for both doses at all time points tested, and although NanoSTING-N20 elicited higher IgG titers than NanoSTING-N10, the difference was not significant. Unlike vaccination with the trimeric NanoSTING-S (early response on day 7), the kinetics of the IgG response were delayed, and responses were only observed on day 14. We assessed SARS-CoV-2-specific antibody responses in the BALF; mean IgG titers were 1:15 and 1:86 for NanoSTING-N10 and NanoSTING-N20, respectively. Consistent with mucosal vaccination, serum IgA was observed for NanoSTING-N10 (titer 1:40) and Nano-STING-N20 (titer 1:53). Together, these results confirm that immunization with the N-protein generates robust humoral immunity in both the serum and the lungs.

[0235] One of the concerns with N protein-based vaccines is that Th2 responses to the N protein could mediate antibody-dependent enhancement (ADE) of viral infection. Therefore, the Th1 and Th2 responses elicited in the lungs and spleens of immunized animals were quantified.

[0236] Nano-STING-N10 and NanoSTING-N20 immunized mice showed robust and significant splenic T cell responses, with an average of 143 and 176 IFN-γ spots / 10 6 Animals immunized with NanoSTING-N10 and NanoSTING-N20 showed elevated T cell responses in the lungs, with an average of 102 and 154 IFN-γ spots / 10 6Unlike the IFN-γ (Th1 / Tc1) response, no measurable IL-4 (Th2) response was observed following immunization with NanoSTING-N10 and NanoSTING-N20. Together, these results suggest that intranasal vaccination elicits a robust Th1 response, but no evidence of a Th2 response.

[0237] CD8+T cell responses can complement antibody-mediated responses and can provide protection independent of antibody responses. The activation and function of N protein-specific memory CD8+T cells in the lung airways, lung parenchyma, and spleen were observed. The effector molecule granzyme B and the activation-inducing marker CD137 were used to identify N protein-reactive CD8+T cells in the spleen and lung. Ex vivo restimulation with the N peptide pool resulted in a substantial increase in the frequency of activated (CD8+CD137+) and cytotoxic (CD8+GzB+) T cells in the spleen of NanoSTING-N10 and NanoSTING-N20 vaccinated mice, and to a lesser extent in the lung. The overall frequency of lung-resident CD103+CD69+CD8+T cells did not differ between immunized animals and controls. Taken together, these results confirm that NanoSTING-N elicits cytotoxic T cell responses in the lungs and spleen.

[0238] The immunogenicity of the N protein based on the alpha variant was also evaluated. Two key mutations within the N protein, R203K and G204R, have been repaired in all subsequent variants. Based on the lyophilized N protein formulation composition, 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 decrease in IgG or IgA was observed. Similarly, N-reactive Th1 responses were retained in the lungs and spleen at day 62. Overall, these mouse studies confirm that NanoSTING-N immunization can elicit strong cellular and humoral immune responses.

[0239] 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 10 4 Immunized hamsters were challenged with the delta variant at TCID50. Animals in both the vaccinated (mean peak weight loss 6.9%) and sham-vaccinated groups exhibited severe weight loss (mean peak weight loss 8.3%). Consistent with the lack of protection from weight loss, there were no differences in infectious virus titers in the lungs or nasal passages on days 2 or 5 between vaccinated and sham-vaccinated animals. Overall, these results from the NanoSTING-N hamster experiment suggest that the N protein as a single antigen is not sufficient to provide protection against the delta strain.

[0240] Example 23: Quantitative modeling of the combined immune response to two proteins predicts synergistic protection

[0241] Results from the NanoSTING-S experiments suggest that the immune response protects against lung disease but is not sufficient to eliminate viral replication in the nasal passages, a surrogate for viral transmission. On the other hand, the NanoSTING-N results suggest that the N protein, while highly immunogenic, does not play a dominant role in combating SARS-CoV-2 infection in all cases. Mathematical modeling was used in conjunction with viral load data to quantify the chances of generating synergistic protection in the nasal compartment by vaccination with both proteins.

[0242] The model was set up to track viral load in the nasal passages by fitting parameters to reflect longitudinal viral titers in infected patients. Vaccine-induced neutralizing antibody responses against the spike protein act as de novo blockers of viral entry and block viral production through effector mechanisms. A range of vaccine efficacy (40% to 100%) was modeled to account for differences in protection specifically in the nasal compartment, and its impact on viral elimination was investigated.

[0243] Cytotoxic T cell responses against the N protein directly kill virus-infected cells, thereby directly reducing the number of cells capable of producing / reproducing virus. For NanoSTING-N vaccines, which do not elicit neutralizing antibodies and cannot block viral entry, our model predicts that T cells will be effective. These estimates exceed the known efficacy of T cell immunity, so it is not surprising that single-antigen N-based vaccines do not provide protection. Combined protection models were constructed by combining S-directed vaccines that provide partial protection in the nasal compartment with cytotoxic T cell responses against the N protein. When a range of S protein vaccine efficacy (40% to 100%) was tested in conjunction with cytotoxic N responses in the nasal compartment, the model predicted that 1-3 cells would need to be eliminated per day, an estimate that is well within the known frequency of T cell killing. Indeed, studies in humans infected with the COVID-19 virus have shown that there are strong and persistent cytotoxic T cell responses in the nasal compartment and that N protein-specific CD8+ T cells can directly inhibit viral replication. When using the virus reduction data from the NanoSTING-S challenge hamster model, the model predicted that the N-directed cytotoxic response would only require killing 1-3 cells / day in the nasal compartment. Together, these quantitative modeling results predict that a combination vaccine targeting both S and N proteins could provide synergistic protection in eliminating viral replication in the nasal compartment.

[0244] Example 24: Single-dose immunization of mice with NanoSTING-NS vaccine produces balanced humoral and cellular immunity against both proteins and produces inactivated immunity

[0245] To validate the prediction of the quantitative model that immune responses to S and N proteins would be synergistic, we formulated vaccines containing both antigens. Initial immunogenicity studies in mice with 10 μg of each N and S protein in NanoSTING adjuvant demonstrated that 100% of animals seroconverted and IgG responses to S protein, while seroconversion rates to N protein were variable (40-80%). A modified alpha N:beta S mass ratio (2:1) was formulated with NanoSTING adjuvant to form NanoSTING-NS. A single intranasal dose of NanoSTING-NS generated strong serum IgG titers to alpha N, full-length alpha S, beta S, and delta S, as well as beta S RBD. Strong antigen-specific, cross-reactive IgG responses were recorded in BALF; cross-reactive IgA responses were observed in serum. In the lungs and spleen, Th1 responses were primarily directed against N protein, but responses to S protein were also evident. Consistent with all other experiments, no Th2 responses were observed in either tissue.

[0246] Based on the favorable immunogenicity data in mice, the protective efficacy of NanoSTING-NS was evaluated in hamsters. Hamsters were vaccinated with two doses of NanoSTING-NS and 10 4 Hamsters were challenged with the delta variant at TCID50. Animals immunized with NanoSTING-NS showed no weight loss at all (mean peak weight loss of 0.8%). Exactly like the NanoSTING-S vaccine, NanoSTING-NS eliminated viral replication in the lungs on day 6 after challenge, suggesting that the S-specific immune response is the primary factor providing immunity in the lungs. Pathology also confirmed that no minimal evidence of inflammatory cell invasion or alveolar damage was found in the vaccinated and challenged animals. In the nasal compartment, NanoSTING-NS showed a significant reduction in infectious viral particles on day 2, even compared to NanoSTING-S, and on day 6, infectious viral particles were completely eliminated in NanoSTING-NS vaccinated animals. In summary, these results suggest that sterilizing immunity can be achieved through synergistic protection of immune responses against N and against the S protein.

[0247] Unless otherwise defined, all technical terms used herein have the same meanings as those commonly understood by ordinary technicians in the field to which the subject belongs. It should be understood that the above general description and the following detailed description are illustrative and explanatory and do not limit the subject matter of the patent protection.

[0248] The section headings used herein are for organizational purposes and should not be construed as limiting the contents. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and treaties, are hereby incorporated by reference in their entirety for any purpose. When one or more of the combined documents and similar materials conflict with the definition of a term in this application, the application shall prevail.

[0249] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. For example, "a" or "an" means "at least one", while the use of "or" means "and / or", unless otherwise specifically stated. As used herein, the term "and / or" includes any one and all combinations of one or more of the associated listed items. It should also be understood that the terms "include" and / or "comprise" or "contain" and / or "containing" used in this specification specifically refer to including the stated features, regions, integers, steps, operations, elements and / or parts, but do not exclude the presence of adding one or more other features, regions, integers, steps, operations, elements, parts and / or combinations thereof. At the same time, unless otherwise specifically stated, terms such as "element" or "component" include elements or components of one unit as well as elements or components including more than one unit.

[0250] A and / or B used herein include one or more of A or B, and combinations thereof, such as A and B. It is understood that, although the terms "first", "second", "third", etc. may be used to describe various elements, components, regions and / or parts here, these elements, components, regions and / or parts should not be limited by these terms. These terms may only be used to distinguish an element, component, region or part from another element, component, region or part. Therefore, in some cases, the first element, component, region or part discussed herein may be referred to as the second element, component, region or part, without departing from the teachings of the present disclosure.

[0251] The term "subject" as used herein includes mammals. Mammals include rats, mice, non-human primates and primates, including humans.

[0252] 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, which does not have an associated antigen molecule. In some cases, the term NanoSTING refers to a composition comprising a modulator, a divalent cation (such as 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 comprising an Antigenic molecule, for example, when described in the context of comprising an Antigenic molecule and / or when the term is modified to reflect an embodiment comprising an Antigenic molecule (e.g., "NanoSTING-monomer", "NanoSTING-trimer", "NanoSTING-RSV", "NanoSTING-S", "NanoSTING-varS", "NanoSTING-ChimS", "NanoSTING-N", "NanoSTING-N10" or "NanoSTING-N20").

[0253] In the present disclosure, various embodiments are presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of any embodiment. Therefore, unless the context clearly stipulates otherwise, the description of the range should be considered to have specifically disclosed all possible sub-ranges and each numerical value within the range, up to one-tenth of the unit of the lower limit. For example, it should be considered that the description of a range such as 1 to 6 has specifically disclosed sub-ranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and each number within the range, such as 1.1, 2, 2.3, 4.62, 5 and 5.9. This is generally applicable and has nothing to do with the breadth of the range. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and they also belong to the scope of the present invention, subject to the limits explicitly excluded by the range. When the range is set to include one or two limits, unless the context clearly stipulates otherwise, the present disclosure also includes a range that excludes one or two of the limits.

[0254] In this specification and claims, unless otherwise indicated, the terms "approximately" and "about" or "substantially" refer to a variation of less than or equal to + / -0.1%, + / -1%, + / -2%, + / -3%, + / -4%, +1-5%, + / -6%, + / -7%, + / -8%, +1-9%, + / -10%, + / -11%, + / -12%, + / -14%, + / -15%, or + / -20%, including increments therein, depending on the embodiment. As an example, approximately 100 nanometers (nm) represents a range of 95 nanometers to 105 nanometers (which is + / -5% of 100 nanometers), 90 to 110 nanometers (which is + / 10% of 100 nanometers), or 85 nanometers to 115 nanometers (which is + / -15% of 100 nanometers), depending on the embodiment.

[0255] Although preferred embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art may make various changes, variations and substitutions without departing from the present invention. It should be understood that various alternative forms of the embodiments of the present invention described herein may be used to implement the present invention. The appended claims define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are encompassed by the present invention.

[0256] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference for all purposes. All publications, patents and patent applications mentioned in this specification are incorporated herein by reference, as if each separate publication, patent or patent application is specifically and individually incorporated herein by reference. The subject matter of the appendix A and B of U.S. Provisional Patent Application No. US 63 / 301,918 and the subject matter of the appendix A and appendix B of U.S. Provisional Patent Application No. US 63 / 329,261, including all embodiments described therein and combinations thereof, are incorporated herein by reference for all purposes. Specifically, the embodiments and combinations thereof of the compositions and / or methods disclosed in U.S. Provisional Patent Application No. US 63 / 301,918 (including its appendix A or appendix B) and / or in U.S. Provisional Patent Application No. US 63 / 329,261 (including its appendix A or appendix B) comprising one or more particles, regulators, antigens, objects and / or experimental examples are incorporated herein by reference for all purposes.

[0257] Although certain embodiments and examples are provided in the foregoing description, the subject matter of the present 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 specific disclosed order. Various operations may be described in sequence as multiple discrete operations, which may be helpful in understanding some embodiments; however, the order of description does not necessarily have to be understood as an order in which the operations are performed. In addition, the structures, systems, and / or devices described herein may be embodied as integrated components or independent components.

[0258] In order to compare various embodiments, certain aspects and advantages of these embodiments are described. It is not necessary that all of these aspects or advantages can be achieved by any specific embodiment. Thus, for example, various embodiments can be carried out in a manner that achieves or optimizes one advantage or a group of advantages taught herein without necessarily achieving other aspects or advantages that may also be taught or suggested herein.

Claims

1. A composition comprising: Lipid-based particles and conditioning agents, The modulator is a pattern recognition receptor agonist, an immune system activator or a combination thereof.

2. The composition of claim 1, wherein the regulator is encapsulated within the lipid-based particles.

3. The composition of claim 1, wherein the lipid-based particles comprise an antigen.

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

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

7. A composition according to claim 6, wherein the lipid-based particles contain a larger number of nucleocapsid protein molecules than spike protein molecules.

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

1.

9. The composition of claim 1, wherein the composition is formulated for intranasal delivery.

10. The composition of claim 1, wherein the modulator is an agonist of the STING pathway.

11. The composition of claim 1, wherein the lipid-based particles comprise DPPC, DPPG, cholesterol, and DPPE-PEG2000 in a ratio of 10:1:1:

1.

12. The composition of claim 3, wherein the antigen is associated with the outer surface of a lipid-based particle.

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

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

15. 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), aminobenzimidazole, a derivative of aminobenzimidazole, a nucleotide modulator, a plasmid DNA modulator, a divalent cation, or a combination thereof.

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

17. A kit comprising a composition as claimed in any one of claims 1 to 16, including instructions for use.

18. A method comprising: Administering to a subject a composition as described in any one of claims 1 to 16, wherein the composition comprises: Lipid-based particles and conditioning agents, The modulator is a pattern recognition receptor agonist, an immune system activator or a combination thereof.

19. The method of claim 18, wherein the subject does not exhibit symptoms of respiratory disease.

20. The method of claim 18, wherein the sample obtained from the subject has detectable levels of a pathogen associated with the respiratory disease.

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

22. The method of claim 18, wherein the subject exhibits symptoms of a respiratory disease.

23. The method of claim 18, wherein the composition is administered in at least one dose.

24. The method of claim 18, wherein the composition is administered in one dose.

25. The method of claim 18, wherein the composition is administered in two doses.

26. The method of claim 18, wherein the composition is administered prior to exposure of the subject to a pathogen associated with the respiratory disease.

27. The method of claim 26, wherein the composition is administered to the subject at least one day prior to exposure of the subject to a pathogen associated with the respiratory disease.

28. The method of claim 26, wherein the composition is administered to the subject at least three days prior to exposure of the subject to a pathogen associated with the respiratory disease.

29. The method of claim 18, wherein the composition is administered by intranasal administration.

30. The method of claim 18, wherein the composition is administered by inhalation.

31. The method of claim 18, wherein the method is used to prevent the occurrence of a disease in the subject, prevent the progression of the disease in the subject, prevent the spread of a disease to a second subject, or a combination thereof.

32. The method of claim 18, wherein the method elicits an innate immune response leading to relevant adaptive immunity.

33. The method of claim 19, wherein the respiratory disease comprises an infection caused by a pathogen.

34. The method of claim 33, wherein the pathogen is a respiratory pathogen.

35. The method of claim 33, wherein the respiratory pathogen is a virus.

36. The method of claim 35, wherein 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.

37. The method of claim 35, wherein the virus is an influenza virus.

38. The method of claim 37, wherein the influenza virus comprises an oseltamivir-susceptible strain, a treatment-resistant strain, or a combination thereof.

39. The method of claim 35, wherein the virus is a coronavirus.

40. The method of claim 39, wherein the coronavirus is the SARS-CoV-2 virus, an alpha variant thereof, a delta variant thereof, an Omicron variant thereof, or a combination thereof.

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