An intranasal nanoparticle STING agonist protects against respiratory viruses in animal models

Abstract

Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2'-3' cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.

Fig. 1. Pharmacokinetic and pharmacodynamic profiling of NanoSTING reveals prolonged delivery of cGAMP and induction of ISGs in the nasal compartment of mice.

A Overall schematic for the synthesis of NanoSTING and intranasal delivery of NanoSTING to mice. Groups of 3–12 BALB/c mice were treated with single doses of NanoSTING (10 µg, 20 µg, or 40 µg) and we euthanized subsets at 6 h, 12 h, 24 h, 36 h, and 48 h followed by collection of blood, nasal turbinates, and lungs. cGAMP ELISA, IFN-β ELISA, CXCL10 ELISA, and qRT-PCR (nasal turbinates) were the primary readouts. B, C ELISA quantification of cGAMP in the nasal turbinates and lungs of mice after treatment with NanoSTING. D–L Fold change in gene expression for NanoSTING-treated (40 µg in green, 20 µg in red, and 10 µg in blue) mice and control mice were quantified using RNA extracted from nasal turbinates by qRT-PCR (Primer sequences are provided in Supplementary Table 2). M Quantification of IFN-β concentration in mouse nasal tissue using quantitative ELISA. N, O Quantification of CXCL10 levels in mouse nasal tissue and lungs using quantitative ELISA. Individual data points represent independent biological replicates taken from separate animals; vertical bars show mean values with error bars representing SEM. Each dot represents an individual mouse. P-values were calculated by a two-tailed Mann–Whitney U-test for (B–O) ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant. Data presented as combined results from (B–O) one independent animal experiment. Gender was not tested as a variable, and only female mice were included in the study. See also Supplementary Figs. 1–3 and Supplementary Table 2. Color codes: 40 µg NanoSTING (green), 20 µg NanoSTING (red), 10 µg NanoSTING (blue) and Control (black). A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Number of animals used: n = 3–12/group. Source data are provided as a Source Data file.

Fig. 2. Uptake of NanoSTING by myeloid populations and epithelial cells in nasal tissue and lungs.

A Overall schematic for tracking the cellular targets of NanoSTING. The liposomes were formulated to encapsulate SRB (red dye) and the liposomes were conjugated to DiD (green dye). The dual-labeled liposomes were administered intranasally to mice, and the single-cell suspensions were analyzed using flow cytometry. The cell types of the murine nasal epithelium are shown schematically. B Quantification of DiD⁺ SRB⁺ cells in lungs and nasal tissue by flow cytometry. C Flow cytometric plots (pseudocolor-smooth) showing uptake of DiD & SRB in nasal tissue. D Quantification of DiD⁺ SRB⁺ epithelial cells (CD45⁻EPCAM⁺), endothelial (CD45⁻CD31⁺), and two myeloid cell subsets-CD45⁺EPCAM⁻CD11b⁺CD11c⁻ and CD45⁺EPCAM⁻CD11c⁺CD11b⁻ in lungs and nasal tissues by flow cytometry. E Flow cytometric plots (pseudocolor-smooth) showing uptake of DiD & SRB by epithelial cells in nasal tissue. F The percentages of epithelial cells (basal cells, secretory cells & ciliated cells) in nasal tissue. Individual data points represent independent biological replicates taken from separate animals; vertical bars show mean values with error bars representing SEM. Each dot represents an individual mouse. Data presented as combined results from (B–F) one independent animal experiment. Gender was not tested as a variable, and only female mice were included in the study. See also Supplementary Fig. 5 (gating strategy), Supplementary Table 4 (list of antibodies or conjugates used). Color codes: Lungs (Black), Nasal tissue (gray). A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Number of animals used: n = 5/group. Source data are provided as a Source Data file.

Fig. 3. Rat toxicology studies based on repeat-dose administration of NanoSTING.

A Groups of Sprague Dawley rats (n = 12) were intranasally administered four doses of 125 µg on the indicated days and euthanized on day 11 for histopathology of the small intestines, lungs, nasal cavity, and stomach. B Representative H & E images of the target organs of the treated rats; all images were acquired at 10×; scale bar, 100 µm. Gender was tested as a variable with an equal number of male and female rats included in the study. See Supplementary Figs. 6, 7. A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Number of animals used: n = 12/group. Source data are provided as a Source Data file.

Fig. 4. RNA-sequencing identifies the activation of IFN-dependent and IFN-independent pathways in the lungs of hamsters treated with NanoSTING.

A Heatmap of the top 50 differentially expressed genes (DEGs) between NanoSTING-treated lungs (marked as green) and control lungs (marked as black). B The volcano plots of DEGs comparing NanoSTING-treated and control animals. C Gene set enrichment analyses (GSEA) of C2 and C7 curated pathways visualized using Cytoscape. Nodes (red and blue circles) represent pathways, and the edges (blue lines) represent overlapping genes among pathways. The size of nodes represents the number of genes enriched within the pathway, and the thickness of edges represents the number of overlapping genes. The color of nodes was adjusted to an FDR q-value ranging from 0 to 0.25. Clusters of pathways are labeled as groups with a similar theme. D The normalized enrichment score (NES) and false-discovery rate (FDR) q values of top antiviral pathways curated by GSEA analysis. E GSEA of IFN-independent activities of STING pathway activated in the lung of NanoSTING-treated animals. The schematic represents the comparison that was made between samples collected from the GSE149744 dataset to generate the pathway gene set. F The expression of genes in lungs associated with IFN-dependent and IFN-independent antiviral pathways between NanoSTING and control groups. Data represents independent biological replicates taken from separate animals. Data presented as combined results from one independent animal experiment. Gender was tested as a variable with an equal number of male and female hamsters included in the study. See also Supplementary Figs. 8 and 9 and Supplementary Table 3. Color codes: Control (Black), NanoSTING (green). Fig. 1A-Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Number of animals used: n = 4/group.

Fig. 5. Quantitative modeling of the dynamics of replication of SARS-CoV-2.

A, B Schematic representing rate constants and equations governing viral dynamics during A natural infection and B in the presence of NanoSTING treatment. C Reduction in the viral area under the curve (AUC) at different NanoSTING efficacies (RIR) compared to natural infection. The treatment is initiated on day 0, and we assume that the effects of NanoSTING treatment only last for 24 h. D Heatmap of viral AUC with varying NanoSTING efficacy and treatment initiation time. The red box represents the combination with close to 100% reduction in viral AUC. See Supplementary Fig. 10, Supplementary Tables 13–15, and Supplementary Note 1. Fig. 5A, B Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en).

Fig. 6. Protective efficacy of NanoSTING against the pathogenic SARS-CoV-2 Delta (B.1.617.2) VOC and IFN evasive SARS-CoV-2 Alpha VOC (B.1.1.7).

A We treated groups of 12 Syrian Golden hamsters, each with a single dose of 120 µg NanoSTING, and later challenged with ∼3 × 10⁴ CCID₅₀ of SARS-CoV-2 Delta VOC on day 0 by the intranasal route. We euthanized half of the hamsters (n = 6) hamsters on day 2 and determined viral titers of lung and nasal tissues. We rechallenged the remaining 6 hamsters on day 28 and tracked the body weight change until day 35. B Percent body weight change compared to the baseline at the indicated time intervals. C Percent body weight change monitored during the primary infection (day 0–day 6). D, E Viral titers measured by endpoint titration assay in nasal tissues and lungs post-day 2 of infection. The dotted line indicates the limit of detection of the assay (LOD). F Percent body weight change monitored after rechallenge (day 28–day 35). G We tested groups of 9 hamsters, each with two different doses of NanoSTING (30 µg and 120 µg) and 24 h later challenged with the ∼3 × 10⁴ CCID₅₀ of SARS-CoV-2 Alpha VOC (B.1.1.7). On day 2, five animals from each group were euthanized for assessing the viral titers and remaining animals used for the histopathology at day 5. No animals were excluded in this study. H Change in body weight of hamsters. I, J Pathology scores and a representative hematoxylin and eosin (H & E) image of the lung showing histopathological changes in lungs of hamsters treated with NanoSTING (30 µg) and PBS; all images were acquired at 10× and 20×; scale bar, 100 µm. K, L Viral titers were quantified in the lung and nasal tissue by endpoint titration assay on day 2 after the challenge. The dotted line indicates the limit of detection of the assay (LOD). Individual data points represent independent biological replicates taken from discrete samples; vertical bars show mean values with error bars representing SEM. Each dot represents an individual hamster. For (D, E, I, K, L), analysis was performed using a two-tailed Mann–Whitney U-test. For (C, H), data was compared via a mixed-effects model for repeated measures analysis. Lines depict group mean body weight change from day 0; error bars represent SEM. Asterisks indicate significance compared to the placebo-treated animals at each time point. Mann–Whitney U-test ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant. For (B), the p-values are as follows: Day 4: p = 6e−3, Day 5: p = 1e−3, and Day 6: p = 5e−5. For (H), the exact p-values comparing the 30 µg NanoSTING group to the Placebo group are Day 3: p = 5e−3, Day 4: p = 6e−7, and Day 5: p = 10e−9. Additionally, for the 120 µg NanoSTING and Placebo-treated group, the p-values are Day 4: p = 2e−5 and Day 5: p = 3.5e−9. Data presented as combined results from two independent experiments [A−F Challenge study with SARS-CoV-2 Delta VOC, G−L challenge study with SARS-CoV-2 Alpha VOC)], each involving one independent animal experiment. Gender was tested as a variable, and an equal number of male and female hamsters were included in the study. See also Supplementary Figs. 11 and 12. Figure 6A, G—Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Number of animals used in the study: n = 12/group (for A−F), n = 9/group (for G–L). Source data are provided as a Source Data file.

Fig. 7. Intranasal administration of NanoSTING limits transmission and viral replication in the lungs and nasal passage of contact hamsters exposed to the SARS-CoV-2 Omicron (BA.5) VOC.

A Experimental setup: For group 1, we challenged groups of 5 hamsters each on day 0 with ∼3 × 10⁴ of SARS-CoV-2 Omicron VOC (BA.5) and after 24 h cohoused index hamsters in pairs with contact hamsters (n = 5) for 4 days in clean cages. In group 2, we pre-treated the hamsters with 120 µg of NanoSTING 24 h prior to infection. In group 3, we treated the contact hamsters with NanoSTING 12 h after the cohousing period began. We euthanized the contact and index hamsters on day 4 of cohousing. Viral titers in the nasal tissue of the index and contact hamsters were used as primary endpoints. B Viral titers were quantified in the lung of the index (infected) and contact hamsters by endpoint titration assay post-day 5 of infection. C Viral titers were quantified in the nasal tissue of index and contact hamsters by endpoint titration assay post-day 5 of infection. The dotted line indicates the limit of detection of the assay (LOD). For (B, C) analysis was performed using a two-tailed Mann–Whitney U-test. Individual data points represent independent biological replicates taken from separate animals; vertical bars show mean values with error bars representing SEM. Each dot represents an individual hamster. Asterisks indicate significance compared to the placebo-treated animals. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant. Data presented as combined results from one (B, C) independent animal experiment. NT Non-treated, NS-Pro Prophylactic treatment with NanoSTING, NS-Tx Post-exposure treatment with NanoSTING. Gender was tested as a variable with an equal number of male and female hamsters included in the study. A and parts of B, C—Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Number of animals used: n = 5/group. Source data are provided as a Source Data file.

Fig. 8. Intranasal administration of NanoSTING limits transmission and viral replication in the nasal passage of contact hamsters exposed to the SARS-CoV-2 Omicron (B.1.1.529) VOC.

A Experimental setup: For group 1, we challenged groups of 8 hamsters each on day 0 with ∼3 × 10⁴ of SARS-CoV-2 Omicron VOC (B.1.1.529) and after 24 h cohoused index hamsters in pairs with contact hamsters (n = 8) for 4 days in clean cages. In group 2, we pre-treated the hamsters with 120 µg of NanoSTING 24 h prior to infection. In group 3, we treated the contact hamsters with NanoSTING 12 h after the cohousing period began. We euthanized the contact and index hamsters on day 4 of cohousing. Viral titers in the nasal tissue of the index and contact hamsters were used as primary endpoints. B, C Infectious viral particles in the nasal tissue of contact hamsters at day 2 and day 5 after viral administration post-infection were measured by endpoint titration assay. The dotted line indicates limit of detection of the assay (LOD). For (B, C), analysis was performed using a two-tailed Mann–Whitney U-test. Individual data points represent independent biological replicates taken from separate animals; vertical bars show mean values with error bars representing SEM. Each dot represents an individual hamster. Mann–Whitney test: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant. Data presented as combined results from one (B, C) independent animal experiment. Gender was tested as a variable with an equal number of male and female hamsters included in the study. See supplementary Fig. 13. Abbreviations- NS-Pro: Prophylactic treatment with NanoSTING; NS-Tx-Post-exposure treatment with NanoSTING. A and parts of B, C—Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en) Number of animals: n = 8/group. Source data are provided as a Source Data file.

Fig. 9. NanoSTING offers protection against Oseltamivir-sensitive and resistant strains of Influenza A.

A Experimental set up: We treated groups of 10 BALB/c mice, each with a single dose of NanoSTING (40 µg) or Oseltamivir (30 mg/kg/day administered twice daily) or placebo and 24 h later challenged with 2 × 10⁴ CCID₅₀ of Influenza A/California/04/2009 (H1N1dpm) strain and monitored for 14 days. Body weight change was used as the primary endpoint. Oseltamivir was used as a control. B Percent body weight change for the different groups of mice. C Experimental set up: We treated groups of 10 BALB/c mice with a single intranasal dose of NanoSTING (40 µg) and 24 h later challenged with 2 × 10⁴ CCID₅₀ of influenza A/Hong Kong/2369/2009 (H1N1)-H275Y [A-H275Y] followed by rechallenge on day 28 and tracked the body weight change until day 35. We evaluated the animals for 41 days and used weight loss as the primary endpoint. On day 15, we evaluated the percent survival of different groups of mice. We conducted IgG and IgA ELISA on day 28. We treated one group of mice with a clinically relevant dose of oseltamivir, twice daily for five days. D Percent weight change compared to the weight at day 0 at the indicated time intervals. E Percent body weight change monitored after rechallenge (day 28–day 41). F Percent body weight change monitored during the primary infection (day 0–day 15). G Percent survival of the different groups of mice. H Humoral immune responses in the serum were evaluated on day 28 using IgG ELISA. I Humoral immune responses in the serum were evaluated on day 28 using IgA ELISA. J Experimental set up: We treated groups of 10 BALB/c mice with a single intranasal dose of NanoSTING (40 µg), and 24 h later challenged with 2 × 10⁴ CCID₅₀ of influenza A/Hong Kong/2369/2009 (H1N1)-H275Y [A-H275Y]. We monitored the animals for 7 days for body weight change and quantified viral titers at the end of the study. We treated one group of mice with oseltamivir, twice daily, for five days. K Weight change of the different groups of mice. L Viral titers were measured by endpoint titration assay in lungs post 7 days after infection. The dotted line indicates the limit of detection of the assay (LOD). For (H, I, L), analysis was performed using a two-tailed Mann–Whitney U-test. Individual data points represent independent biological replicates taken from separate animals; vertical bars show mean values with error bars representing SEM. Each dot represents an individual mouse. For (B, F, K), weight data was compared via a mixed-effects model for repeated measures analysis. Lines depict group mean body weight change from day 0; error bars represent SEM. For (B, K), asterisks indicate statistically significant differences between the NanoSTING-treated group and placebo-treated animals, whereas, the pound sign shows statistically significant differences between the Oseltamivir-treated group and placebo-treated animals. For (F), asterisks indicate statistically significant differences between the NanoSTING-treated group and non-challenged animals, whereas, pound sign indicate statistically significant differences between the Oseltamivir-treated group and non-challenged animals. For (G) we compared survival percentages between NanoSTING-treated and Oseltamivir-treated animals using the Log-Rank Test (Mantel–Cox). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant. For (B) the exact p-values comparing the 40 µg NanoSTING group to the Placebo group are as follows: Day 2: p = 5e−3, Day 3: p = 5.5e−4, Day 4: p = 3e−4, Day 5: p = 2e−5, Day 6: p = 3e−7, Day: 8: p = 1e−4, Day 9: p = 4e−3, Day 10: p = 2.5e−2, Day 11: p = 1e−2, Day 12: p = 6e−3, Day 13: p = 6e−3, Day 14: p = 8e−3 and Day 15: p = 2e−2. Additionally, for the Oseltamivir and Placebo-treated group, the p-values are as follows: Day 2: p = 1e−2. For (F) the exact p-values comparing the 40 µg NanoSTING group to the Placebo group are as follows: Day 9: p = 5e−3. Additionally, for the Oseltamivir and Placebo-treated group, the p-values are as follows: Day 3: p = 6e−6, Day 4: p = 2e−8, Day 5: p = 10e−10, Day 6: p = 2e−10, Day 9: p = 6e−8, Day 10: p = 1e−3, Day 11: p = 1e−2. For (K) the exact p-values comparing the 40 µg NanoSTING group to the Placebo group are as follows: Day 3: p = 4e−2, Day 4: p = 5e−3, Day 5: p = 9e−4, Day 6: p = 7e−6, Day 7: p = 1e−5. The data combines results from three independent animal studies: Study 1 (A, B), Study 2 (C–I), and Study 3 (J–L), each involving one independent experiment. Gender was tested as a variable with an equal number of male and female mice included in the study. See also Supplementary Fig. 14. A, J—Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en) Number of animals used: n = 10/group. Source data are provided as a Source Data file.

Fig. 10. NanoSTING activates innate immunity in upper airways in Rhesus macaques.

A Experimental set up: We administered one group (n = 4/group) of Rhesus macaques (RM’s) with two doses of NanoSTING (0.1 mg/kg-range: 0.06–0.14 mg/kg) administered intranasally on day 0 and day 2, and we monitored the animals until day 4 for changes in body weight, body temperature, and nasal area temperature. We euthanized one of the animals on day 4 to assess the histopathological changes in the lungs and trachea. B Percent body weight change for the RM’s at indicated time intervals. C Body temperature change for RM’s at indicated time intervals. D Monitoring of nasal area temperature pre and post-nasal wash collection/NanoSTING treatment. E Quantification of CXCL10 levels in the nasal wash of animals using quantitative ELISA. F, G Representative hematoxylin and eosin (H & E) images of the lungs and trachea of RM’s treated with two doses of NanoSTING (0.1 mg/kg-range: 0.06–0.14 mg/kg); all images were acquired at 2×; scale bar, 100 µm. For (B, C, D), the analysis was performed using Kruskal–Wallis test. For (E), we performed Mann–Whitney U-test. Individual data points represent independent biological replicates taken from separate animals. Kruskal–Wallis test, Mann–Whitney U-test ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns not significant. Data presented as combined results from one (B–G) independent animal experiment. 3 female and 1 male RM’s were taken for the study. See also Supplementary Figs. 15 and 16. A-Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license (https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en). Number of animals: n = 4/group Source data are provided as a Source Data file.