Compositions and methods of lung surfactant-biomimetic nanoparticles

Abstract

Disclosed are compositions and methods containing pulmonary surfactant (PS) biomimetic nanoparticles. Specifically, the present disclosure relates to compositions containing nanoparticles having an average size of 200-400 nm, the nanoparticles comprising a plurality of pulmonary surfactant biomimetic molecules, wherein the nanoparticles are negatively charged; and one or more cargo molecules encapsulated by the nanoparticles, wherein the molecular weight of the cargo molecules is less than 1200 Da.

Description

[0001] Federally Sponsored Research or Development

[0002] This invention was made with U.S. Government support under Grant Nos. AI089779, AI070785, and AI097696 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Technical Field

[0003] Described herein are compositions comprising pulmonary surfactant-biomimetic nanoparticles such as PS-GAMP, and methods of making and using pulmonary surfactant-biomimetic nanoparticles such as PS-GAMP. Background Art

[0004] Current influenza vaccines protect against viral infection primarily by inducing neutralizing antibodies specific for viral surface hemagglutinin (HA) and neuraminidase (NA). However, these surface proteins continue to undergo antigenic drift / shift, greatly limiting the efficacy of these vaccines (1). + Resident memory T(T RM An explanation for this limitation may be provided by studies of the integral role of pulmonary CD8 T cells in heterosubtypic immunity (2,3). Fully induced by natural viral infection, these cells not only recognize highly conserved internal proteins shared among heterosubtypic influenza viruses but are also able to clear virus from sites of entry when viral numbers are low (4-6). Similarly, live vector-engineered and attenuated influenza vaccines induce pulmonary CD8 T cells. + T RM Cells (7,8), but a delicate balance must be struck between their safety and immunogenicity. Furthermore, these replicating vaccines are often affected by pre-existing immunity and are therefore only suitable for certain populations (9). In contrast, non-replicating influenza vaccines induce poor T cell immunity in the respiratory tract, requiring potent mucosal adjuvants to overcome the immune regulatory mechanisms of the respiratory mucosa. Summary of the invention

[0005] Described herein are safe and potent mucosal adjuvants that can be used, for example, to boost influenza vaccines.

[0006] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

[0007] In one aspect, the present disclosure relates to a composition containing nanoparticles with an average size of 200-400 nm, wherein the nanoparticles include a plurality of lung surfactant biomimetic molecules, wherein the nanoparticles are negatively charged; and one or more cargo molecules encapsulated by the nanoparticles, wherein the molecular weight of the cargo molecules is less than 1200 Da.

[0008] In some embodiments, the lung surfactant-biomimetic molecule includes 50 wt%-90 wt% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 5 wt%-15 wt% negatively charged lipids, and / or 5 wt%-15 wt% neutral lipids.

[0009] In some embodiments, the negatively charged lipid is 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) and the neutral lipid is cholesterol.

[0010] In some embodiments, the nanoparticles further include a plurality of polyethylene glycols (PEGs) having an average molecular weight of 500-5000 Da. In some embodiments, the polyethylene glycols are attached to the outer surface of the nanoparticles.

[0011] In some embodiments, the nanoparticles further include 5-15 wt % of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000).

[0012] In some embodiments, the cargo molecule is a stimulator of interferon genes (STING) agonist.

[0013] In some embodiments, the STING agonist is or comprises cyclic guanosine monophosphate [GMP]-adenosine monophosphate [AMP] (cGAMP).

[0014] In some embodiments, cGAMP is present at a concentration of 10-100 μg / ml.

[0015] In some embodiments, the cargo molecule is a long-acting β2-agonist (LABA) (e.g., formoterol, salmeterol, or vilanterol); a corticosteroid (ICS) (e.g., budesonide, fluticasone propionate, or fluticasone furoate); furoate); leukotriene pathway modulators (e.g., montelukast or zileuton); inhibitors of targeted kinases (e.g., spleen tyrosine kinase, p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Janus kinase (Jak), or phosphodiesterase-4 (PDE4)); agonists or antagonists of receptors (e.g., chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2), chemokine receptor 2 (CCR2)); ion channels (e.g., GABA receptor, transient receptor potential cation channel, subfamily A, member 1); 1, TRPA1), or voltage-gated sodium channels); inducers of IFN-α; long-acting muscarinic antagonists / anticholinergics (LAMAs); inhibitors of IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; CXCR2 antagonists; molecules that block proinflammatory cytokines (e.g., TNF-α, TNF-β, or IL-6); molecules that block IL-17 / T H17; macrolides; molecules that activate HDAC2; STAT6 inhibitors (e.g., AS1517499); antiviral small molecule drugs (e.g., oseltamivir (Tamiflu), Relenza, or zanamivir); Favipiravir (T705); agonists of intracellular Toll-like receptor (TLR) TLR3 (e.g., imiquimod, resiquimod (R848), imidazoquinoline (IMQ), motolimod, CU-CPT4a, IPH-3102, or Rintatolimod); agonists of Nodinitib (NOD1), NOD2, NLPR3, or NPLRC3 (e.g., muramyl dipeptide (MDP), FK5 65, or FK156; TLR7 or TLR8 agonists (e.g., Isatoribine, Loxoribine, gardiquimod, AZD8848, IMO-8400, ANA773, IMO-3100, SM360320, or 852A); TLR8 agonists (e.g., VTX-1463, VTX-2337, IMO-8400 , or 2,3-diamino-furano[2,3-c]pyridine); and / or a TLR9 agonist (IMO-8400, IMO-3100, SAR-21609, AZD1419, SD-101, IMO-2055, IMO-2125, QAX-935, AVE0675, DIMS0150, MGN-1703, MGN-1706, ISS1018, or Agatolimod).

[0016] In one aspect, the disclosure relates to a method of promoting an immune response to an antigen, the method comprising administering to a subject an effective amount of a composition as described herein; and administering to the subject the antigen.

[0017] In some embodiments, the subject is a mammal.

[0018] In some embodiments, the antigen is encapsulated within the nanoparticle; the nanoparticle and the antigen are administered in a single composition; or the nanoparticle and the antigen are administered in separate compositions.

[0019] In one aspect, the disclosure relates to a method of treating a subject having influenza, the method comprising administering to the subject a therapeutically effective amount of a composition as described herein; and administering to the subject an antigen.

[0020] In some embodiments, the cargo molecule is cGAMP and the antigen is influenza vaccine.

[0021] In some embodiments, the subject is a human and the antigen is a human influenza vaccine.

[0022] In one aspect, the disclosure relates to a method of treating a subject having an airway disease, the method comprising administering to the subject a therapeutically effective amount of a composition as described herein.

[0023] In some embodiments, the cargo molecule is a long-acting β2-agonist (LABA) (e.g., formoterol, salmeterol, or vilanterol); a corticosteroid (ICS) (e.g., budesonide, fluticasone propionate, or fluticasone furoate); a leukotriene pathway modulator (e.g., montelukast or zileuton); an inhibitor of a targeted kinase (e.g., spleen tyrosine kinase, p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Janus kinase (Jak), or phosphodiesterase-4 (PDE4)); a receptor (e.g., a chemoattractant expressed on Th2 cells); agonists or antagonists of ion channels (e.g., GABA receptors, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or voltage-gated sodium channels); inducers of IFN-α; long-acting muscarinic antagonists / anticholinergics (LAMAs); inhibitors of IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; CXCR2 antagonists; molecules that block proinflammatory cytokines (e.g., TNF-α, TNF-β, or IL-6); molecules that block IL-17 / T H 17; macrolides; molecules that activate HDAC2; STAT6 inhibitors (e.g., AS1517499); antiviral small molecule drugs (e.g., oseltamivir (Tamiflu), Relenza, or zanamivir); and / or favipiravir (T705).

[0024] In some embodiments, the subject is a human and the airway disease is one or a combination of asthma, chronic obstructive pulmonary disease (COPD), allergies, or a pneumoviral infection.

[0025] In one aspect, a method of treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition as described herein. In some embodiments, the cargo molecule is a chemotherapeutic agent.

[0026] In some embodiments, the subject is a mammal.

[0027] In some embodiments, the cancer is lung cancer and the chemotherapeutic agent is gefitinib, erlotinib, crizotinib, everolimus, afatinib, crizotinib, doxorubicin, etoposide, nivolumab (Opdivo), and / or methotrexate (Trexall).

[0028] In some embodiments, the cancer is nasopharyngeal carcinoma and the chemotherapeutic agent is Cisplatin, Carboplatin, Gemcitabine, Doxorubicin, and / or D5-fluorouracil (5-FU).

[0029] In some embodiments, the cancer is tracheal cancer and the chemotherapeutic agent is etoposide, cisplatin, and / or carboplatin.

[0030] In some embodiments, the cancer is bronchial carcinoma and the chemotherapeutic agent is etoposide, cisplatin, carboplatin, 5-FU, docetaxel, paclitaxel, and / or epirubicin.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are exemplary only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail. Other features and advantages of the present invention will be apparent from the following detailed description and drawings, as well as from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) (or more) will be provided by the Office upon request and payment of the necessary fee.

[0033] Figures 1A-1J.PS-GAMP uptake by AM requires SP-A and SP-D. (A) Schematic diagram of PS-liposomes labeled with SRB and DiD. (BE) Free SRB (20 μg) or SRB-DiD-nano4 or SRB-DiD-nano5 (20 μg SRB) were intranasally administered to mice, followed by SRB 12 hours later. + and / or DiD + Flow cytometric analysis of lung cells. Analysis (B) and quantification (C and D) were also performed for CD11c + AM (red) or CD11c - SRB of AEC (blue) + % of cells (n=4). (E) Schematic overlay of flow cytometry images of AMs and AECs stained for DiD and SRB. (F) AMs were isolated from MHC II-GFP mice and cultured with DiD-nano4 or DiD-nano5 for 4 h after pre-incubation with (lower panel) or without (upper panel) PS for 30 min. Scale bar: 10 μm. Alternatively, AMs were isolated from wild-type (WT) mice and cultured with WT or Sftpa1 - / - Sftpd - / - DiD-nano4 pretreated with PS for 30 min was cultured for 4 h (I). Cells were imaged by fluorescence microscopy, and DiD fluorescence intensity in individual cells was quantified using Image J (G and I). n = 18-36. (H) Lungs were visualized by fluorescence microscopy 12 h after receiving DiD-nano4 or DiD-nano5. Scale bar: 50 μm. (J) DiD-nano4 was intranasally administered to WT or Sftpa1 - / - Sftpd - / - Mice. 12 hours later, CD11c + CD11b - CD24 - AM Analysis DiD + . n=6. Each symbol represents a single mouse in (C, D, and J) or a cell in (G and I). Results are expressed as mean ± SEM. Statistical analysis was one-way ANOVA for (C, D, G, and I) and Student t test for (J). **p<0.01 and ***p<0.001 between the indicated groups. All experiments were repeated three times with similar results.

[0034] Figure 2A-2LAdjuvant properties of PS-GAMP. (A and B) Swiss Webster mice were immunized intranasally with VN04 H5N1 vaccine plus 20 μg of free cGAMP or PS-GAMP containing the indicated amounts of cGAMP. Two weeks later, Ag-specific serum HAI (A) and BALF IgA (B) titers were determined. n = 8. (C to E) C57BL / 6 mice were immunized intranasally with VN04 H5N1 vaccine in the presence or absence of PS-GAMP (20 μg cGAMP) on day 0 and boosted on day 14. Serum was collected on day 14 (primary) or day 21 (boost) and Ag-specific IgG (C), IgG2c (D), and IgG1 (E) titers were determined. n = 4. (F to L) C57BL / 6 mice were immunized intranasally with CA09 H1N1 vaccine with or without 20 μg of PS-GAMP or poly IC. After 2 weeks, serum IgG (F), BALF IgA (G), and serum HAI (H) titers were determined. (IJ) Splenocytes were isolated 7 days after immunization and stimulated with CA09 H1N1 vaccine. CD8 T cells producing IFN-γ after viral Ag stimulation were determined by flow cytometry. + (I) and CD4 + (J) T cells. (K and L) Survival curves (K) and body weight changes (L) of unimmunized mice (black) or mice that received a single vaccine (green) and were treated with 10×LD 50 CA09 H1N1 virus challenge combined with vaccine with polyIC (blue) or PS-GAMP (red). n=6. Results are expressed as mean ± SEM. Each symbol represents a single mouse in (A to J). Statistical analysis, one-way ANOVA for (A to J), two-way ANOVA for (L), and log-rank test for (K). *p<0.05, **p<0.01, and ***p<0.001 in the presence or absence of PS-GAMP. ns, not significant. All experiments were repeated twice with similar results.

[0035] Figures 3A-3I CD8 induced by PS-GAMP + T cell responses. (A) CD4 T cells in lung (upper panel) and MLN (lower panel) were analyzed by flow cytometry on the indicated days after intranasal administration of PS-GAMP to mice. + and CD8 + T cells, NK cells, and CD11b + and CD11b - The number of DCs. n = 4. (B) After intranasal immunization with PS-GAMP or with 1×LD 50Quantification of CD11b in lung and MLN by flow cytometry on the indicated days after CA09 H1N1 virus infection of mice + mono-DC and CD11b + tDC. n=4. (C to E) Mice were vaccinated intranasally with OVA-AF647 with or without PS-GAMP. 36 hours after immunization, OVA-capturing DCs were counted in the MLN (C). n=6. The mean fluorescence intensity (MFI) of CD40 (E) or CD86 (F) on these DCs was quantified by flow cytometry. n=4. (F and G) Mice were immunized intranasally with CA09 H1N1 vaccine with or without PS-GAMP, or with PBS alone as a non-immunized control. At the indicated days after immunization, mice were vaccinated intranasally with NP 366-374 Tetramer staining for CD8 in lung and MLN + T cells were analyzed for their Ag-specificity. n = 4-8. (H) Mice were immunized as described in (F and G) and 2 days later were treated with 10×LD 50 CA09 H1N1 virus challenge. GB counts of BALF and lung cells were performed on the indicated days after immunization. + CD8 + T cells. n = 4. (I) Mice were immunized and challenged similarly to (H) except that 20 μg of poly IC or Pam2CSK4 was used instead of PS-GAMP for immunization. GB were counted as described in (H) 4 days after challenge + CD8 + T cells. n=4. Each symbol represents a single mouse in (A, CE, and I). Results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA for (A, B, and I), two-way ANOVA for (F to H), and Student t test for (C to E). *p<0.05; **p<0.01, and ***p<0.001 compared to day 0 (A and B), influenza vaccine alone (F to H), or between the specified groups. All experiments were repeated twice with similar results.

[0036] Figures 4A-4J .PS-GAMP mediated early protection. (AB) 10×LD 50Survival of immunized C57BL / 6 mice after CA09 H1N1 virus challenge. (A) Mice were immunized intranasally with CA09 H1N1 vaccine (0.5 μg HA) and PS-GAMP (20 μg cGAMP) 2, 4, 6, 8, or 14 days before virus challenge as described in Figure 28A. n = 6-11. (B) Mice were immunized and challenged on the same day (0) or 2 days after immunization (-2). n = 6. (C) Two days later, mice were immunized and challenged as described in (A). CD8 was depleted in some mice by injecting anti-CD8 antibodies 2 days before vaccination and 0, 2, and 4 days after vaccination. + T cells. n = 4. (D) As described in Figure 28A, 10×LD 50 Survival of mice immunized with VN04 H5N1 vaccine plus PS-GAMP at the indicated days prior to day 0 of rgVN04 H5N1 virus challenge. n = 4-8. (E) Survival of mice immunized with VN04 H5N1 vaccine, PS-GAMP, or vaccine plus free cGAMP, CT, or PS-GAMP followed by rgVN04 H5N1 virus challenge 2 days later. n = 4-8. (F) Survival of mice immunized intranasally with H7-Re1 H7N9 vaccine and 20 μg of PS-GAMP or poly IC and challenged 2 days later with 40×LD 50 (G to J) Ferrets were immunized intranasally with CA09 H1N1 vaccine (9 μg) with or without 200 μg of PS-GAMP and 2 days later with 10 6 TCID 50 CA09 H1N1 virus challenge. Body weight (G), disease score (H), temperature (I), and virus titer in nasal washes (J) were monitored for 12 hours. n=4. Results are expressed as mean ± SEM. *p<0.05, **p<0.01, and ***p<0.001 compared to day 0 (A, D), vaccine alone (B, E, and F), or in the presence or absence of anti-CD8 antibodies (C). The mouse experiment was repeated twice with similar results. For ferrets, * indicates significance between PBS and vaccine + PS-GAMP, and # indicates significance between vaccine and vaccine + PS-GAMP. *, #p<0.05; **, ##p<0.01; and ***, ###p<0.001. Statistical analysis, two-way ANOVA for (C, G, I, and J), Kruskal Wallis test for (H), and log-rank test for (A, B, and DF).

[0037] Figures 5A-5N.AECs make an indispensable contribution to the adjuvant activity of PS-GAMP. (A) Mice were intraperitoneally administered CBX once a day for 3 consecutive days, followed by intranasal administration of SRB-nano4. SRB was analyzed 12 hours later. + AM (red) and AEC (blue), the percentages of these cells are shown in (B and C). n=6. (D) Mice were intraperitoneally administered CBX, Tonabersat, or Meclofenamate and immunized intranasally with CA09 H1N1 vaccine with or without 20 μg of poly IC or PS-GAMP. After 14 days, sera were collected and analyzed for IgG2c. n=6. (E) Mice were immunized with CA09 H1N1 vaccine and PS-GAMP in the presence or absence of CBX as described in (D). After 24 hours, lung CD11b + DC. n = 4. (F and G) Mice immunized with the indicated gap junction inhibitors as described in (D) and treated 2 hours later with 10×LD 50 CA09 H1N1 virus challenge. GB in BALF (F) and lung (G) was analyzed by flow cytometry. + CD8 + T cells. n = 4. (H) Schematic diagram of the generation of chimeric mice. Mice were subjected to lethal irradiation before bone marrow (BM) cell transfer. Chimerism was confirmed 3 months later (Figure 32), and chimeras were immunized and challenged as described in (F). GBs were counted in BALF (I) and lungs (J) by flow cytometry 4 days after challenge + CD8 + T cells and body weight changes relative to day 0 (K), and lung virus titers were determined in these mice (L). n = 4-7. (M and N) GB was determined by regression analysis + CD8 + Correlation between the number of T cells and viral titer. Results are expressed as mean ± SEM. Each symbol represents a single mouse. Statistical analysis, one-way ANOVA for (D, F, G, and IL), Student t test for (B, C, and E). *p<0.05, **p<0.01, and ***p<0.001. All experiments were repeated twice with similar results.

[0038] Figure 6A-6OPS-GAMP enhances cross-protection against heterologous subtype influenza A viruses. (A to H) Mice were immunized intranasally with CA09 H1N1 vaccine in addition to SH09 H1N1 vaccine or vaccine plus PS-GAMP as in (G and H) and then treated 2 days (first row of graphs) or 2 weeks later (second row of graphs) with 5×LD 50 Challenge with different PR8 H1N1 viruses (A and B) and heterologous subtype Aichi H3N2 (C and D), rgVN04 H5N1 (E and F), or SH13 H7N9 viruses (G and H). n = 6-7 for (A to F), n = 8-13 for (G and H). (I) Mice were immunized as described in (A) and 2 days later with 10×LD 50 Oseltamivir-resistant NC09 H1N1 virus challenge. Unimmunized mice were treated with oseltamivir (20 mg / kg / day) 6 hours before challenge and every day after virus challenge until the end of the study. 50 Mice challenged with CA09 H1N1 or NC09 H1N1 virus. n = 6. (J) Mice were immunized with 2018-19 trivalent seasonal influenza vaccine (SIV18-19) alone or with PS-GAMP and then immunized with 5×LD 50 Mismatched GZ89 H3N2 virus challenge. n = 6-12. (K) Mice were immunized with CA09 H1N1 vaccine alone or with PS-GAMP and then immunized with 5×LD 50 Heterologous subtype rgVN04 H5N1 virus challenge. Alternatively, 1×LD 50 Mice were infected with PR8H1N1 virus and 6 months later were treated with 5×LD 50 Mice surviving infection were re-challenged with rgVN04 H5N1 virus for comparison (pre-infection). n = 6-7. (L to O) Ferrets were immunized intranasally with inactivated Perth H3N2 vaccine (15 μg) with or without PS-GAMP (200 μg). 30 days after immunization, 10 6 TCID 50Ferrets were challenged with heterologous subtype Michigan 15 H1N1 virus. Body weight (L), disease score (M), temperature (N), and virus titer in nasal washes (O) were monitored for 12 days. Results are expressed as mean ± SEM. Mice: *p<0.05, **p<0.01, and ***p<0.001 compared to non-immunized mice. The experiment using mice was repeated twice with similar results. For ferrets, * indicates significance between PBS and vaccine + PS-GAMP, and # indicates significance between vaccine and vaccine + PS-GAMP. *, #p<0.05; **, ##p<0.01; and ***, ###p<0.001. Statistical analysis was two-way ANOVA for (L, N, and O), Kruskal-Wallis test for (M), and log-rank test for (A to K).

[0039] Figures 7A-7K .PS-GAMP preparation and characterization. (A) Schematic diagram of PS-GAMP preparation. Liposomes were synthesized based on mammalian PS components, which are typically composed of 90% lipids and 10% protein and are evolutionarily conserved. The lipids contain 8-10% cholesterol, 60-70% zwitterionic phosphatidylcholine (PC), mainly dipalmitoylphosphatidylcholine (DPPC), up to 8-15% anionic phosphatidylglycerol (DPPG), and a relatively small portion of other lipids (17). PEG2000 was used instead of hydrophilic proteins and cationic DPTAP was used instead of DPPG in nano3 and nano5 to determine the importance of charge. These PS lipids and PEG2000 formed liposomes with a single lipid bilayer that encapsulated cGAMP by reverse phase evaporation as described in detail in Materials and Methods. (B to E) Swiss Webster mice were immunized intranasally with VN04 H5N1 vaccine (1 μg HA content) plus 10 μg of free cGAMP or an equivalent amount of cGAMP packaged in the indicated liposomes. Serum IgG (B) and bronchoalveolar lavage fluid (BALF) IgA (C) were measured 2 weeks later, body weight was monitored for 7 days after immunization (D), and the area under the curve (AUC) (E) was calculated from (D) by PRISM software. n = 5. The size (F), entrapment efficiency (G), and zeta potential (J) of the indicated liposomes were determined. (H and I) Free cGAMP or cGAMP-encapsulated liposomes were incubated with BMDCs (H) and BMMs (I) at a final cGAMP concentration of 10 μg / ml for 8 h, after which IFN-β (Ifnb1) was measured by real-time RT-PCR. n = 4. (K) STING-deficient mice (Sting - / -)(red) or wild-type (WT) (blue) control mice, and serum IgG titers were determined as described above 2 weeks later. n=4. Results are expressed as mean ± SEM. Each symbol represents a single mouse in (B, C, E, and K) or independent replication in (G, H, and I). Statistical analysis was one-way ANOVA for (B, C, E, and HK) and two-way ANOVA for (D). *p<0.05, **p<0.01, and ***p<0.001 compared to the vaccine group alone or between the specified groups. All experiments were repeated twice with similar results.

[0040] Figures 8A-8F . Kinetics of nanoparticle uptake in different tissues. Mice were intranasally administered with PBS, free SRB, SRB-encapsulated and DiD-labeled nano4 (Nano4-SRB), or SRB-encapsulated and DiD-labeled nano5 (Nano5-SRB) and analyzed by flow cytometry at different times. (A) SRB in brain (upper panel), nasal tissue (middle panel), and MLN (lower panel) + Representative flow cytometry of cells. Data are representative of two separate experiments each tested in triplicate. (B and C) SRB in the lungs of mice receiving nano4-SRB (B) or nano5-SRB (C) + Cells. Figures 9A-9D The alveolar macrophages (AM), interstitial macrophages (IM), CD11b + DC, and CD11b - DC gating. (DF) SRB in MLN (D), brain (E), or nasal tissue (F) of mice receiving nano4-SRB or nano5-SRB + Cells. n=4. Results are expressed as mean ± SEM. Statistical analysis, two-way ANOVA for (BF). *p<0.05, **p<0.01, and ***p<0.001. The experiment was repeated twice with similar results.

[0041] Figures 9A-9D . Gating strategy for flow cytometric analysis of cells isolated from the indicated tissues. (A) In lung cells, NK1.1 + and CD3 - Identify NK cells and CD3 + CD4 + and CD8 + T cells. Lung CD11c - Cells are classified into neutrophils such as CD11b + Ly6C + 6G + , however, inflammatory monocytes were identified as CD11b+ Ly6C hi 6G - Based on CD11c + The cell gates were set to identify four populations using CD24 and CD11b markers, of which AM was CD24 - CD11b - Siglec F + , IM is CD24 - CD11b + , CD24 + CD11b - DC is CD103 + MHC II + , and tissue-resident CD24 + CD11b + DCs also express MHCII but not CD103. (B) CD11b + DCs can be divided into monocyte-derived DCs (Mono-DCs) and tissue-resident DCs (tDCs). hi , and MHC II expression varies with their activation state. On the other hand, tDCs are Ly6C lo MHC II hi (C and D) CD11b in MLN (C) + DC and CD11b - Gating strategies for DCs or DCs in nasal tissue (D). The gating strategies for T cells, NK cells, neutrophils, and monocytes in MLN, nasal tissue, and brain were similar to those in lung (A).

[0042] Figures 10A-10B Analysis of cells capturing PS-liposomes in the lung. (A) Nano4-SRB prepared as shown in Figure 1A was intranasally administered to mice. Twelve hours later, CD11c + SRB + Cells were mainly characterized by CD24 - CD11b - AM, CD11c - SRB + EpCAM + CD11b - AEC, which are also positive for MHC II. (B) In mice receiving nano4-SRB, AM, CD103 + DC, and CD11b + DC analysis of direct nanoparticle uptake (SRB + D+ ). About half of the AM uptake of nano4-SRB is shown as SRB + D + , however, DCs rarely captured nanoparticles. Results are expressed as mean ± SEM. n = 4 mice. The experiment was repeated twice with similar results.

[0043] Figures 11A-11C Nano4 delivers cGAMP to AM. (A) Schematic diagram of DiD-labeled empty PS-mimicking nanoparticles (DiD-PS) and PS-mimicking nanoparticles encapsulating cGAMP (DiD-PS-GAMP). (B) Intranasal inoculation of DiD-PS or DiD-PS-GAMP (20 μg cGAMP). Analysis of DiD in lung cells by flow cytometry after 12 or 36 h in mice receiving DiD-PS (red) or DiD-PS-GAMP (blue) + CD11c + Cells. These cells were also evaluated for CD40 expression to verify STING activation in the cells. A typical histogram of CD40 expression is given in the middle, and the mean fluorescence intensity (MFI) is summarized in the right graph. n=4. (C) DiD expressing CD40 was analyzed similarly to (B). + (blue) or DiD - AMs (red). n=4. Results are expressed as mean ± SEM. Each symbol represents an individual mouse in the right panel of B and C. Statistical analysis, t-test for (B and C). **p<0.01 and ***p<0.001 in the presence or absence of cGAMP. The experiment was repeated twice with similar results.

[0044] Fig.12 Positively charged nano5 is captured by PS in ex vivo culture. DiD-labeled Nano4 or nano5 was incubated with PS for 30 min. Nanoparticle aggregates on PS were visualized by confocal microscopy. The area outlined in the second column is magnified on the right. BF, bright field. Scale bars, 100 μm in columns 1 and 2, 10 μm in columns 3 and 4. Data represent 10 similar results from two separate experiments.

[0045] Figures 13A-13C. Uptake of Nano4 by AM isolated from non-human primates (NHP). AM and PS were isolated from rhesus macaques. (A) DiD-nano4 or DiD-nano5 were cultured with rhesus PS for 30 minutes. Nano5, but not nano4, aggregated on PS and visualized by confocal microscopy. The outlined area is enlarged in the corresponding figure on the right. Scale bar, 10 μm. Data represent 5 similar results. (B) Monkey AM was isolated and cultured for 3 hours with DiD-nano4 (upper figure) or DiD-nano5 (lower figure) pretreated with or without nanoparticles PS and imaged by fluorescence microscopy. Live cells were stained with Calcein-AM and cell nuclei were stained with Hoechst. Scale bar, 50 μm. DiD fluorescence intensity in cells was quantified by Image J (C). n=221-275. Each symbol represents a single cell. Results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA for (C). ***p<0.001 between indicated groups. We thank Prof. Wanli Liu, Dr. Junyi Wang and Ms. Shaoling Qi for their help in the NHP study.

[0046] Fig.14 .AM captures nano4 in the lung. 12 hours after mice received DiD-nano4 intranasally, lungs were collected and frozen thin sections were stained for the AM-specific marker Siglec F and visualized by fluorescence microscopy. Scale bar, 30 μm. The square in the second panel is enlarged in the right panel. Data represent 6 similar results from two separate experiments.

[0047] Fig.15 .TEM of nanoparticle distribution in the lung. As shown in Figure 7A, nanogold was encapsulated in nano4 (nano4-gold) and nano5 (nano5-gold). Mice were intranasally administered with nanoparticles. Lungs were collected 6 hours later and prepared for TEM. Note: nano4-gold was trapped in cell vesicles within the AM (red arrows), and some nanogold was observed in the cell vesicles (hollow red arrows, lower figure). In contrast, nano5-gold was mainly present on the surface of the alveoli (blue arrows). The areas outlined in the upper figure are enlarged in the corresponding lower figure. Scale bar, 2μm for the upper figure and 500nm for the lower figure.

[0048] Fig.16 .In nano4 uptake, from Sftpa1 / Sftpd - / -Mouse AMs have similar capabilities to WT AMs. SP-A / D are large hydrophilic proteins and have been established as the first line of innate defense. Both collectins can integrate into PS-encapsulated bacteria, viruses, cell debris, apoptotic cells, and various nanoparticles to promote their endocytosis or phagocytosis by AMs (20). To test whether this might be the mechanism of nano4 uptake by AMs, we isolated WT or Sftpa1 / Sftpd from the mouse AMs. - / - AMs were isolated from mice and incubated with DiD-nano4 pretreated with WT PS for 30 min. DiD fluorescence in cells was captured by confocal microscopy and quantified by Image J software. - / - Mouse AMs took up nano4 as efficiently as WT AMs. Fig.16 ), but not in the presence of SP-A / D-deficient PS ( FIG. 11 ). n=25-32. Each symbol represents a single cell. Statistical analysis, one-way ANOVA. *p<0.05 and **p<0.01, ns, not significant between the indicated groups. The experiment was repeated twice with similar results.

[0049] Figures 17A-17C .PS-GAMP does not induce significant inflammation in the lungs, nose, and central nervous system (CNS). Swiss Webster mice were intranasally administered with PBS, PS-GAMP, VN04 H5N1 vaccine, or vaccine plus PS-GAMP or CT. (A) Histological examination of the lungs (1st and 2nd rows of figures), nose (3rd and 4th rows of figures), and CNS (5th and 6th rows of figures) 2 days after immunization. The alveoli and bronchi of the lungs, the nose-associated lymphoid tissue of the nose, and the olfactory bulb area of ​​the brain tissue are outlined by dotted rectangles and enlarged in the corresponding lower figures. The olfactory bulb area of ​​the brain tissue is directly connected to the olfactory nerves in the nasal cavity. The data represent two separate experiments each tested in triplicate. Scale bars for the lungs and nose, 400 μm in the upper figure and 100 μm in the lower figure. Scale bars for the CNS, 800 μm in the upper figure and 200 μm in the lower figure. (B) Each mouse was analyzed for eosinophil infiltrates, epithelial damage, and necrosis as previously reported (48). Numbers indicate the number of mice with (+) or without (-) eosinophil infiltrates, epithelial damage, and necrosis. n = 6 mice. (C) Two days after immunization, the expression of the indicated cytokines and chemokines in the CNS of mice receiving VN04 H5N1 vaccine in the presence or absence of PS-GAMP or CT was determined by real-time RT-PCR. n = 2-8. Each symbol represents an individual mouse. Results are expressed as mean ± SEM.

[0050] Figure 18A-18V.Changes in inflammation and immune cells after PS-GAMP administration or viral infection. C57BL6 mice were intranasally administered with CA09 H1N1 vaccine plus 20 μg of PS-GAMP (blue), or with 1×LD 50 CA09 H1N1 influenza virus infected C57BL6 mice (red). Neutrophils, NK cells, CD4 T cells, and leukocytes in the lung (A to G) or MLN (H to N) were analyzed by flow cytometry at the indicated days post infection or immunization (dpi). + , and CD8 + T cells, monocytes, and CD11b + and CD11b - DC. Similarly, neutrophils, NK cells, CD4 + , and CD8 + T cells, monocytes and DCs (O to T) or neutrophils and monocytes in the brain (U and V). n=4. Results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA. *p<0.05, **p<0.01, and ***p<0.001 compared with day 0. The experiment was repeated twice with similar results.

[0051] Figures 19A-19C In contrast to viral infection, PS-GAMP did not induce significant inflammation in the lungs. Mice were immunized intranasally with CA09 H1N1 vaccine plus 20 μg of PS-GAMP (A) or with 1×LD 50 CA09H1N1 influenza virus infected mice (B). Lungs were analyzed by H&E staining at indicated days after immunization or infection. Data represent two separate experiments each tested in triplicate. Scale bar, 400 μm for the upper panel and 100 μm for the lower panel. (C) Pulmonary inflammation was quantified according to the standard scoring system shown on the right (49). n=6. Results are expressed as mean ± SEM. *p<0.05 and ***p<0.001 compared to day 0 by nonparametric test.

[0052] Fig. 20 PS-GAMP induces transient production of immune mediators in the lungs. Mice were intranasally administered 20 μg of PS-GAMP (blue) or 1×LD 50 CA09 H1N1 influenza virus infected mice (red). The mRNA levels of the indicated mediators were determined by real-time RT-PCR at various time points and normalized to untreated mice. n=4. Results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA. *p<0.05, **p<0.01, and ***p<0.001 compared to day 0. The experiment was repeated twice with similar results.

[0053] Figures 21A-21C PS-GAMP transiently increased IFN-β protein in BALF. Mice were intranasally administered 20 μg of PS-GAMP (blue) or 1×LD 50 CA09 H1N1 influenza virus infected mice (red). Protein levels of IFN-β (A), TNF-α (B), and IL-10 (C) in BALF were determined by ELISA at various time points. n=4. Results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA. *p<0.05 and ***p<0.001 compared to day 0 (before treatment). The experiment was repeated twice with similar results.

[0054] Figures 22A-22G .PS-GAMP does not induce any inflammation systemically. Mice were immunized intranasally with CA09 H1N1 vaccine plus 20 μg of PS-GAMP. Body weight (A) and temperature (B) were monitored for 6 days. Mice receiving PBS were used as controls. n=5. Serum IFN-β (C), TNF-α (D), IFN-γ (E), IL-6 (F), and IL-10 (G) were also monitored by ELISA for 6 days. n=4. Results are expressed as mean ± SEM. The experiment was repeated twice with similar results.

[0055] Figures 23A-23D PS-GAMP increases CD11b uptake of extracellular Ag in lung and MLN + DC numbers. (A) Mice were vaccinated intranasally with OVA-AF647 with or without 20 μg of PS-GAMP. + Cells were analyzed for CD11b and CD24 expression. Numbers in the figure are mean percentages ± SEM of individual cell subsets. (B) Mice receiving OVA (non-fluorescent) + PS-GAMP were used as controls to gate out autofluorescence associated with cell activation. After 36 hours, OVA uptake was analyzed based on gating on DC prepared from MLN, revealing that OVA + DCs are mainly CD11b + (C) As shown by the reduced CD11c when mice were intranasally dosed with 20 μg of DiD-PS-GAMP and analyzed similarly. + D + As shown in Figure 5, DCs do not directly take up PS-GAMP in MLN. (D) DiD was also tracked in MLN from 0 to 60 hours after PS-GAMP administration. + Cells. n = 4. Results are expressed as mean ± SEM. The experiment was repeated twice with similar results.

[0056] Figures 24A-24E.PS-GAMP does not enhance Ag uptake or processing in vivo. AF647-labeled OVA and DQ-OVA were used to evaluate whether PS-GAMP affects Ag uptake or Ag processing. DQ-OVA is OVA conjugated with a BODIPY fluorescent dye (DQ) and remains self-quenched until OVA is proteolytically processed to produce DQ-green fluorescence, which is commonly used to evaluate Ag processing. To this end, mice were intranasally dosed with PBS (grey) or AF647-OVA and DQ-OVA together in the presence (red) or absence (blue) of PS-GAMP and were euthanized 24 hours later for flow cytometric analysis (A). Based on the DC11b + DCs were gated to analyze AF647-OVA, which was further quantified based on DQ-green fluorescence for OVA cleavage. + CD11b + The percentage and cell number of DCs are summarized in (B) and (C). The AF647 and DQ-green MFI in these cells are given in (D) or (E), respectively. Each symbol represents an individual mouse in B to E. Results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA for (BE). *p<0.05, **p<0.01, and ***p<0.001 in the presence or absence of PS-GAMP. ns, not significant. The experiment was repeated twice with similar results. Note: AF647 was significantly higher in the presence or absence of PS-GAMP than in the presence of PS-GAMP. + CD11b + There was no difference in the MFI of DQ-green fluorescence or OVA in DCs (D and E). However, in the presence of PS-GAMP, CD11b positive for OVA + The percentage and number of DCs increased significantly (B and C), which was mainly attributed to the increased number of CD11b secondary to immune mediators induced by PS-GAMP. + DC.

[0057] Figures 25A-25D.PS-GAMP enhances Ag cross-presentation. (A) Mice were vaccinated intranasally with 60 μg of OVA with or without 20 μg of PS-GAMP. One day later, OT-I cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were transferred to the vaccinated mice. Lungs and MLNs were collected 3 days after cell transfer. (B) OT-I cells were analyzed for Ag-specific proliferation by the gradual decrease in CFSE fluorescence. Insets in the first two figures (PBS and OVA): reduced scale bar on the y-axis to show CFSE reduction. Highly divided (≥6,hi) cells were gated. The number of highly divided cells in the lung (C) and MLN (D) is summarized. n=4-6. Each symbol represents a single mouse in C and D. Statistical analysis, one-way ANOVA for (C and D). Results are expressed as mean ± SEM. **p<0.01 and ***p<0.001 in the presence or absence of PS-GAMP. The experiment was repeated twice with similar results.

[0058] Figures 26A-26C .CD8 in spleen, lung and MLN + T cell responses. C57BL / 6 mice were immunized intranasally with CA09 H1N1 vaccine plus 20 μg of PS-GAMP. Mice receiving PBS served as controls. (A) Splenocytes were isolated 7 days after immunization and stimulated with CA09 H1N1 vaccine. IFN-γ producing CD4 + and CD8 + Representative cell count profiles of T cells. (B) NP in the lung 4 days after immunization 366-374 + CD8 + Representative cell count profiles of T cells. (C) Based on the CD3 + CD8 + Gating of T cells to determine PA 224-233 (blue) or PB1 703-711 (Red) Percentage of positive cells. Each figure represents 4 similar results in the same group. n=4. Data are expressed as mean ± SEM. The experiment was repeated twice with similar results.

[0059] Figures 27A-27C .Early virus-specific GB + CD8 + T cells and BALF antibodies. (A) C57BL / 6 mice were immunized with either CA09 H1N1 vaccine alone or with vaccine plus 20 μg of PS-GAMP, followed by 10×LD 50 CA09 H1N1 virus challenge. Lungs were collected 4 days after infection. (B) Based on GB + CD8 +Gating of T cells to obtain NP 366-374 Tetramer (blue), PB1 703-711 (red), or PA 224-233 (Green) Percentage of positive cells. CD8 + T cells served as negative control (grey). n=3. (C) Ag-specific IgA and IgM titers were analyzed in BALF 6 days after immunization. n=4. Data are presented as mean ± SEM. The experiment was repeated twice with similar results.

[0060] Figures 28A-28I . Figures 4A-4J Supplementary data. (A) Schematic diagram of vaccination and virus challenge schedule. (B to F) Weight change (B, C, E, and F) or survival (D) of mice corresponding to those described in Figures 4A-4E, respectively. (G and H) Mice were immunized intranasally with H7-Re1 H7N9 vaccine alone or with 20 μg of PS-GAMP or poly IC and challenged with clinically isolated SH13 H7N9 virus 14 days later. n=10-13. (I) Weight change of mice described in Figure 4F. Statistical analysis, two-way ANOVA for (B, C, E, F, H, I) and log-rank test for (D and G). *p<0.05, **p<0.01, ***p<0.001, and #p<0.05. All experiments were repeated at least twice with similar results.

[0061] Figures 29A-29B .In vivo SRB + AM vs SRB + AEC is inversely correlated with time, while DiD + AM maintained the same percentage. SRB-DiD-nano4 was intranasally inoculated into mice. (A) After inoculation, SRB was tracked over time. + AM and SRB + The percentage change of AEC relative to the total number of lung cells. n = 4. (B) DiD was analyzed by flow cytometry 12 and 18 hours after nanoparticle administration. + AM. n = 4. Results are expressed as mean ± SEM. Statistical analysis, t test. *p < 0.05 and **p < 0.01 compared between 18 and 12 hours. All experiments were repeated twice with similar results.

[0062] Figures 30A-30D.cGAMP enters AEC from AM. (A) Mice were injected intraperitoneally with the gap junction inhibitor CBX or PBS for 3 consecutive days, followed by intranasal administration of 20 μg of PS-GAMP. AM and AEC were sorted 12 hours later and analyzed for Ifnb1 (B) and Gmcsf (C) expression by real-time RT-PCR. mRNA levels were first normalized to Gapdh and then to the corresponding cells isolated from naive mice. n=4. (D) Unsorted lung cells were also analyzed similarly for comparison. n=8. Results are expressed as mean ± SEM. Each symbol represents an individual mouse in B to D. Statistical analysis, t-test. *p<0.05 and ***p<0.001 in the presence or absence of CBX. ns, not significant. All experiments were repeated twice with similar results.

[0063] Figures 31A-31E .Tissue and cellular distribution of poly IC. Mice received 20 μg of rhodamine-labeled poly IC intranasally. (A) Twelve hours later, lungs were dissected and digested for CD11c + and CD11c - Flow cytometric analysis of poly IC uptake by subpopulations. + poly IC + The cells were further confirmed to be CD24 - CD11b - Then, based on the expression of EpCAM + AEC (B) or CD11c + CD24 + DC (C) was gated to analyze poly IC uptake. MLN (D) and nasal epithelial and lymphoid tissues (E) were also prepared as single cell suspensions to determine poly IC uptake. Data are representative of two separate experiments each tested in triplicate.

[0064] Figures 32A-32B .Cell reconstitution efficacy after bone marrow (BM) cell transfer. Mice were pretreated with lethal radiation before infusion with BM cells isolated from mice carrying reciprocal CD45 alleles CD45.1 and CD45.2 (surface biomarkers of all leukocytes). Donor cells were distinguished from recipients by specific antibodies for CD45.1 or CD45.2. After 3 months of infusion, transfer efficacy was analyzed by quantifying CD45.1 or CD45.2 expression on leukocytes in different tissues in the recipient (A) and summarized in (B). Each symbol represents a single mouse in (B). n=5-7. The experiment was repeated twice with similar results.

[0065] Figures 33A-33K.Supplementary data for cross protection study. (A to K) correspond to the weight changes of mice described in Figures 6A-6K, respectively. Results are expressed as mean ± SEM. Statistical analysis, two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001, and #p<0.05. All experiments were repeated twice with similar results.

[0066] Figures 34A-34B Vaccination with trivalent seasonal influenza vaccine and PS-GAMP induces cross-protective immunity against mismatched influenza B viruses. (A) Schematic diagram of the vaccination / sampling schedule. BALB / c mice were immunized with trivalent seasonal influenza vaccine (2018-19) (SIV) alone or together with 20 μg of PS-GAMP and 1 month later with 4 × 10 5 TCID 50 Mismatched Florida06 B virus challenge. (B) Lungs were isolated 4 days after immunization and analyzed for viral titers. Each symbol represents an individual mouse in (B). n=8-9. Results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA. **p<0.01 in the presence or absence of PS-GAMP. ns, not significant. The experiment was repeated twice with similar results.

[0067] Figures 35A-35E .PS-GAMP / inactivated influenza vaccine induces virus-specific lung CD8 + T RM (A) Schematic representation of the vaccination / sampling timeline for the OT-I mouse model. Mice were transferred with OT-I cells and 1 hour later, they were immunized intranasally with OVA in the presence or absence of PS-GAMP. 35 days later, mice were intravenously injected with anti-CD8β antibody to deplete circulating CD8 + T cells, and the mice were then sacrificed for flow cytometry analysis. (B) + and CD8α + Total CD8 + T cells were gated (not shown), and lung OT-I cells were identified as CD45.2 + and CD8β - (Antibody intravenous injection) (first two pictures). RM Phenotypic identification of OT-I cells as CD103 + CD69 + CD49a + OT-I cells in the spleen were used as a control (grey). RMThe number of cells is summarized in (C). n = 6. (D and E) Mice were immunized intranasally with CA09 H1N1 vaccine in the presence or absence of PS-GAMP. Lungs were isolated 6 months later for flow cytometry. 366-374 + CD8 + T cell gating and verification of CD103 and CD69 expression (D), NP 366-374 + CD8 + T RM The number of cells is summarized in (E). n = 4. NP in spleen 366-374 + CD8 + T cells were used as controls. Results are expressed as mean ± SEM. Statistical analysis was performed by t-test for (C) and one-way ANOVA for (E). ***p<0.001 in the presence or absence of PS-GAMP. All experiments were repeated twice with similar results.

[0068] Figures 36A-36D FTY720 did not affect cross-protection elicited by influenza vaccine / PS-GAMP. (A and B) Mice were immunized intranasally with CA09 H1N1 vaccine alone or with 20 μg of PS-GAMP and then treated with 5×LD 50 GZ89 H3N2 virus challenge. (C and D) Mice were immunized and challenged as described in A and B, except that mice additionally received daily injections of FTY720 (1 mg / kg / day) from days -2 to 14 after challenge. n=6-8. Statistical analysis, two-way ANOVA for (B and D) and log-rank test for (A and C). *p<0.05, **p<0.01, and ***p<0.001 compared to vaccine alone. All experiments were repeated twice with similar results.

[0069] Figures 37A-37E .Safety and efficacy of PS-GAMP in ferrets. Ferrets were immunized intranasally with an inactivated virus vaccine (Perth H3N2 15 μg) with or without 200 μg of PS-GAMP. The weight (A) and temperature (B) of the animals were monitored for 6 days. (C) Serum was collected 4 weeks after immunization and tested for Perth H3N2-specific IgG titers (C). HAI titers were also determined for Perth H3N2 (D) or Michigan H1N1 (E) virus strains. n=4. Each symbol represents a single animal in C to E. The results are expressed as mean ± SEM. Statistical analysis, one-way ANOVA for (C and D). **p<0.01 in the presence versus absence of PS-GAMP comparison. DETAILED DESCRIPTION

[0070] The cGAS-cGAMP-STING pathway is an important immune surveillance pathway activated in the presence of cytoplasmic DNA, for example, due to microbial infection or pathological-physiological conditions including cancer and autoimmune disorders. Cyclic GMP-AMP synthase (cGAS) belongs to the nucleotidyl transferase family and is a universal DNA sensor that is activated to produce the signal molecule cyclic GMP-AMP (or 2'-3'-cGAMP or cyclic guanosine monophosphate-adenosine monophosphate) when bound to cytosolic dsDNA. As a second messenger during microbial infection, 2'-3'-cGAMP binds to and activates STING, leading to the production of type I interferons (IFNs) and other co-stimulatory molecules that trigger immune responses. In addition to its role in infectious diseases, the cGAS / STING pathway presents a promising new target for autoimmune diseases and cancer immunotherapy. It is proposed that DNA fragments present in the tumor microenvironment activate cGAS in dendritic cells (DCs), followed by IFN-induced DC maturation and activation of a powerful and beneficial immune response against cancer cells. In additional contexts, dysregulation of the cGAS / STING pathway has been implicated in self-DNA-triggered inflammatory and autoimmune disorders such as systemic lupus erythematosus (SLE) and Aicardi-Goutieres syndrome.

[0071] Despite decades of research, effective mucosal adjuvants are still lacking. 2'-3'-cGMP-AMP (cGAMP), a natural agonist of stimulator of interferon genes (STING), is a second messenger produced in response to DNA viral infection or tissue damage (10,11). It stimulates the production of type I interferon (IFN-I), which helps determine the strength of the T-helper 1 (Th1) immune response, especially CD8 + STING agonists are potent adjuvants that elicit potent antitumor immunity and boost intradermal influenza vaccines after intratumoral administration (13,14). However, using these small, water-soluble agonists as mucosal adjuvants is challenging. They must be delivered to the cytosol of antigen (Ag) presenting cells (APCs) and / or alveolar epithelial cells (AECs) without disrupting the integrity of the pulmonary surfactant (PS) layer, a mixture of lipids and proteins secreted by type II AECs. This PS layer forms a strong barrier that separates the outside air from the inner alveolar epithelium in the alveoli and prevents nanoparticles and hydrophilic molecules from entering the AECs (15,16).

[0072] It is highly desirable to develop a "universal" influenza vaccine that confers protection not only against intra-subtype variants, but also against other subtypes of influenza virus. However, it remains unclear whether such a universal influenza vaccine is achievable. It has long been recognized in humans and animal models that viral infection can stimulate CD8+ T cell-mediated heterosubtypic immunity (2,3,6). Here, a single immunization with an inactivated H1N1 vaccine adjuvanted with PS-GAMP conferred protection against lethal challenge with H1N1, H3N2, H5N1, or H7N9 viruses as early as 2 days (d) after immunization. This cross-protection lasted for at least 6 months, with persistent virus-specific CD8 + T RM This is mainly due to the fact that PS-GAMP-adjuvanted influenza vaccines stimulated viral infection-induced immunity characterized by AEC activation, rapid CD11b + DC recruitment and differentiation, and potent CD8 in the respiratory system + T cell response. PS-GAMP is a stand-alone adjuvant that is compatible not only with inactivated influenza virus vaccines, but also with other vaccines such as those that include a cocktail of multiple B and T cell epitopes or influenza vaccine subunits. The ability of PS-GAMP to enhance non-replicating influenza vaccines for strong heterologous subtype immunity makes it a promising adjuvant for "universal" influenza vaccines, if its efficacy is shown in humans. As such, it may confer significant advantages over "replicating" vaccines.

[0073] Unlike conventional vaccine adjuvants that primarily target APCs, PS-GAMP activates both AMs and AECs; without wishing to be bound by theory, AEC activation appears to be crucial for adjuvantity, as blockade of gap junctions and STING deficiency in AECs significantly attenuate adjuvantity, whereas STING deficiency in myeloid cells does not. The critical role played by AECs in coordinating innate and adaptive immune responses compared to AMs is consistent with what has been described during the early stages of influenza virus infection (24). The ability of cGAMP to enter AECs without disrupting the PS layer is attributed to SP-A / D-receptor-mediated endocytosis after integration of SP-A and SP-D into PS-biomimetic liposomes, which is not feasible in any non-PS-biomimetic liposomes (39-41). In addition, the adjuvant can induce strong protection within only 2 days after immunization, in stark contrast to existing influenza vaccines that require at least 10-14 days to be effective. Early cross-protection is very important for protecting first responders and high-risk individuals, especially when antiviral-resistant viruses or highly pathogenic viruses such as H5N1 and H7N9 viruses present as pandemics. Because viral transmission can accelerate exponentially after an epidemic expands into a pandemic, early protection during an epidemic will be the most effective means of limiting viral spread and minimizing or preventing an epidemic from becoming a pandemic, saving millions of lives (42).

[0074] Pulmonary surfactant (PS)-biomimetic nanoparticles

[0075] Provided herein are compositions containing nanoparticles having an average size of 200-400 nm. The nanoparticles include a variety of lung surfactant-biomimetic molecules, wherein the nanoparticles are negatively charged; and more than one cargo molecule encapsulated by the nanoparticles, wherein the molecular weight of the cargo molecule is less than 1200 Da.

[0076] Provided herein are methods for promoting an immune response to an antigen. The method comprises administering to a subject an effective amount of a composition as described herein; and administering to the subject an antigen.

[0077] Provided herein are methods for treating a subject suffering from influenza. The methods include administering to the subject a therapeutically effective amount of a composition as described herein; and administering to the subject an antigen. In some embodiments, the cargo molecule is cGAMP and the antigen is an influenza vaccine.

[0078] 1. Provided herein is a method for treating a subject with an airway disease. The method comprises administering to the subject a therapeutically effective amount of a composition as described herein, wherein the cargo molecule is a long-acting β2-agonist (LABA) (e.g., formoterol, salmeterol, or vilanterol); a corticosteroid (ICS) (e.g., budesonide, fluticasone propionate, or fluticasone furoate); a leukotriene pathway modulator (e.g., montelukast or zileuton); a targeted kinase (e.g., spleen tyrosine kinase, p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Janus kinase (Jak), or phosphodiesterase-4 (PDE4)) inhibitor; a receptor (e.g., Th2 agonists or antagonists of chemoattractant receptor homolog (CRTH2), chemokine receptor 2 (CCR2) expressed on cells; agonists or antagonists of ion channels (e.g., GABA receptors, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or voltage-gated sodium channels); inducers of IFN-α; long-acting muscarinic antagonists / anticholinergics (LAMA); inhibitors of IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; CXCR2 antagonists; molecules that block proinflammatory cytokines (e.g., TNF-α, TNF-β, or IL-6); molecules that block IL-17 / T H 17; macrolides; molecules that activate HDAC2; STAT6 inhibitors (e.g., AS1517499); antiviral small molecule drugs (e.g., oseltamivir (Tamiflu), Relenza, or zanamivir); and / or favipiravir (T705).

[0079] Provided herein are methods of treating a subject having cancer. The methods include administering to the subject a therapeutically effective amount of a composition as described herein. In some embodiments, the cargo molecule is a chemotherapeutic agent.

[0080] The methods described herein can provide at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold improvement in the delivery efficiency of the cargo molecules described herein compared to a similar method performed without the use of PS-bionic nanoparticles.

[0081] Nanoparticles

[0082] In some embodiments, the nanoparticle is a liposome, a vesicle, an emulsion, or a micelle.

[0083] In some embodiments, the nanoparticles may include more than one surfactant, including a detergent, a wetting agent, an emulsifier, a foaming agent, or a dispersant. In some embodiments, the surfactant includes at least one hydrophobic end and / or at least one hydrophilic end. In some embodiments, the surfactant is positively charged, neutral, or negatively charged.

[0084] In some embodiments, the surfactant is a lipid. In some embodiments, the surfactant is a phospholipid. In some embodiments, the nanoparticles may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 layers or more of surfactant. In some embodiments, the nanoparticles are water-in-oil-in-water emulsions.

[0085] In some embodiments, the percentage of surfactant in the nanoparticles can be from 0 wt % to 100 wt %, from 5 wt % to 100 wt %, from 10 wt % to 100 wt %, from 15 wt % to 100 wt %, from 20 wt % to 100 wt %, from 25 wt % to 100 wt %, from 30 wt % to 100 wt %, from 35 wt % to 100 wt %, from 40 wt % to 100 wt %, from 45 wt % to 100 wt %, from 50 wt % to 100 wt %, from 55 wt % to 100 wt %, from 60 wt % to 100 wt %, from 65 wt % to 100 wt %, from 70 wt % to 100 wt %, from 75 wt % to 100 wt %, from 80 wt % to 100 wt %, from 85 wt % to 100 wt %, from 90 wt % to 100 wt %, from 95 wt % to 100 wt %, from 100 wt % to 100 wt %, from 110 wt % to 100 wt %, from 120 wt % to 100 wt %, from 130 wt % to 100 wt %, from 140 wt % to 100 wt %, from 150 wt % to 100 wt %, from 160 wt % to 100 % to 100 wt %, from 50 wt % to 100 wt %, from 55 wt % to 100 wt %, from 60 wt % to 100 wt %, from 65 wt % to 100 wt %, from 70 wt % to 100 wt %, from 75 wt % to 100 wt %, from 80 wt % to 100 wt %, from 85 wt % to 100 wt %, from 90 wt % to 100 wt %, or from 95 wt % to 100 wt %. In some embodiments, the percentage of surfactant in the nanoparticles can range from 0 wt % to 95 wt %, from 0 wt % to 90 wt %, from 0 wt % to 85 wt %, from 0 wt % to 80 wt %, from 0 wt % to 75 wt %, from 0 wt % to 70 wt %, from 0 wt % to 65 wt %, from 0 wt % to 60 wt %, from 0 wt % to 55 wt %, from 0 wt % to 50 wt %, from 0 wt % to 45 wt %, from 0 wt % to 40 wt %, from 0 wt % to 35 wt %, from 0 wt % to 30 wt %, from 0 wt % to 25 wt %, from 0 wt % to 20 wt %, from 0 wt % to 15 wt %, from 0 wt % to 10 wt %, or from 0 wt % to 5 wt %. In some embodiments, the percentage of surfactant in the nanoparticles can be 0 weight %, about 1 weight %, about 2 weight %, about 3 weight %, about 4 weight %, about 5 weight %, about 10 weight %, about 15 weight %, about 20 weight %, about 25 weight %, about 30 weight %, about 35 weight %, about 40 weight %, about 45 weight %, about 50 weight %, about 55 weight %, about 60 weight %, about 65 weight %, about 70 weight %, about 75 weight %, about 80 weight %, about 85 weight %, about 90 weight %, about 95 weight %, or about 100 weight %.

[0086] In some embodiments, nanoparticles as described herein may have an average size of from 200nm to 210nm, from 210nm to 220nm, from 220nm to 230nm, from 230nm to 240nm, from 240nm to 250nm, from 250nm to 260nm, from 260nm to 270nm, from 270to 280nm, from 280nm to 290nm, from 290nm to 300nm, from 300nm to 310nm, from 310nm to 320nm, from 320nm to 330nm, from 330nm to 340nm, from 340nm to 350nm, from 350nm to 360nm, from 360nm to 370nm, from 370nm to 380nm, from 380nm to 390nm, or from 390nm to 400nm.

[0087] Pulmonary surfactant (PS)

[0088] Pulmonary surfactant is a surface-active lipoprotein complex (phospholipid protein) formed by type II alveolar cells. The proteins and lipids that constitute the surfactant have both hydrophilic and hydrophobic regions. The main lipid component of the surfactant, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), reduces surface tension by adsorption to the air-water interface of the alveoli, with the hydrophilic head group in water and the hydrophobic tail facing the air.

[0089] Pulmonary surfactant is typically composed of 90% lipid and 10% protein and is evolutionarily conserved. The lipids include 8-10% cholesterol, 60-70% zwitterionic phosphatidylcholine (PC), mainly dipalmitoylphosphatidylcholine (DPPC), up to 8-15% anionic phosphatidylglycerol (DPPG), and relatively small amounts of other lipids (17).

[0090] Pulmonary surfactant (PS)-biomimetic nanoparticles

[0091] In some embodiments, the PS-bionic nanoparticles can be nanoparticles comprising a plurality of PS-bionic molecules, including but not limited to, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), cholesterol, polyethylene glycol (e.g., PEG2000), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000), phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, and / or lysophospholipids.

[0092] In some embodiments, the PS-biomimetic molecule is a lipid, a protein, a lipoprotein, a phospholipid, or a phospholipoprotein.

[0093] In some embodiments, the PS-bionic molecule is a domain, a moiety, a portion, or the entire molecule of a pulmonary surfactant. In some embodiments, the PS-bionic molecule is a natural product. In some embodiments, the PS-bionic molecule is artificially synthesized.

[0094] In some embodiments, the PS-bionic molecule is positively charged, neutrally charged, or negatively charged. In some embodiments, the PS-bionic molecule has at least one hydrophobic end and / or at least one hydrophilic end.

[0095] In some embodiments, PS-bionic molecules include more than one fatty acid group or its salt, and / or more than one head group. In some embodiments, the fatty acid group may include digestible, long chain (e.g., C8-C50), substituted or unsubstituted hydrocarbons. In some embodiments, the fatty acid group may be a C10-C20 fatty acid or its salt. In some embodiments, the fatty acid group may be a C15-C20 fatty acid or its salt. In some embodiments, the fatty acid group may be a C15-C25 fatty acid or its salt. In some embodiments, the fatty acid group may be unsaturated. In some embodiments, the fatty acid group may be monounsaturated. In some embodiments, the fatty acid group may be polyunsaturated. In some embodiments, the double bond of the unsaturated fatty acid group may be a cis conformation. In some embodiments, the double bond of the unsaturated fatty acid may be a trans conformation. In some embodiments, the fatty acid group is palmitic acid. In some embodiments, the head group is phosphatidylcholine.

[0096] Cargo molecules of PS-biomimetic nanoparticles

[0097] The cargo molecules that can be carried in nanoparticles as described herein may include those that have therapeutic or preventive effects on cells of the lung, such as alveolar epithelial cells (AEC) and / or alveolar macrophages (AM). Examples include agents (immunostimulants) that enhance the immune response to co-administered antigens, such as adjuvants to stimulate immune responses; agents (anti-inflammatory drugs or immunosuppressants) that block signal pathways associated with inflammation, such as those particularly used to suppress inflammation-related lung diseases, including allergies, asthma, and chronic obstructive pulmonary disease (COPD); and anticancer drugs, including chemotherapeutic agents. The cargo molecules can be completely encapsulated by PS (e.g., contained in a PS film forming the outer surface of the nanoparticle), can be mixed into PS (e.g., in solid nanoparticles), or can be on / in / attached to the film.

[0098] In some embodiments, cargo molecules can be transferred through gap junctions present between AM and AEC, and are limited to those small molecules that are small enough to pass through gap junctions. Detailed descriptions can be found in references 29 and 30. Thus, in some embodiments, cargo molecules can have a molecular weight ranging from 10Da to 1200Da, from 50Da to 1200Da, from 100Da to 1200Da, from 200Da to 1200Da, from 300Da to 1200Da, from 400Da to 1200Da, from 500Da to 1200Da, from 600Da to 1200Da, from 700Da to 1200Da, from 800Da to 1200Da, from 900Da to 1200Da, from 1000Da to 1200Da, or from 1100Da to 1200Da. In some embodiments, the cargo molecule may have a molecular weight ranging from 10 Da to 50 Da, from 10 Da to 100 Da, from 10 Da to 200 Da, from 10 Da to 300 Da, from 10 Da to 400 Da, from 10 Da to 500 Da, from 10 Da to 600 Da, from 10 Da to 700 Da, from 10 Da to 800 Da, from 10 Da to 900 Da, from 10 Da to 1000 Da, from 10 Da to 1100 Da, or from 10 Da to 1200 Da. In some embodiments, the cargo molecule may have a molecular weight of about 10 Da, 20 Da, 50 Da, 100 Da, 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, 1100 Da, or 1200 Da.

[0099] In some embodiments, the cargo molecule can be an immunostimulatory agent (used as an adjuvant), for example, a stimulator of interferon genes (STING) agonist (e.g., cGAMP, CDN, MK-1454, ADU-S100, E7766); an agonist for intracellular Toll-like receptors including TLR3, TLR7, TLR8, or TLR9 (e.g., imiquimod, resiquimod (R848), imidazoquinoline (IMQ), motomod, CU-CPT4a, IPH-3102, or ampligen); and / or an agonist for Nodinitib (NOD1), NOD2, NLPR3, or NPLRC3 (e.g., muramyl dipeptide (MDP), FK565, or FK156).

[0100] In some embodiments, the cargo molecule can be an anti-inflammatory drug for airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), or allergies), such as a long-acting β2-agonist (LABA) (e.g., formoterol, salmeterol, or vilanterol); a corticosteroid (ICS) (e.g., budesonide, fluticasone propionate, or fluticasone furoate); a leukotriene pathway modulator (e.g., montelukast or zileuton); an inhibitor of a targeted kinase (e.g., spleen tyrosine kinase, p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), Janus kinase (Jak), or phosphodiesterase-4 (PDE4)); a receptor (e.g., a chemoattractant receptor homolog expressed on Th2 cells); molecules (CRTH2, chemokine receptor 2 (CCR2)); agonists or antagonists of ion channels (e.g., GABA receptors, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or voltage-gated sodium channels); inducers of IFN-α; long-acting muscarinic antagonists / anticholinergics (LAMAs); inhibitors of IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; CXCR2 antagonists; molecules that block proinflammatory cytokines (e.g., TNF-α, TNF-β, or IL-6); molecules that block IL-17 / TH17; macrolides; molecules that activate HDAC2; and / or STAT6 inhibitors (e.g., AS1517499). Detailed descriptions can be found in Barnes, "Therapeutic approaches to asthma–chronic obstructive pulmonary disease overlap syndromes." Journal of Allergy and Clinical Immunology 136.3 (2015): 531-545; Glossop et al. "Small-molecule anti-inflammatory drug compositions for the treatment of asthma: a patent review (2013–2014)." Expert opinion on therapeutic patents 25.7 (2015): 743-754, each of which is incorporated herein by reference in its entirety.

[0101] In some embodiments, the cargo molecule can be an antiviral small molecule drug for treating pneumovirus infections such as influenza A and B viruses, respiratory syncytial virus (RSV), rhinovirus, parainfluenza virus, or severe acute respiratory syndrome (SARS) coronavirus. Antiviral small molecules include oseltamivir (Tamiflu), Relenza, and zanamivir for inhibiting the neuraminidase of influenza virus; and Favipiravir (T705) for treating various pneumovirus infections.

[0102] In some embodiments, the cargo molecule is a chemotherapeutic agent directed against cancer, for example, gefitinib, erlotinib, everolimus, afatinib, and / or crizotinib for non-small cell lung cancer; doxorubicin, etoposide, nivolumab, and / or methotrexate for small cell lung cancer; cisplatin, carboplatin, gemcitabine, doxorubicin, and / or D5-fluorouracil (5-FU) for nasopharyngeal carcinoma; etoposide, cisplatin, and / or carboplatin for tracheal cancer; etoposide, cisplatin, carboplatin, 5-FU, docetaxel, paclitaxel, and / or epirubicin for bronchial cancer.

[0103] In some embodiments, the cargo molecule is a labeling agent, for example, the nanoparticle can include more than one detectable moiety, for example, in addition to the cargo molecule, such as a fluorescent dye, for example, carbocyanine, indocarbocyanine, oxacarbocyanine, thuicarbocyanine, merocyanine, polymethine, coumarin, rhodamine, sulforhodamine B (SRB), xanthene, fluorescein, boron-dipyrromethene (BODIPY) dye, or a derivative thereof, including but not limited to, BODIPY FL, BODIPY R6G, BODIPY TR, BODIPY TMR, BODIPY 581 / 591, BODIPY 630 / 650, and BODIPY 650 / 665, Cy5, Cy5.5, Cy7, VivoTag-680, VivoTag-S680, VivoTag-S750, AlexaFluor660, AlexaFluor680, AlexaFluor700, AlexaFluor750, AlexaFluor790, Dy677, Dy676, Dy682, Dy752, Dy780, DyLight547, Dylight647, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IR800 (dimethyl{4-[1,5,5-tris(4-dimethylaminophenyl)-2,4-pentadienyl]-2,5-cyclohexadien-1-yl}ammonium perchlorate), IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, ADS832WS, 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate (DiD), 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiI, also known as DiIC18 (3)), or any other detectable moiety known in the art. The detectable moiety can be, for example, inside or outside the nanoparticle (e.g., in or attached to the outer surface membrane).

[0104] In some embodiments, the cargo molecule is a small molecule or an antibody fragment, eg, an antigen-binding fragment of an antibody.

[0105] STING agonists

[0106] The stimulator of interferon genes (STING) agonist can be any suitable agonist. In some embodiments, the STING agonist is a nucleic acid, a protein, a peptide, or a small molecule.

[0107] In some embodiments, the STING agonist can be a nucleotide STING agonist or a non-nucleotide STING agonist.

[0108] Nucleotide STING agonists include natural cyclic dinucleotides (CDNs), such as cGAMP; or synthetic CDNs, such as the 'disulfide' analog ADU-S100 (sulfur-modified phosphodiester bonds on the c-di[AMP] scaffold), or MK-1454. Non-nucleotide STING agonists include vascular disruptors, such as 5,6-dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA, also known as vadimezan or ASA404); or aminobenzimidazole STING agonists (see WO2019069270A). Other STING agonists are described in WO2015185565A1 (including fluorinated derivatives) and WO2019079261A1, which are incorporated herein by reference. A detailed description can be found in Marloye et al. "Current patent and clinical status of stimulator of interferon genes (STING) agonists for cancer immunotherapy." (2019): 87-90, which is incorporated herein by reference.

[0109] cGAMP

[0110] As used herein, "cGAMP," or cyclic GMP-AMP, or 2'-3'-cGMP-AMP refers to cyclic guanosine monophosphate-adenosine monophosphate.

[0111] antigen

[0112] In some embodiments, the nanoparticle comprises, or is co-administered with, an antigen. In some embodiments, the antigen is a viral antigen.

[0113] In some embodiments, the antigen is a respiratory syncytial virus (RSV) antigen. In some embodiments, the antigen is a RSV F protein antigen. In some embodiments, the antigen is a SARS coronavirus (CoV) antigen. In some embodiments, the antigen is the spike (S) protein of SARS-CoV. In some embodiments, the antigen is a rhinovirus antigen. In some embodiments, the antigen is a parainfluenza antigen.

[0114] In some embodiments, the antigen is an influenza virus antigen. In some embodiments, the antigen is an influenza B virus antigen. In some embodiments, the antigen is an influenza virus nucleocapsid protein (NP), RNA polymerase PB1, PB2, PA, hemagglutinin (HA), or neuraminidase (NA), alone or in various combinations of proteins.

[0115] Chemotherapeutic agents

[0116] As used herein, "chemotherapeutic agent" is a cytotoxic drug or cytotoxic mixture of drugs intended to destroy malignant cells and tissues. Non-limiting examples of chemotherapeutic agents include more than one alkylating agent; anthracyclines; cytoskeletal disruptors (taxanes); epothilones; histone deacetylase inhibitors; inhibitors of topoisomerase I; inhibitors of topoisomerase II; kinase inhibitors; nucleotide analogs and precursor analogs; peptide antibiotics; platinum-based agents; retinoic acid; and / or vinca alkaloids and derivatives; or any combination thereof. In some embodiments, the chemotherapeutic agent is a nucleotide analog or precursor analog, for example, azacitidine; azathioprine; capecitabine; cytarabine; doxifluridine; fluorouracil; gemcitabine; hydroxyurea; mercaptopurine; methotrexate; or thioguanine. Other examples include cyclophosphamide, embicin, chlorabucil, melphalan, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, etoposide, teniposide, tafluposide, bleomycin, carboplatin, cisplatin, oxaliplatin, all-trans retinoic acid, vinblastine, vincristine, vindesine, vinorelbine, and bevacizumab (or its antigen-binding fragment). Additional examples of chemotherapeutic agents are known in the art.

[0117] In some embodiments, chemotherapeutic agents can be used for cancer treatment, for example, gefitinib, erlotinib, everolimus, afatinib, and / or crizotinib for non-small cell lung cancer; doxorubicin, etoposide, nivolumab, and / or methotrexate for small cell lung cancer; cisplatin, carboplatin, gemcitabine, doxorubicin, and / or D5-fluorouracil (5-FU) for nasopharyngeal carcinoma; etoposide, cisplatin, carboplatin for tracheal cancer; etoposide, cisplatin, carboplatin, 5-FU, docetaxel, paclitaxel and / or epirubicin for bronchial cancer.

[0118] Preparation method of PS-bionic nanoparticles

[0119] Nanoparticles as described herein can be prepared using methods known in the art. For example, in some embodiments, depending on the cargo molecule, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1'-rac-glycerol) (DPPG), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) -2000] (DPPE-PEG2000) and cholesterol can be mixed, for example, in a mass ratio of about 10: 1: 1: 1, or 5-12: 0.5-1.5: 0.5-1.5: 0.5-1.5.

[0120] In some embodiments, more than one surfactant may be mixed in any mass ratio known in the art.

[0121] The mixture may be dissolved in chloroform, dichloromethane, trichloroethylene, methyl chloroform, or other organic solvents known in the art.

[0122] In some embodiments, the mixture of lipids is dissolved in a solvent and mixed with a cGAMP solution. The volume ratio between the solvent and the cGAMP solution can be about 1:5, about 1:4, about 1:3, about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1 or more.

[0123] In some embodiments, the concentration of cGAMP in the nanoparticle solution may be about 0.1 μg / ml, about 0.5 μg / ml, about 1 μg / ml, about 5 μg / ml, about 10 μg / ml, about 20 μg / ml, about 30 μg / ml, about 40 μg / ml, about 50 μg / ml, about 60 μg / ml, about 70 μg / ml, about 80 μg / ml, about 90 μg / ml, about 100 μg / ml, about 200 μg / ml, about 300 μg / ml, about 500 μg / ml, about 1 mg / ml, about 5 mg / ml, about 10 mg / ml, about 50 mg / ml, about 100 mg / ml or more.

[0124] In some embodiments, trehalose can be added to the nanoparticle suspension at a final concentration of about 1%, about 2%, about 2.5%, about 3%, about 5%, or about 10%.

[0125] Pharmaceutical compositions and methods of administration

[0126] The methods described herein include the use of pharmaceutical compositions containing the nanoparticles described herein as active ingredients.

[0127] Pharmaceutical compositions generally include a pharmaceutically acceptable carrier. As used herein, the language "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial agents, antiviral agents, and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are compatible with drug administration. Supplementary active compounds may also be mixed into the composition, for example, additional adjuvants.

[0128] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include nasal (e.g., inhalation).

[0129] Methods for preparing suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions, powders, or suspensions for intranasal inhalation or spraying may include the following components: a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be encapsulated in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0130] For intranasal administration or administration by inhalation, the nanoparticles can be delivered, for example, from a pump spray container squeezed or pumped by the subject in the form of a solution, powder, aerosol, or suspension, or from a pressurized container or nebulizer optionally with a suitable propellant as an aerosol spray. Formulations suitable for intranasal administration can be in the form of dry powders from a dry powder inhaler (alone, or as a mixture, for example, as a dry blend with a carrier such as lactose, or as mixed component particles, for example, mixed with a phospholipid such as phosphatidylcholine), or as an aerosol spray from a pressurized container, pump, nebulizer, spray bottle (atomizer) (e.g., using electrohydrodynamics to produce a fine mist), or nebulizer with or without a suitable propellant such as 1,1,1,2-tetrafluoroethane or 1,1,1,2,3,3,3-heptafluoropropane.

[0131] The pharmaceutical compositions may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for nasal delivery.

[0132] The pharmaceutical compositions may be included in a container, pack, or dispenser, such as an inhaler, nebulizer, or dropper, optionally with instructions for administration for use in the methods described herein.

[0133] How to use PS-bionic nanoparticles

[0134] In some embodiments, PS-bionic nanoparticles can be used to promote a protective immune response to an antigen, such as as part of a vaccine, such as to treat or reduce the risk of developing a viral or bacterial infection, such as influenza (or flu), such as in the lungs. In some embodiments, PS-bionic nanoparticles can be used to treat asthma, respiratory allergies, or chronic obstructive pulmonary disease (COPD), or reduce one or more symptoms thereof.

[0135] In some embodiments, the PS-bionic nanoparticles can be administered to a mucosa (eg, nasal or lung tissue). In some embodiments, the PS-bionic nanoparticles can be administered intranasally (eg, via an inhaler, nebulizer).

[0136] In some embodiments, PS-biomimetic nanoparticles can be used to increase immune responses (e.g., activate innate immunity in the lungs; elicit CD8 + T cell response; protection against the virus, e.g., intrasubtypic protection against influenza virus; or heterosubtypic protection against influenza virus).

[0137] In some embodiments, PS-biomimetic nanoparticles can be used as chemotherapeutic adjuvants to treat cancers such as lung cancer. In these methods, the cargo is a chemotherapeutic agent, and the method includes administering a therapeutically effective amount of nanoparticles, such as an amount sufficient to cause a reduction in tumor size, tumor number, tumor growth rate, or metastasis.

[0138] Example

[0139] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0140] Materials and methods

[0141] PS-GAMP Synthesis

[0142] All lipids were purchased from Avanti Polar Lipids, including 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DPPE-PEG2000). Cholesterol was obtained from Sigma Aldrich. The mass ratio of nano4 and nano6 was 10:1:1:1 DPPC / DPPG / DPPE-PEG / Chol. Lipids were dissolved in 3 ml of chloroform and mixed with 1 ml of cGAMP solution (200 μg cGAMP, 13.7 mM NaCl, 0.27 mM KCl, 0.43 mM Na 2 HPO 4 , and 0.147 mM KH 2 PO 4 ) were mixed. Optionally, cGAMP was replaced with SRB (Sigma Aldrich) and / or 0.5 μmol DiD dye (Life Technologies) was added to the lipid mixture to label the cargo or liposome membrane, respectively. Liposomes were synthesized by the reverse phase evaporation method (43). Briefly, the liposomes were heated at 50 °C under N 2 The mixture of lipid and cGAMP was sonicated for 30 minutes at 40 ° C to obtain a water-in-oil emulsion, followed by gentle removal of the solvent by rotary evaporation at a speed of 220 rpm. Excess buffer was added to the mixture and continued to rotate for another 5 minutes at 50 ° C. The resulting liposomes were extruded through 400- and 200-nm membranes (Avanti Polar Lipids) at 50 ° C. The size and zeta potential of the liposomes were determined by Zetasizer (Malvern). In Nanodrop (Life Technologies), the encapsulation efficiency was determined by UV absorption of cGAMP at 260 nm and confirmed by liquid chromatography-mass spectrometry (LC-MS) (Agilent). Free cGAMP was removed by size exclusion column G-50 (GE Healthcare). In order to stabilize the liposomes, trehalose was added to the liposome suspension at a final concentration of 2.5%. The resulting suspension was frozen in a dry ice / ethanol bath and then lyophilized by Freezone 4.5 (Labconco) under vacuum at -45 ° C. Lyophilized liposomes (PS-GAMP) were stored at -20°C until use and were used for all in vivo studies unless specifically stated otherwise.

[0143] animal

[0144] C57BL / 6J and BALB / c mice were purchased from Jackson Laboratories or Shanghai SLAC Laboratory Animal Co., Ltd. Sting-deficient mice (C57BL / 6J-Tmem173gt / J), Sftpa1 - / - Sftpd - / - Mice (B6.Cg-Sftpa1tm2Haw Sftpdtm2Haw / J), C57BL / 6CD45.1 mice (B6.SJL-Ptprca Pepcb / BoyJ), and Swiss Webster mice were obtained from Jackson Laboratory or Charles River Laboratories. MHC II-EGFP mice expressing MHC class II molecules fused to enhanced green fluorescent protein (EGFP) were a kind gift of Dr. H. Ploegh of the Massachusetts Institute of Technology. Influenza-free 4-month-old female ferrets were purchased from Marshall BioResources. Healthy naive 6-year-old male rhesus monkeys were purchased from Beijing Institute of Xieerxin Biology Resource, China. Animals were maintained in pathogen-free animal facilities at Massachusetts General Hospital (MGH) or Fudan University in accordance with institutional, hospital, and NIH guidelines. Studies were evaluated and approved by the MGH or Fudan University Institutional Animal Care and Use Committee.

[0145] Flu viruses and vaccines

[0146] SH13 H7N9 virus (A / Shanghai / 4664T / 2013), SH09 H1N1 virus (A / Shanghai / 37T / 2009), and rgGZ89 H3N2 virus composed of H3 and N2 of A / Guizhou / 54 / 1989 H3N2 virus and A / Puerto Rico / 8 / 1934 (PR8) virus backbone were obtained from Fudan University. Pandemic CA09 H1N1 virus was requested from American Type Culture Collection (ATCC, #FR-201). PR8 (NR-348), A / Aichi / 2 / 68H3N2 (Aichi, NR-3177), rgPerth H3N2 [A / Perth / 16 / 2009H3N2×PR8 (NR-3499)], and B / Florida / 4 / 2006 (Florida06, NR-9696) virus strains were obtained from BEI Resources, NIAID. Reverse genetics (rg) modified VN04 (rgVN04) H5N1 virus was a kind gift from Dr. R. Webby of St. Jude Children's Research Hospital, which includes H5 and N1 genes from A / Vietnam / 1203 / 2004H5N1 virus and PR8 virus backbone. A / Michigan / 45 / 2015H1N1 (Michigan15, FR-1483) and antiviral resistant A / North Carolina / 39 / 2009H1N1 virus (NC09, FR-488) were obtained from International Reagent Resources, CDC. Viruses were amplified in 10-day-old embryonated chicken eggs (Charles River Laboratories) at 35°C for 3 days, harvested, purified by sucrose gradient ultracentrifugation, and frozen at -80°C. To challenge mice, viruses were adapted in mice for 3 cycles of intranasal instillation-lung homogenate preparations and were administered according to standard protocols, by 50% lethal dose (LD 50 ) to test their infectivity in mice.

[0147] The monovalent CA09 H1N1 vaccine (NR-20347, Sanofi Pasteur, Inc.) and the inactivated H5N1 vaccine (NR-12148, Baxter AG) were obtained from BEI Resources, NIAID. The H7-Re1 H7N9 inactivated vaccine was a gift from the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The trivalent seasonal influenza vaccine 2018-2019 (SIV 18-19) was obtained from Hualan Biological Bacterin Co., Ltd., China. SH09 H1N1 and Perth H3N2 inactivated vaccines were prepared by inactivating the virus with 0.02% formalin at 37°C and purifying as described above. Ag concentration was quantified based on HA content, by BCA protein assay and SDS-PAGE.

[0148] Mouse immunization and challenge

[0149] Mice were sedated with ketamine / xylazine and inoculated intranasally with 30 μl (15 μl in each nostril) of the designated influenza vaccine or a mixture of vaccine and adjuvant. VN04 H5N1, SIV 18-19, and CA09 H1N1 SV vaccines were used at the corresponding doses of 1 μg (HA content), 1 μg, or 0.5 μg per mouse, respectively, whereas H7-Re1 and SH09 H1N1 vaccines were each administered at 0.25 μg or 3 μg per dose, respectively. Poly IC (Invivogen), Pam2CSK4 (Invivogen), and cholera toxin (Sigma) were administered at 20, 20, or 10 μg per mouse, respectively. To block gap junctions, CBX, tonabosat, and meclofenamic acid were obtained from Sigma Aldrich and injected intraperitoneally into individual mice at the corresponding doses of 25, 10, or 20 mg / kg / day, respectively, for 4 consecutive days (from 2 days before immunization to 1 day after immunization) (31, 32). To deplete CD8 +T cells, anti-CD8α (53-6.7, BioLegend) antibodies were administered to mice at a dose of 200 μg / day 2 days before immunization and 0, 2, and 4 days after immunization. C57BL / 6 mice were used for challenge studies, except for Aichi H3N2 virus, Florida06 B influenza virus, and GZ89 virus, which challenged Swiss Webster mice or BALB / c mice as an alternative, unless otherwise indicated, because C57BL / 6 mice are relatively insensitive to these viruses. To verify the antiviral resistance of NC09 virus, non-immunized mice were treated with oseltamivir (20 mg / kg / day) 6 hours before challenge and every day thereafter until the end of the study. On the specified days after immunization, 10×LD 50 Mouse-adapted homologous viruses were used to challenge immunized and control mice, except that H7N9 virus was 40×LD 50 However, each is 5×LD 50 Heterologous viruses were used for challenge, except that the dose of Florida06 influenza B virus was 4 × 10 5 TCID 50 , because the virus is not lethal to mice. After challenge, body weight and survival were monitored daily for 12 days.

[0150] Ferret Immunity and Attack

[0151] Four-month-old female ferrets that were negative for anti-influenza antibodies were anesthetized with ketamine / xylazine / atropine and immunized intranasally with vehicle, influenza vaccine, or a mixture of vaccine and PS-GAMP. To test for early protection, 10 6 TCID 50 Each ferret received 9 μg of CA09 H1N1 vaccine alone or with 200 μg of PS-GAMP. To evaluate cross protection, each ferret was immunized intranasally with 15 μg of Perth H3N2 vaccine in the presence or absence of 200 μg of PS-GAMP and 30 days after immunization, the ferrets were re-immunized with 10 μg of Perth H3N2 vaccine. 6 TCID 50 Heterologous subtype Michigan 15 H1N1 virus challenge. Body temperature was monitored by two microchips implanted in each animal (BioMedicData Systems), and clinical symptoms were scored according to published protocols (Table 1) (44). Two weeks after virus challenge, animals were humanely euthanized by sedation and intracardiac injection of 0.5 ml of Euthanasia-III.

[0152] Table 1. Clinical symptom scores of ferrets (44)

[0153]

[0154] Tissue processing and flow cytometry

[0155] Lungs, nasal tissues, MLNs, and spleens were excised from designated mice and processed into single cell suspensions for flow cytometry analysis. Specifically, lung and nasal tissues were minced into 1-mm 2 The BALF was collected by complete perfusion of mice with ice-cold PBS, followed by intratracheal lavage with 0.5% BSA in PBS. Single cell suspensions of spleen and MLN were prepared by passing the tissue directly through a 40-μm cell strainer. After removing red blood cells in ACK buffer, the remaining cells were washed, blocked with anti-CD16 / CD32 antibodies (clone 93, 10 μg / ml, BioLegend) for 20 minutes, and stained with fluorescent conjugated antibodies on ice for 30 minutes or with NPs on ice. 366-374 ,PA 224-233 PB1 703-711 MHC I tetramer staining was performed for 1 hour. Activated T cells were fixed and permeabilized after surface staining, followed by intracellular staining with anti-granzyme B (GB) antibody overnight at 4°C. Stained cells were acquired on a FACSAria II (BD) and analyzed using FlowJo software (Tree Star). Cell populations and subpopulations in the mouse respiratory system were gated and analyzed as described (18). Information on various antibodies is shown in Table 2.

[0156] Table 2. Antibodies and tetramers used in flow cytometry

[0157]

[0158]

[0159] Cytokine and chemokine assays

[0160] C57BL / 6 mice were intranasally administered 20 μg of PS-GAMP or 1×LD 50CA09 H1N1 virus infected C57BL / 6 mice. Lungs were harvested at designated times and prepared for total RNA extraction using an RNA purification kit (Roche). To determine cytokines in the brain, mice were intranasally administered VN04 H5N1 vaccine alone (1 μg HA) or with PS-GAMP (20 μg) or CT (10 μg), and mice were killed 48 hours later to collect brain tissue for RNA extraction as described above. RNA was reverse transcribed (Life technologies) and amplified by real-time PCR using a SYBRGreen PCR kit (Roche). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. All primers used are listed in Table 3. Mouse GM-CSF (eBioscience), IFN-β (Invivogen), TNF-α (BioLegend), IFN-γ (eBioscience), IL-6 (eBioscience), and IL-10 (BioLegend) levels in BALF and serum were determined by specific ELISA kits.

[0161] Table 3. Primers used for real-time PCR

[0162]

[0163] Histology

[0164] Siwss Webster mice were intranasally administered PBS, PS-GAMP (20 μg), H5N1 vaccine (1 μg HA), or vaccine plus PS-GAMP or CT (5 μg). Some mice were infected with CA09 H1N1 virus (250 PFU) as positive controls. At the designated days after immunization or infection, lungs, nasal tissues, and brains were excised, fixed, and stained using standard H&E procedures. Sections were scanned and analyzed using NanoZoomer (Hamamatsu).

[0165] Confocal microscopy

[0166] To track DiD-labeled liposomes in the lung, C57BL / 6 mice were intranasally administered with equal amounts of DiD-nano4 or DiD-nano5. Lungs were excised 12 h later, embedded in optimal cutting temperature (OCT) compound (Sakura Finetek), and cut into 5-μm cryosections. Sections were mounted with ProLong Antifade Mountant (Life Technologies) containing DaPI and imaged by confocal microscopy (Olympus FV1000, UPLSAPO 60XW). To visualize the ex vivo AM uptake of nanoparticles, mouse lungs were lavaged 6 times with 1 ml of PBS containing 0.5% BSA and 5 mM EDTA. The lung lavage fluids were combined and centrifuged at 200 × g. Cells were collected, washed extensively with PBS, and cultured in RPMI 1640 medium for 45 min, followed by removal of non-adherent cells. Adherent cells were collected as AMs and cultured in medium at 2 × 10 5 Cells / ml were suspended and added to a 96-well plate at 200 μl / well. To purify PS, lung lavage fluid was prepared by washing the lungs 6 times with 1 ml of PBS and centrifuged at 220 × g for 10 minutes to remove cell debris, and then centrifuged at 100,000 × g for 1 hour to pellet the PS. The supernatant (6 ml) was concentrated to 200 μl by a 3-kDa Amicon ultracentrifugal filter unit (Merk Millipore) and mixed with the PS pellet prepared above. Then, 4 × 10 4 The obtained PS (100 μg total protein) was mixed with DiD-nano4 or DiD-nano5 (12 μg lipid content in the nanoparticles) for 30 min before AM cell culture of 100 cells. 2 After 4 hours of incubation at 4°C, cells were stained with the vital dye Calcein-AM (Life Technologies). Liposome uptake was quantified by confocal microscopy (Olympus FV1000, UPLSAPO 60XW), followed by ImageJ software analysis.

[0167] Statistical analysis

[0168] Two-tailed Student t test was used to analyze the differences between the two groups. ANOVA or Kruskal-Wallis test was used to analyze the differences between multiple groups by PRISM software (GraphPad). p < 0.05 was considered statistically significant. The sample size was determined based on preliminary experiments with a statistical power of 0.8. Most experiments were repeated at least twice with similar results. The investigators were not blinded to which experiments were performed under highly standardized and predetermined conditions, except for microscopic images and H&E slide examinations, which were evaluated in a researcher-blind manner.

[0169] Hemagglutination inhibition (HAI) test

[0170] Serum samples were collected from immunized and control animals at designated times and treated with receptor-destructive enzyme (RDE) (Denka Seiken, Tokyo, Japan) for 20 hours at 37°C, followed by heat inactivation at 56°C for 30 minutes. The resulting serum samples were serially diluted and incubated with 4 hemagglutination units (HAU) of the designated influenza virus at 37°C for 1 hour. The serum-treated viruses were incubated with 0.5% chicken erythrocytes (for H1N1 and H7N9) or horse erythrocytes (for H5N1) at room temperature for 30 minutes. The HAI titer was defined as the reciprocal of the highest serum dilution that inhibited 4 HAU of a given virus.

[0171] Enzyme-linked immunosorbent assay (ELISA)

[0172] Influenza-specific IgG, IgG1, IgG2a, IgA, and IgG2c antibody titers were determined by ELISA. Briefly, 1 μg / ml of recombinant HA was coated on a plate in NaHCO 3 Buffer, pH 9.6 ELISA plate overnight, to which serially diluted serum samples were added. Antibody subtypes were quantitatively determined by HRP-conjugated goat anti-mouse IgG (NA931V, GE healthcare, dilution 1: 6000), IgG1 (1073-05, Southern Biotech, 1: 4000), IgG2c (1079-05, Southern Biotech, 1: 4000), IgA (A90-103P, Bethyl, 1: 10000), IgM (ab97230, 1: 20000) or IgG2a (1083-05, Southern Biotech, 1: 4000). The titer of specific antibody subtypes was quantitatively determined by using SIGMAFASTM OPD as substrate and reading the reaction at A490 on a plate reader (Molecular Devices).

[0173] Cellular immune response

[0174] Splenocytes were isolated from mice 7 days after immunization by passing the spleen through a 40 μm filter, followed by lysis of erythrocytes with ACK (ammonium-chloride-potassium) buffer on ice for 4 minutes. 1×10 6 The cells were grown overnight at 4% 4% TNF-α and 1% TNF-α. Golgi-Plug (BD Pharmingen) was added to the culture and cultured for another 5 hours. First, cells stimulated by fluorescence-conjugated antibody staining for CD3, CD4, and CD8 were followed by intracellular staining with anti-IFN-γ antibodies. All antibodies are listed in Table S2. Dyed cells were obtained on FACSAria II (BD) and analyzed using FlowJo software (Tree Star).

[0175] Generation of chimeric mice by bone marrow transplantation

[0176] Chimeric mice were generated by bone marrow (BM) transplantation as described (33). Briefly, BM cells were harvested from the femurs and tibias of sex- and age-matched donor mice that differed in CD45 alleles. Recipient mice were lethally irradiated with a 137Cs gamma irradiator (Mark I, 30 J.L. Shepherd) at a dose of 1100 rad in two divided doses, 3 hours apart. After the second irradiation, 5 × 10 6 Donor BM cells were injected intravenously into recipient mice. BM cells from STING-deficient mice (Sting- / - or ST) were transferred to age- and sex-matched WT mice, or vice versa. WT mice receiving WT BM cells or ST mice receiving ST BM cells were also prepared in parallel. Mice were supplied with antibiotic-containing water from 5 days before irradiation to 14 days after irradiation and were raised for 3 months to complete the complete reconstruction of the donor population, which was confirmed by flow cytometric analysis of lungs, MLNs, spleens, and peripheral blood mononuclear cells (PBMCs) after staining with anti-CD45.1 (clone A20, BioLegend, 2 μg / ml) or anti-CD45.2 (clone 104, BioLegend, 2.5 μg / ml) antibodies.

[0177] BM-derived dendritic cells (BMDCs) and BM-derived macrophages (BMMs)

[0178] BMDCs and BMMs were prepared as described previously (45). Briefly, BM cells were harvested from the tibia and femur of 4- to 6-week-old C57BL / 6 mice. 1 × 10 6BMDCs or BMMs were generated by culturing cells at a concentration of 100 cells / ml for 7 days. CD11c+ BMDCs were further purified by high-speed cell sorting in a FACSAria II (BD).

[0179] PS is required for AM uptake of nano4 in nonhuman primates (NHPs)

[0180] Lungs were surgically removed after euthanasia of rhesus monkeys, filled with 150 ml of cold RPMI 1649 culture medium supplemented with antibiotics, immersed in cold culture medium, and transported to the laboratory on ice. AM and PS were separated as described in (46). In brief, RPMI 1640 culture medium filled with lungs was collected and centrifuged at 200 × g to remove cell debris, and then centrifuged at 8000 × g for 20 minutes to pelletize PS. The supernatant (30 ml) was concentrated to 1 ml by a 10 kDa Amicon ultracentrifugal filter unit (Merk Millipore) and mixed with the PS pellets prepared above to obtain concentrated PS with both lipids and surfactant proteins. AM was separated by washing the lungs 6 times with 100 ml of PBS containing 0.5 mM EDTA. Lung lavage fluid was combined and centrifuged at 200 × g to collect cells. The cells were fully washed with PBS and cultured in RPMI 1640 for 20 minutes, followed by removal of non-adherent cells. Concentrated PS with a total protein content of 2 mg was mixed with DiD-nano4 or DiD-nano5 (48 μg lipid content) for 30 min and then incubated at 37°C under 5% CO 2 In 1 ml of culture medium, 1.6 × 10 5 AMs were cultured. AMs were stained with the vital dyes calcein-AM (Life Technologies) and Hoechst (Sigma). AM uptake was evaluated by confocal microscopy (Olympus FV3000, UPLSAPO40×) and analyzed by ImageJ software.

[0181] Transmission Electron Microscopy (TEM)

[0182] To determine the ultrastructural localization of nano4 and nano5 in the alveoli, gold nanoparticles (5 nm, Alfa Aesar) were encapsulated into nano4 or nano5 by reverse phase evaporation as described (47). Mice were intranasally administered with equal amounts of gold nanoparticles-nano4 or nano5, and lungs were isolated 12 h later, fixed in Karnovsky fixative overnight at 4°C, and then fixed in 0.1 M sodium cacodylate buffer in 1% OsO 4The samples were post-fixed for 1.5 hours, dehydrated in a graded alcohol series, infiltrated with a gradient mixture of s-propylene oxide / Epon t812, and embedded in Epon t812 (Tousimis). Ultrathin sections were cut at 80 nm on an ultramicrotome (Reichert-Jung Ultracut E), collected on 100-mesh copper grids, stained with 2% uranyl acetate and lead citrate (2.66% lead nitrate, 3.52% sodium citrate), and examined on a CM-10 transmission electron microscope (Philips). Digital TEM images were obtained by an AMT-XR41M4.0Megapixel Cooled sCMOS camera (Advanced Microscopy Techniques).

[0183] Example 1. PS-GAMP made from PS ingredients

[0184] Based on the PS component (17), we synthesized a series of liposomes to encapsulate cGAMP (Figure 7A). In terms of lipid composition and charge, the negatively charged nano4 is close to the PS. Compared with the vaccine control alone, when introduced intranasally (in) with the whole inactivated A / Vietnam / 1203 / 2004 (VN04) H5N1 vaccine, only the liposomes strongly stimulated the production of serum IgG and bronchoalveolar lavage fluid (BALF) IgA without weight loss (Figures 7B-7E). In contrast, neutral liposomes (e.g., nano1), liposomes with cationic 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) replacing anionic phosphatidylglycerol (DPPG) (e.g., nano3 or nano5), or liposomes lacking PEG2000 (e.g., nano2 and nano3) showed significantly less adjuvantity and caused significant weight loss (Figures 7A-7E). Although their size and encapsulation efficiency are similar to nano4 (Fig. 7F-7G). Therefore, negative charge and PEG2000 seem to play an important role in the function and safety of liposomes. Unexpectedly, compared with the stimulation with negatively charged liposomes (nano2 and nano4), the cGAMP in vitro stimulation of bone marrow-derived dendritic cells (BMDC) encapsulated in positively charged liposomes (nano3 or nano5) expressed higher levels of Ifnb1 (Fig. 7H). When bone marrow-derived macrophages (BMM) were stimulated with positively or negatively charged liposomes encapsulated with cGAMP, similar patterns (Fig. 7I) appeared. This emphasizes the necessity of in vivo evaluation of the safety and efficacy of nanoparticles. Trehalose was then added to the liposome suspension to increase nano4 stability (Fig. 7A) before lyophilization. The resulting nano6 liposomes, which we call PS-GAMP, were stable at -20°C for at least 6 months and exhibited similar zeta potential, size, function, and safety to freshly prepared nano4 (Figures 7A, 7F, 7J, and 7B-7E). In addition, influenza vaccines adjuvanted by PS-GAMP induced high Ag-specific IgG titers in wild-type (WT) mice, but not in STING-deficient mice, confirming that cGAMP, rather than any other components, was responsible for the adjuvant properties of PS-GAMP (Figure 7K).

[0185] Example 2: PS-GAMP uptake by alveolar macrophages requires surfactant proteins A and D

[0186] Then, the cellular targets of nano4 and its cargo were investigated by labeling nano4 and nano5 membranes with DiD, a fluorescent lipophilic carbocyanine, and packaging another fluorescent dye (sulforhodamine B, SRB) with a molecular mass and net negative charge comparable to cGAMP within the liposomes (Figure 1A). Liposomes were intranasally administered to mice, and their nasal tissues, brain, mediastinal lymph nodes (MLN), and lungs were analyzed by flow cytometry at various time points. Compared with controls, the lungs were the site of SRB. + Here, nano4 is expressed by CD11b - CD11c + CD24 - Direct uptake by alveolar macrophages (AMs) and expression of CD11b - CD11c - EpCAM + MHC II + AEC (Figures 1B-1D and 10A) indirectly take up (18). Therefore, AEC is SRB + Instead of DiD - , however, most SRB + AM is still DiD + , indicating that the cells directly engage nano4 (Figure 1E). Greater than 95% of CD11c + SRB + Cells identified as AM (Fig. 10A, 2nd panel) or 44% of total AM in the lungs took up liposomes shown as SRB + D + (Figure 10B, first panel). SRB + AM and SRB + The proportion of AECs reached peak values ​​at 12 and 18 hours, respectively, and returned to the basal level within 36 hours (Figure 8B). + Dendritic cells (DC) (<2%) and CD11b + DC (<2%) is DiD + and SRB + , which precludes direct uptake of liposomes by these cells (Figure 10B). +The ability of PS-GAMP to deliver cGAMP to AMs was functionally confirmed by upregulation of CD40 in AMs. The same nanoparticles (DiD-PS) lacking cGAMP had no effect on CD40 expression (Figures 11A-11B) (19). Therefore, AM activation appears to be directly caused by PS-GAMP uptake rather than through a bystander effect (Figure 11C). In contrast to nano4, nano5 was not significantly associated with AMs or AECs when compared to free SRB (Figures 1B-1D and Figure 8C).

[0187] Surprisingly, AMs isolated from lung lavage fluid did not effectively uptake nano4 in vitro. In fact, AMs took up more nano5 than nano4, as confirmed by higher DiD fluorescence (Figures 1F-1G), which complements our earlier observations that nano5 induces higher Ifnb1 expression in BMDCs and BMMs (Figures 7H-7I). The difference between the in vivo and ex vivo uptake of these liposomes may be due to the lack of PS in the ex vivo culture. Therefore, we purified PS from BALF and cultured PS with nanoparticles for 30 minutes before adding them to AMs. Nano4 uptake increased significantly, while nano5 uptake decreased (Figures 1F-1G). Note that positively charged nano5 aggregates on negatively charged PS, explaining its poor entry into AMs (Figures 1F-1G). Fig.12 ). Under similar conditions, no such aggregates were formed when PS was incubated with nano4 ( Fig.12 Similar results were obtained when AM and PS were isolated from non-human primates (NHPs) ( Figures 13A-13C ). Therefore, PS may play an evolutionarily conserved role in PS-GAMP endocytosis. Consistent with these in vitro observations, DiD-nano4 was localized in single cells positive for Siglec F, a biomarker for AM, after intranasal administration (Figures 1H and Fig.14 ). In contrast, positively charged nano5 electrostatically interacted and fused with negatively charged PS, showing diffuse staining along the alveolar surface (Figure 1H). The different localization of nano4 and nano5 was confirmed by transmission electron microscopy (TEM) using nanogold-labeled nano5 and nano4 ( Fig.15 ). In vitro verification of efficient uptake of nano4 only in the presence of PS implicates surfactant proteins (SP)-A and -D (called “collagens”) in this uptake. Indeed, compared with controls, nano4 uptake from Sftpa1 - / - Sftpd - / - In stark contrast to PS isolated from WT mice, PS isolated from WT mice were unable to enhance nano4 uptake in vitro by WT AM (Figure 1I). - / - Sftpd- / - mice (Figure 1J), which was not due to Sftpa1 - / - Sftpd - / - Any defects in AM, because after pre-incubation with WT PS in vitro, Sftpa1 - / - Sftpd - / - AM uptakes nano4 ( Fig.16 ).

[0188] Example 3: PS-GAMP transiently activates innate immunity in the lung

[0189] The dependence of SP-A and SP-D in Nano4 uptake suggests that a natural and molecularly specific mechanism of particle clearance in the lung is involved, which would be the best way to maintain the integrity of PS and the alveolar epithelial barrier (20). Indeed, 2 days after intranasal administration of PS-GAMP, whole-killed VN04H5N1 vaccine, or a combination of both, mouse lung, nasal tissue, and brain were histologically indistinguishable from PBS controls (Figures 17A-17B). There was no cell death, damage to the epithelial barrier, or significant infiltration of inflammatory cells in these tissues ( Figures 17A-17C ). Only a moderate and transient infiltration of mononuclear cells was found in the lungs on day 3, which was significantly less severe than the mononuclear cells responding to viral infection (Figure 18E). We did not observe any significant cytokine production in the brain compared to controls (Figure 17C). In sharp contrast, the VN04H5N1 vaccine formulated with cholera toxin (CT) induced a large number of inflammatory cell infiltrates in the lungs and measurable cytokine mRNA expression in the brains of some mice (Figure 17C). Figures 17A-17C ).

[0190] Despite the lack of overt lung inflammation histologically over time ( FIG. 19A ), PS-GAMP was found to rapidly and robustly, but only transiently, activate innate immunity. Ifnb1, Gmcsf, and Tnf as well as Ccl2, Ccl3, Ccl5, and Cxcl10 mRNA expression peaked at 12 hours after stimulation and resolved within 48 hours ( Fig. 20 ). In contrast, low-dose infection with CA09 H1N1 influenza virus induced significantly higher levels of these mediators ( Fig. 20 ), causing lung inflammation that worsened during the course of viral infection despite robust Il10 expression ( Figures 19B-19C and 20 ). Protein levels in BALF also confirmed transient IFN-β production, but TNF-α and IL-10 were beyond the detection limit ( Figures 21A-21C In sharp contrast, as infection progressed, production of these cytokines was significantly higher from day 2 to day 6 ( Figures 21A-21C). The transient activation of innate immunity was limited to the lungs, as serum IFN-β, IFN-γ, IL-6, IL-10, and TNF-α levels were unchanged compared to controls (Figures 22C-22G). This is consistent with the lack of adjuvant side effects in terms of mouse body weight and temperature (Figures 22A-22B).

[0191] Example 4: PS-GAMP is a potent adjuvant for both humoral and cellular immune responses

[0192] Although PS-GAMP activated innate immunity only transiently, the effect appeared to be sufficient to enhance humoral and cellular immune responses, consistent with our previous findings that long-term activation of innate immunity is not required for strong adaptive immunity (13, 21, 22). PS-GAMP increased serum hemagglutination inhibition (HAI) antibodies and BALF IgA titers in a dose-dependent manner (Figures 2A-2B). The adjuvant was potent in both the primary and booster immune responses, increasing serum Ag-specific IgG1 by 10-fold, IgG by more than 100-fold, and IgG2c by ~1,000-fold compared to VN04 H5N1 vaccine alone (Figures 2C-2E). In addition to the whole-killed VN04 H5N1 vaccine, PS-GAMP also exhibited strong adjuvant properties when combined with split virion (SV) vaccines such as A / California / 7 / 2009 (CA09) H1N1 vaccine. Compared with SV vaccine alone, the adjuvant enhanced HAI titer 10-fold, BALFIgA 60-fold, and IgG 10,000-fold (Figures 2F-2H). Under similar conditions, poly IC showed 5-fold, 30-fold, and 100-fold lower efficacy in enhancing HAI titer, BALFIgA, and serum IgG, respectively (Figures 2F-2H). PS-GAMP not only enhanced humoral immune responses, but also significantly enhanced cellular immune responses. PS-GAMP-adjuvanted CA09 H1N1 vaccine increased IFN-γ compared with vaccine alone. + CD8 + T cells 24-fold, or IFN-γ compared to vaccines formulated with poly IC + CD8 + The duo also induced the highest amount of IFN-γ in all vaccination groups. + CD4 + T cells (Figure 2J and Figure 26A). A strong immune response translated to 10×LD 50Complete protection from CA09 H1N1 virus challenge was accompanied by only moderate to no weight loss (Figures 2K-2L). In contrast, the poly IC-adjuvanted CA09 H1N1 vaccine conferred only partial (33%) protection against virus challenge with severe weight loss.

[0193] Example 5: PS-GAMP induces potent CD8 + T cell response

[0194] We investigated which DC subsets are involved in PS-GAMP-mediated adjuvantity and found that after intranasal administration of PS-GAMP, CD11b + DC, not CD11b - DCs increased 14-fold and 36-fold on day 3 in lung (upper panel) and MLN (lower panel), respectively, relative to day 0 (Fig. 3A). + In DCs, the expression of MHC II and Ly6C distinguishes monocyte-derived CD11b + DC (Mono-DC) and tissue-resident CD11b + DC (tDC) (Figure 3B) (23,24). MHC II hi CD11b + tDCs have been shown to be the most competent lung DCs for cross-presentation during influenza virus infection (23). Significantly, after PS-GAMP administration, these cells accumulated in large numbers, similar to those in the early stages of viral infection (first 3 days), whereas proinflammatory mono-DCs increased only slightly during the same experimental period, albeit significantly relative to day 0 (Figure 3B). Thereafter, CD11b + tDCs were decreased in lungs and MLNs receiving PS-GAMP, consistent with infection for more than 3 days, and these CD11b + The continuous accumulation of DCs in both the lung and MLN was in sharp contrast (Figure 3B). Figure 18A-18V The changes in other immune cell types in the lung, MLN, nasal tissue, and brain after immunization or infection are described in detail in . In addition to DCs, natural killer (NK) cells and CD4 + T cells were transiently elevated in the lungs for 1 or 2 days, whereas other immune cells did not change during the experimental period (Fig. 3A).

[0195] These CD11b + DCs appear to be efficient in Ag cross-presentation and can induce potent CD8 + When fluorescently labeled ovalbumin (OVA) was administered intranasally, very few lung CD11b +DCs (0.3%) showed OVA uptake. However, in the presence of PS-GAMP, the proportion of these DCs that took up OVA increased significantly from 3% at 12 hours to 26% at 36 hours (Figure 23A). This translates to a higher OVA uptake compared to mice that received OVA alone. + DCs have a major CD11b + The expression of α-amyloid proliferator-activated protein kinase (AEP) in MLN increased by 10-fold in MLN DCs (Fig. 3C and Fig. 23B). These DCs matured and activated, as indicated by upregulation of CD40 and CD85 (Fig. 3D-3E). This effect is presumably secondary to AEC and AM activation, given that the majority of MLN DCs were negative for PS-GAME (Fig. 23C-23D). Ag-specific CD11b + The increase in DCs was not due to altered Ag processing or Ag uptake, as OVA uptake or its proteolytic cleavage was not affected by PS-GAMP (Figures 24D-24E). Therefore, the robust proliferation of OT-I cells in the presence of PS-GAMP may be due to the expression of CD11b + In turn, these cells resulted in a more than 6-fold increase in highly proliferative OT-I cells in the lungs and MLNs when OVA and PS-GAMP were introduced together compared to OVA alone ( Figures 25A-25D ).

[0196] As early as 4 days after immunization with PS-GAMP-adjuvanted influenza vaccine, abundant nucleoprotein (NP) was observed in the lungs 366-374 -Specific CD8 + T cells and to a lesser extent MLN (Figures 3F-3G and Figure 26B). NP 366-374 Is the main CD8 + T cell epitopes, and for other epitopes such as PA 224-233 or PB1 703-711 CD8 + T cells were undetectable in these animals, likely due to the low copy numbers of these proteins in the inactivated influenza vaccine (Fig. 26C) (25). These virus-specific CD8 + T cells expressed the early activation biomarker granzyme B (GB) under viral challenge (Figure 27A) (26). After receiving the PS-GAMP adjuvanted CA09 H1N1 vaccine, GB + CD8 + T cells increased significantly in BALF at 4 days and in lung at 6 days (Figure 3H). More than 65% of these GB + CD8 + T cells for NP 366-374 However, only a small number of cells were positive for PA 224-233or PB1 703-711 In a similar situation, the vaccine alone could not significantly amplify GB + CD8 + CD8 T cells stimulated by PS-GAMP were found + T cell responses were superior to those of poly IC or the TLR2 agonist Pam2CSK4 (27,28) (Figure 3I). Despite the induction of T cell immune responses shortly after immunization, Ag-specific BALF IgA and IgM were undetectable at these early time points (Figure 27C). Thus, PS-GAMP plays a key role in the induction of CD8 + T cell induction mimics key events of viral infection without causing excessive lung inflammation or immunopathology ( Figures 17A-17C , 18A-18V , 19A-19C, 20, 21A-21C, and 22A-22G).

[0197] Example 6: PS-GAMP provides strong protection as early as 2 days after immunization

[0198] CD8 + The rapid induction of T cells prompted us to determine how quickly PS-GAMP can achieve protection. To this end, as shown in Figure 28A, mice were attacked at 0, 2, 4, 6, 8, or 14 days after immunization. Including PS-GAMP in vaccination fully protected mice from homologous virus attack as early as 2 days after immunization (Figure 4A). At all early attack time points (-2, -4, and -6 days), mice only experienced slight weight loss (<10%), and all mice survived (Figure 4A and Figure 28B). When attacked on the 8th day after immunization, mice did not suffer any weight loss, 100% survival (Figure 4A and Figure 28B). This early protection is not directly caused by innate immunity, because giving a single PS-GAMP on day 0 or 2 days before does not confer any protection (Figure 4B and Figure 28C). In order to determine the CD8 + To determine whether T cells are responsible for early protection, CD8 T cells were depleted by intraperitoneal (ip) injection of anti-CD8 antibody every other day, starting 2 days before and ending 4 days after immunization. + T cells. CD8 +Depletion of T cells abolished early protection, as demonstrated by dramatic weight loss and 100% mortality similar to those of mice receiving the vaccine alone (Fig. 4C and Fig. 28D). To rule out that this early protection was unique to the CA09 H1N1 vaccine, we extended our studies to the H5N1 vaccine, which is a weakly immunogenic antibody compared to the CA09 H1N1 vaccine. Again, for mice immunized 2-8 days previously in a manner dependent on an adaptive immune response, the presence of PS-GAMP conferred 75%-100% protection against rgVN04 H5N1 virus challenge (Fig. 4D and Fig. 28E), as no protection was obtained with PS-GAMP alone (Fig. 4E and Fig. 28F). Under similar conditions, the vaccine combined with CT did not provide any early protection (Fig. 4E and Fig. 28F), convincingly arguing that the exacerbated inflammation is not necessarily associated with a strong adaptive immune response ( Figures 17A-17C ). In addition to rgH5N1 virus, mice were significantly or completely protected from lethal challenge with clinical isolates of the previously pandemic A / Shanghai / 4664T / 2013 (SH13) H7N9 virus 2 or 14 days after immunization with the inactivated H7N9 vaccine (H7-Re1) adjuvanted with PS-GAMP (Fig. 4F and Fig. 28G-28I). Under similar conditions, vaccines adjuvanted with poly IC did not provide any benefit compared to vaccines alone (Fig. 4F and Fig. 28I).

[0199] The ability of PS-GAMP to quickly establish protection was also demonstrated in the FDA-approved ferret model. When infected with the homologous CA09H1N1 virus, ferrets that had received the PS-GAMP adjuvanted CA09 H1N1 vaccine 2 days earlier experienced <5% weight loss, accompanied by moderate to no clinical symptoms, and only a brief fever on the 2nd day after the virus challenge (Figures 4G-4I). Virus shedding was significantly reduced from the 4th day and thereafter (Figure 4J). However, compared to the control, the CA09H1N1 vaccine alone could not prevent animals from weight loss after a similar virus challenge, and did not improve clinical symptoms or reduce virus shedding, although it moderately reduced body temperature (Figures 4G-4J).

[0200] Example 7: AEC is essential for PS-GAMP-mediated adjuvantity

[0201] It has been well documented that cGAMP is readily transferred through gap junctions present between AM and AEC (29, 30). Dynamic flow from AM to AEC was demonstrated by the progressive loss of SRB in AM from 12 to 18 hours after intranasal administration of SRB-nano4, accompanied by a continuous gain of SRB in AEC (Figure 29A). The loss of SRB in AM could not be attributed to the loss of liposomes because DiD+ The number of cells did not change until 18 hours later (Figure 29B). Blocking SRB entry into AECs by the gap junction blocker (29) carbenoxolone (CBX) did not affect SRB uptake by AMs (Figures 5A and 5C). In AMs and AECs sorted from lungs receiving PS-GAMP, CBX greatly attenuated the transcription of Ifnb1 and Gmcsf in AECs, while increasing Ifnb1 transcription in AMs (Figures 30B-30C). This is most likely the result of elevated cGAMP levels in cells. Therefore, there is a gap junction-mediated flow of cGAMP from AMs to AECs. On the other hand, poly IC remained predominant (>97%) in AMs after intranasal immunization (Figure 31A). In the lungs, only 0.4% of total AECs and 4% of total DCs took up poly IC (Figures 31B-31C). MLNs and nasal tissue DCs as well as nasal epithelial cells rarely internalized poly IC (Figures 31D-31E).

[0202] PS-GAMP induced 100-fold higher IgG2c titers than poly IC (Fig. 5D). However, adjuvantity was significantly attenuated when mice were treated with CBX or two other gap junction inhibitors, tonabolsa and meclofenamic acid, before and during immunization (Fig. 5D) (31,32). In contrast, these inhibitors had a smaller effect on poly IC-mediated adjuvantity (Fig. 5D), consistent with the inability of poly IC, a large molecule, to enter adjacent cells through gap junctions (Fig. 31A). Blockade of cGAMP entry into AECs reduces CD11b + DC recruitment was 50% (Figure 5E) and showed expression of early CD8 + Furthermore, in BALF and lung, including Sting-deficient ( - / - Chimeric mice containing ST or ST) bone marrow (BM) cells and WT AECs (ST→WT) had similar levels of CD8 + T cells (Figures 5H-5J and Figures 32A-32B )(33). In contrast, mice with STING deficiency in AECs, generated by transferring WT BM cells into Sting-deficient mice (WT→ST mice), produced significantly lower levels of Ag-specific CD8 + T cells (Figures 5I-5J). These WT→ST mice showed poor protection from the PS-GAMP-adjuvanted CA09 H1N1 vaccine, as indicated by weight loss and high lung virus titers, in contrast to the protection similarly observed between ST→WT and WT→WT mice (Figures 5K-5L). We also found that GB in BALF and lung + CD8+ The number of T cells is inversely correlated with the viral titer, further supporting the GB + CD8 + T cells play a key role in controlling infection (Figure 5M-5N). Therefore, AECs, rather than AMs, appear to be necessary to determine the efficacy of PS-GAMP, consistent with their key role in coordinating innate and adaptive immune responses in the respiratory system during viral infection (24, 34-36).

[0203] Example 8: PS-GAMP extends protection against heterologous subtypes of influenza virus

[0204] Powerful CD8 induced by PS-GAMP + T cell immunity prompted us to investigate its role in heterosubtypic protection, an issue hotly debated in the field of universal influenza vaccines. Mice receiving either CA09H1N1 vaccine (Figures 6A-6F) or A / Shanghai / 37T / 2009 (SH09)H1N1 vaccine (Figures 6G-6H), together with PS-GAMP, were highly protected from lethal challenge with different PR8H1N1 viruses and heterosubtypic A / Aichi / 2 / 1968 (Aichi)H3N2, rgVN04 H5N1, or highly pathogenic SH13 H7N9 viruses, whether animals were infected 2 days (Figures 6A, 6C, 6E, and 6G) ​​or 14 days (Figures 6B, 6D, 6F, and 6H) after immunization (Figures 33A-33H). Vaccination also protected against an oseltamivir-resistant A / NorthCarolina / 39 / 2009 H1N1 virus (NC09) with the H275Y mutation (NC09) (FIG. 6I and FIG. 33I), which emerged during the 2009 H1N1 pandemic and H7N9 epidemic (37, 38). The virus's resistance to oseltamivir was confirmed by the ability of oseltamivir to effectively control CA09 H1N1 but not NC09 virus infection (FIG. 6I). Under similar conditions, the H1N1 vaccine alone provided no or low protection against the challenge of these heterologous subtype variants (FIG. 6A-6I and FIG. 33A-33I). In contrast to PS-GAMP, the poly IC-adjuvanted SH09 H1N1 vaccine did not induce significant heterologous subtype protection against the H7N9 virus (FIG. 6G-6H and FIG. 33G-33H). In addition to the monovalent vaccines, PS-GAMP enhanced the immunity induced by the trivalent 2018-2019 seasonal influenza vaccine (SIV18-19) against the mismatched recombinant A / Guizhou / 54 / 1989H3N2 (rgGZ89) virus ( Figures 6J and 33J ) or the Florida / 4 / 2006B influenza virus from the Yamagata lineage ( Figures 34A-34BThese findings suggest that PS-GAMP can simultaneously boost multiple influenza vaccines and is equally effective against both influenza A and B virus vaccines.

[0205] Long-lived Ag-specific memory CD8 + T cells can be rapidly recalled in the event of a viral infection and are essential for adequate control of viral replication in the lung (2,3). In mice receiving OT-I cells, after immunization with OVA combined with PS-GAMP, compared with OVA alone, the expression of CD103 + CD49a + CD69 + Lung CD8 marker + T RM The number of cells increased 20-fold (Figures 35A-35C). In addition, 6 months after a single immunization, the PS-GAMP-adjuvanted CA09 H1N1 vaccine completely protected mice from heterologous subtype rgVN04 H5N1 virus challenge (Figures 6K and 33K). This long-term cross-protection is associated with persistent influenza-specific CD8 + T RM These CD8 + T RM cells, not circulating memory CD8 + T cells contribute to the observed long-term protection because their function is not affected by the T cell egress inhibitor FTY720 ( Figures 36A-36D )(3).

[0206] Heterologous subtype immunity was further confirmed in ferrets by immunization with PS-GAMP together with inactivated rgPerthH3N2 vaccine. When compared with those receiving PBS or vaccine alone, the weight and temperature of the animals were not affected by immunization, indicating the good safety of PS-GAMP in ferrets (Figures 37A-37B). Compared with the vaccine alone at 28 days after immunization, immunization induced 40-fold higher serum IgG titers and 5-fold higher HAI titers for homologous Perth H3N2 virus (Figures 37C-37D), but no HAI antibodies were detected for heterologous subtype A / Michigan / 45 / 2015H1N1 (Michigan H1N1) virus, as expected (Figure 37E). In the case of challenge with Michigan H1N1 virus, ferrets receiving vaccine and PS-GAMP showed significantly less weight loss and milder clinical symptoms, especially in the late stages of infection (>7 days), and normalized their body temperature more quickly (Figures 6L-6N) compared to animals receiving PBS or vaccine alone. Animals also shed significantly less virus 2 days after infection (Figure 6O). The ability of vaccination to inhibit viral replication led to a significant improvement in clinical outcomes, and the acceleration of weight recovery may be a result of the primary T cell immunity in the animals.

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[0257] Other Implementations

[0258] It should be understood that although the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims

1. A composition for promoting an immune response to an antigen, comprising Negatively charged nanoparticles, the nanoparticles comprising A plurality of pulmonary surfactant-biomimetic molecules, wherein the pulmonary surfactant-biomimetic molecules include polyethylene glycol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) and cholesterol; and One or more cargo molecules are encapsulated by the nanoparticles, wherein the one or more cargo molecules include cyclic GMP-AMP.

2. The composition of claim 1, wherein the composition comprises a plurality of nanoparticles, each nanoparticle comprising a plurality of lung surfactant-mimetic molecules.

3. The composition according to claim 1, wherein the lung surfactant-bionic molecule further comprises: More than one phospholipid; or One or more molecules having at least one hydrophobic end and / or at least one hydrophilic end.

4. The composition of claim 1, wherein the pulmonary surfactant-biomimetic molecule comprises: 50% to 90% by weight of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 5% to 15% by weight of 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), and / or 5 wt% - 15 wt% cholesterol.

5. The composition of claim 1, wherein the cyclic GMP-AMP is present at a concentration of 10 μg / ml-50 mg / ml. The composition according to claim 1 , wherein the average molecular weight of the polyethylene glycol is 500-5000 Da.

7. The composition of claim 1, wherein the polyethylene glycol is attached to the outer surface of the nanoparticle.

8. The composition of claim 1, wherein the polyethylene glycol is polyethylene glycol 2000.

9. The composition of claim 8, wherein the lung surfactant-bionic molecule further comprises 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, wherein the polyethylene glycol 2000 is covalently linked to 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine as 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000].

10. The composition of claim 1, wherein the pulmonary surfactant-biomimetic molecule further comprises 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin and / or lysophospholipids.

11. The composition of claim 9, wherein the composition is formulated for intranasal administration or administration by inhalation.

12. The composition of claim 1, wherein the composition further comprises trehalose.

13. The composition of claim 1, wherein the composition is a suspension.

14. The composition of claim 1, wherein the composition is lyophilized or suitable for lyophilization.

15. The composition of claim 1, wherein the nanoparticles comprise liposomes.

16. The composition of claim 1, wherein the composition is formulated for intranasal administration or administration by inhalation.

17. The composition according to claim 1, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

18. The composition of claim 1, further comprising an antigen.

19. The composition of claim 18, wherein the antigen is encapsulated within the nanoparticle.

20. The composition of claim 9, wherein the nanoparticles are prepared by a method comprising the steps of: A mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] and cholesterol is combined with cyclic guanosine monophosphate-adenosine monophosphate to produce the nanoparticles.

21. Use of a composition according to any one of claims 1-20 in the preparation of a medicament for treating a subject suffering from influenza by the following method, the method comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 1-20; and administering to the subject an antigen, wherein the antigen is an influenza vaccine.

22. The use according to claim 21, wherein the subject is a human and the antigen is a human influenza vaccine.

23. Use of a composition according to any one of claims 1-20 in the preparation of a medicament for treating a subject suffering from an airway disease by a method comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 1-20.

24. The use according to claim 23, wherein the subject is a human and the airway disease is one or a combination of asthma, chronic obstructive pulmonary disease, allergy, or pneumoviral infection.

25. The use according to claim 21 or 23, wherein the subject is a mammal.

26. The use according to any one of claims 21-24, wherein the composition is administered to the subject intranasally or by inhalation.

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