Compositions and methods of pulmonary surfactant-biomimetic nanoparticles

Abstract

The present invention relates to compositions and methods of pulmonary surfactant-biomimetic nanoparticles. Compositions and methods containing pulmonary surfactant (PS) biomimetic nanoparticles are disclosed. 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 1200 Da or less.

Description

[0001] This application is a divisional application of the patent application "Compositions and Methods of Pulmonary Surfactant-Biomimetic Nanoparticles" with an international filing date of October 6, 2020, application number 202080084627.3 (International Application No. PCT / US2020 / 054377).

[0002] Federally Sponsored Research or Development

[0003] This invention was made with government support under Grant Nos. AI089779, AI070785, and AI097696 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0004] Compositions are described herein that include pulmonary surfactant-biomimetic nanoparticles such as PS-GAMP, as well as methods of making and using pulmonary surfactant-biomimetic nanoparticles such as PS-GAMP. Background Art

[0005] Current influenza vaccines primarily protect against viral infection by inducing neutralizing antibodies specific for viral surface hemagglutinin (HA) and neuraminidase (NA). However, these surface proteins continuously undergo antigen drift / shift, greatly limiting the efficacy of these vaccines (1). Studies indicating the indispensable role of lung CD8 + resident memory T (T RM ) cells in heterosubtypic immunity may provide an explanation for this limitation (2, 3). Induced sufficiently by natural viral infection, these cells not only recognize highly conserved internal proteins shared among heterosubtypic influenza viruses but also are capable of clearing virus at the site of virus entry when viral numbers are low (4 - 6). Similarly, live vector-engineered and attenuated influenza vaccines can induce lung CD8 + T RM cells (7, 8), but a delicate balance must be achieved between their safety and immunogenicity. In addition, these replicating vaccines are generally affected by pre-existing immunity and are thus only suitable for some populations (9). In contrast, non-replicating influenza vaccines induce poor T cell immunity in the respiratory tract and require potent mucosal adjuvants to overcome the immunoregulatory mechanisms of the respiratory mucosa. Summary of the Invention

[0006] Safe and potent mucosal adjuvants are described herein that can be used, for example, to enhance influenza vaccines.

[0007] Other features and advantages of the present invention will be apparent from the following detailed description, the accompanying drawings, and the claims.

[0008] In one aspect, the present disclosure relates to a composition comprising nanoparticles having an average size of 200 - 400 nm, the nanoparticles comprising 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 cargo molecules have a molecular weight of 1200 Da or less.

[0009] In some embodiments, the lung surfactant - biomimetic molecules comprise 50 wt% - 90 wt% of 1,2 - dipalmitoyl - sn - glycero - 3 - phosphocholine (DPPC), 5 wt% - 15 wt% of negatively charged lipids, and / or 5 wt% - 15 wt% of neutral lipids.

[0010] 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.

[0011] In some embodiments, the nanoparticles further comprise a plurality of polyethylene glycols (PEGs) having an average molecular weight of 500 - 5000 Da. In some embodiments, the polyethylene glycol is attached to the outer surface of the nanoparticles.

[0012] In some embodiments, the nanoparticles further comprise 5 - 15 wt% of 1,2 - dipalmitoyl - sn - glycero - 3 - phosphoethanolamine - N - [methoxy(polyethylene glycol)-2000] (DPPE - PEG2000).

[0013] In some embodiments, the cargo molecule is a stimulator of interferon genes (STING) agonist.

[0014] In some embodiments, the STING agonist is cyclic guanosine monophosphate [GMP] - adenosine monophosphate [AMP] (cGAMP) or comprises cyclic guanosine monophosphate [GMP] - adenosine monophosphate [AMP] (cGAMP).

[0015] In some embodiments, cGAMP is present at a concentration of 10 - 100 μg / ml.

[0016] 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)); an agonist or antagonist of a receptor (e.g., chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), chemokine receptor 2 (CCR2)); an agonist or antagonist of an ion channel (e.g., GABA receptor, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or voltage-gated sodium channel); an inducer of IFN-α; a long-acting muscarinic antagonist / anticholinergic (LAMA); an inhibitor against IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; a CXCR2 antagonist; a molecule that blocks a pro-inflammatory cytokine (e.g., TNF-α, TNF-β, or IL-6); a molecule that blocks IL-17 / T HMolecules of 17; 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), FK565, 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-furo[2,3-c]pyridine); and / or TLR9 agonists (IMO-8400, IMO-3100, SAR-21609, AZD1419, SD-101, IMO-2055, IMO-2125, QAX-935, AVE0675, DIMS0150, MGN-1703, MGN-1706, ISS1018, or Agatolimod).

[0017] In one aspect, the present disclosure relates to a method for promoting an immune response against an antigen, the method comprising administering to a subject an effective amount of a composition as described herein; and administering an antigen to the subject.

[0018] In some embodiments, the subject is a mammal.

[0019] In some embodiments, the antigen is encapsulated within a nanoparticle; the nanoparticle and the antigen are administered as a single composition; or the nanoparticle and the antigen are administered as separate compositions.

[0020] In one aspect, the present disclosure relates to a method for treating a subject suffering from influenza, the method comprising administering to the subject a therapeutically effective amount of a composition as described herein; and administering an antigen to the subject.

[0021] In some embodiments, the cargo molecule is cGAMP and the antigen is an influenza vaccine.

[0022] In some embodiments, the subject is a human and the antigen is a human influenza vaccine.

[0023] In one aspect, the present 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.

[0024] 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 target 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)); an agonist or antagonist of a receptor (e.g., chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), chemokine receptor 2 (CCR2)); an agonist or antagonist of an ion channel (e.g., a GABA receptor, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or voltage-gated sodium channel); an inducer of IFN-α; a long-acting muscarinic antagonist / anticholinergic (LAMA); an inhibitor against IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; a CXCR2 antagonist; a molecule that blocks a pro-inflammatory cytokine (e.g., TNF-α, TNF-β, or IL-6); a molecule that blocks IL-17 / T H 17; a macrolide; a molecule that activates HDAC2; a STAT6 inhibitor (e.g., AS1517499); an antiviral small molecule drug (e.g., oseltamivir (Tamiflu), Relenza, or zanamivir); and / or favipiravir (T705).

[0025] In some embodiments, the subject is a human and the airway disease is one or a combination of asthma, chronic obstructive pulmonary disease (COPD), allergy, or a pulmonary viral infection.

[0026] 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.

[0027] In some embodiments, the subject is a mammal.

[0028] In some embodiments, the cancer is lung cancer and the chemotherapeutic agent is Gefitinib, Erlotinib, Crizotinib, Everolimus, Afatinib, Crizotinib, Doxorubicin, etoposide, Opdivo, and / or Trexall.

[0029] In some embodiments, the cancer is nasopharyngeal cancer and the chemotherapeutic agent is Cisplatin, Carboplatin, Gemcitabine, Doxorubicin, and / or 5-FU.

[0030] In some embodiments, the cancer is tracheal cancer and the chemotherapeutic agent is etoposide, Cisplatin, and / or Carboplatin.

[0031] In some embodiments, the cancer is bronchial cancer and the chemotherapeutic agent is etoposide, Cisplatin, Carboplatin, 5-FU, docetaxel, paclitaxel, and / or epirubicin.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the present invention will be apparent from the following detailed description and the drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings (more than one) will be provided by the Patent Office upon request and payment of the necessary fees.

[0034] Figure 1A - 1J. Uptake of PS-GAMP by AM requires SP-A and SP-D. (A) Schematic of PS-liposomes labeled with SRB and DiD. (B-E) Free SRB (20 μg) or SRB-DiD-nano4 or SRB-DiD-nano5 (20 μg SRB) was administered intranasally to mice, followed by flow cytometry analysis of SRB + and / or DiD + in lung cells 12 h later. Analysis (B) and quantification (C and D) were also for CD11c + AM (red) or CD11c - AEC (blue) SRB + cell percentages (n = 4). (E) Schematic overlay flow cytometry plots of DiD and SRB-stained AM and AEC. (F) AM were isolated from MHC II-GFP mice and cultured with DiD-nano4 or DiD-nano5 for 4 h after pre-culture with (bottom panels) or without (top panels) PS for 30 min. Scale bar: 10 μm. Optionally, AM were isolated from wild-type (WT) mice and cultured with DiD-nano4 pretreated with WT or Sftpa1 - / - Sftpd - / - PS for 30 min for 4 h (I). Cells were imaged by fluorescence microscopy and the 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 administered intranasally to WT or Sftpa1 - / - Sftpd - / - mice. 12 h later, CD11c + CD11b - CD24 - AM were analyzed for DiD + . n = 6. Each symbol represents an individual mouse in (C, D, and J) or a cell in (G and I). Results are presented as mean ± SEM. Statistical analysis was one-way ANOVA for (C, D, G, and I) and Student's t-test for (J). **p < 0.01 and ***p < 0.001 between the indicated groups. All experiments were repeated three times with similar results.

[0035] Figure 2A - 2L.Adjuvanticity of PS-GAMP. (A and B) Swiss Webster mice were intranasally immunized 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 intranasally immunized with VN04 H5N1 vaccine on day 0 in the presence or absence of PS-GAMP (20 μg cGAMP) 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 intranasally immunized with CA09 H1N1 vaccine with or without 20 μg of PS-GAMP or poly IC. Two weeks later, serum IgG (F), BALF IgA (G), and serum HAI (H) titers were determined. (I-J) Spleen cells were isolated 7 days after immunization and stimulated with CA09 H1N1 vaccine. CD8 + (I) and CD4 + (J) T cells were determined by flow cytometry. (K and L) Survival curves (K) and body weight changes (L) of non-immunized mice (black) or singly immunized mice receiving the vaccine alone (green), challenged with 10×LD 50 CA09 H1N1 virus 28 days after vaccination with a vaccine combined with polyIC (blue) or PS-GAMP (red). n = 6. Results are presented as mean ± SEM. Each symbol represents an individual mouse in (A to J). Statistical analysis was 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.

[0036] Figure 3A - 3I .CD8 + T cell responses induced by PS-GAMP. (A) At the indicated days after intranasal administration of PS-GAMP to mice, the numbers of CD4 + and CD8 + T cells, NK cells, and CD11b + and CD11b - DCs in the lung (upper panel) and MLN (lower panel) were analyzed by flow cytometry. n = 4. (B) After intranasal immunization with PS-GAMP or with 1×LD 50On the designated days after CA09 H1N1 virus-infected mice, CD11b in the lungs and MLN was quantified by flow cytometry + mono-DC and CD11b + tDC. n = 4. (C–E) Mice were intranasally vaccinated with OVA-AF647 with or without PS-GAMP. Thirty-six hours after immunization, DCs capturing OVA 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 intranasally immunized with CA09 H1N1 vaccine with or without PS-GAMP, or with PBS alone as an unimmunized control. On the designated days after immunization, CD8 366-374 T cells in the lungs and MLN were analyzed for their Ag-specificity by staining with NP + tetramers. n = 4–8. (H) Mice were immunized as described in (F and G) and challenged with 10× LD 50 of CA09 H1N1 virus 2 days later. On the designated days after immunization, GB + CD8 + T cells were counted in the BALF and lung 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. Four days after challenge, GB + CD8 + T cells were counted as described in (H). n = 4. Each symbol represents an individual mouse in (A, C–E, and I). Results are presented as mean ± SEM. Statistical analysis was one-way ANOVA for (A, B, and I), two-way ANOVA for (F–H), and Student t test for (C–E). *p < 0.05; **p < 0.01, and ***p < 0.001 compared with day 0 (A and B), influenza vaccine alone (F–H), or between the designated groups. All experiments were repeated twice with similar results

[0037] Figure 4A - 4J . PS-GAMP-mediated early protection. (A–B) 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) on days 2, 4, 6, 8, or 14 before virus challenge as described in Fig. 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 in (A). CD8 + T cells were depleted in some mice by injecting anti-CD8 antibody 2 days before vaccination and at days 0, 2, and 4 after vaccination. n = 4. (D) Survival of mice immunized with VN04 H5N1 vaccine plus PS-GAMP on the indicated days before day 0 of challenge with 10×LD 50 of rgVN04 H5N1 virus as described in Fig. 28A. n = 4 - 8. (E) Survival of mice immunized with VN04 H5N1 vaccine, PS-GAMP, or vaccine plus free cGAMP, CT, or PS-GAMP and challenged 2 days later with rgVN04 H5N1 virus. n = 4 - 8. (F) Mice were immunized intranasally with H7-Re1 H7N9 vaccine and 20 μg of PS-GAMP or poly IC and challenged 2 days later with a clinically isolated SH13 H7N9 virus at 40×LD 50 . n = 8 - 12. (G to J) Ferrets were immunized intranasally with CA09 H1N1 vaccine (9 μg) with or without 200 μg of PS-GAMP and challenged 2 days later with 10 6 TCID 50 of CA09 H1N1 virus. Body weight (G), disease score (H), temperature (I), and virus titer in nasal wash (J) were monitored for 12 h. n = 4. Results are presented 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 antibody (C). Mouse experiments were 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. For statistical analysis, two-way ANOVA was used for (C, G, I, and J), Kruskal Wallis test for (H), and log-rank test for (A, B, and D - F).

[0038] Figure 5A - 5N.AEC makes an indispensable contribution to the adjuvanticity of PS-GAMP. (A) Mice were intraperitoneally administered CBX once a day for 3 consecutive days, and then SRB-nano4 was intranasally administered to the mice. SRB was analyzed 12 hours later. + AM (red) and AEC (blue), and 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 the CA09H1N1 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) As described in (D), in the presence or absence of CBX, mice were immunized with the CA09 H1N1 vaccine and PS-GAMP. 24 hours later, lung CD11b + DCs were counted. n = 4. (F and G) Mice receiving the indicated gap junction inhibitor were immunized as described in (D) and challenged with 10×LD 50 CA09 H1N1 virus 2 hours later. GB + CD8 + T cells in BALF (F) and lung (G) were analyzed by flow cytometry. n = 4. (H) Schematic diagram of generating chimeric mice. Mice were subjected to lethal irradiation before bone marrow (BM) cell transfer. Chimerism was confirmed 3 months later (Figure 32), and the chimeras were immunized and challenged as described in (F). 4 days after challenge, GB + CD8 + T cells in BALF (I) and lung (J) were counted by flow cytometry and the change in body weight relative to day 0 (K) was measured, and the lung virus titer (L) was determined in these mice. n = 4 - 7. (M and N) The correlation between the number of GB + CD8 + T cells and the virus titer was determined by regression analysis. Results are expressed as mean ± SEM. Each symbol represents an individual mouse. Statistical analysis was one-way ANOVA for (D, F, G, and I - L) and 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.

[0039] Figure 6A - 6O. PS-GAMP enhances cross-protection against heterologous subtype influenza A viruses. (A to H) Mice were immunized intranasally with the CA09 H1N1 vaccine and challenged 2 days (first row of figures) or 2 weeks (second row of figures) later with 5×LD 50 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). For (A to F), n = 6 - 7; for (G and H), n = 8 - 13. (I) Mice were immunized as described in (A) and challenged 2 days later with 10×LD 50 with the oseltamivir-resistant NC09 H1N1 virus. Unimmunized mice were treated with oseltamivir (20 mg / kg / day) 6 hours before challenge and daily after virus challenge until the end of the study. Mice treated were challenged with 10×LD 50 of the CA09 H1N1 or NC09 H1N1 virus. n = 6. (J) Mice were immunized with the 2018 - 19 trivalent seasonal influenza vaccine (SIV18 - 19) alone or together with PS-GAMP and challenged 1 month later with 5×LD 50 with the mismatched GZ89 H3N2 virus. n = 6 - 12. (K) Mice were immunized with the CA09 H1N1 vaccine alone or together with PS-GAMP and challenged 6 months later with 5×LD 50 with the heterologous subtype rgVN04 H5N1 virus. Optionally, mice were infected with 1×LD 50 of the PR8H1N1 virus and challenged 6 months later with 5×LD 50 of the rgVN04 H5N1 virus again for comparison (pre-infected) in mice that survived the infection. n = 6 - 7. (L to O) Ferrets were immunized intranasally with the inactivated Perth H3N2 vaccine (15 μg) with or without PS-GAMP (200 μg). 30 days after immunization, they were challenged with 10 6 TCID 50Ferrets were challenged with a heterologous subtype Michigan15 H1N1 virus. Body weight (L), disease score (M), temperature (N), and viral 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. Experiments with mice were repeated twice with similar results. For ferrets, * indicates significance between PBS and vaccine + PS-GAMP, # indicates significance between vaccine and vaccine + PS-GAMP. *, #p<0.05; **, ##p<0.01; and ***, p<0.001. For statistical analysis, two-way ANOVA was used for (L, N, and O), Kruskal-Wallis test for (M), and chronometric test for (A to K).

[0040] Figure 7A -7K. Preparation and characterization of PS-GAMP. (A) Schematic of PS-GAMP preparation. Liposomes were synthesized based on mammalian PS components, which typically consist of 90% lipid and 10% protein and are evolutionarily conserved. Lipids contain 8-10% cholesterol, 60-70% zwitterionic phosphatidylcholine (PC), mainly dipalmitoylphosphatidylcholine (DPPC), up to 8-15% anionic phosphatidylglycerol (DPPG), and a relatively small fraction of other lipids (17). PEG2000 was used in place of hydrophilic protein and cationic DPTAP in nano3 and nano5 in place of DPPG to determine the importance of charge. By the reverse-phase evaporation method detailed in Materials and Methods, these PS lipids and PEG2000 form liposomes with a single lipid bilayer encapsulating cGAMP. (B to E) Swiss Webster mice were immunized intranasally with VN04 H5N1 vaccine (1 μg HA content) plus 10 μg 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 area under the curve (AUC) was calculated from (D) by PRISM software (E). n = 5. The size (F), encapsulation efficiency (G), and zeta potential (J) of the indicated liposomes were determined. (H and I) Free cGAMP or liposomes encapsulating cGAMP were cultured with BMDC (H) and BMM (I) at a final cGAMP concentration of 10 μg / ml for 8 hours, followed by determination of IFN-β (Ifnb1) 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 measured as described above 2 weeks later. n = 4. Results are expressed as mean ± SEM. Each symbol represents an individual mouse in (B, C, E, and K), or an independent replicate in (G, H, and I). For statistical analysis, one-way ANOVA was used for (B, C, E, and H-K), and two-way ANOVA was used for (D). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the group of vaccine alone or the designated group. All experiments were repeated twice with similar results.

[0041] Figure 8A - 8F . Kinetics of nanoparticle uptake in different tissues. Mice were administered PBS, free SRB, SRB- and DiD-labeled nano4 (Nano4-SRB) encapsulated, or SRB- and DiD-labeled nano5 (Nano5-SRB) encapsulated intranasally and analyzed by flow cytometry at different times. (A) SRB in the brain (upper panel), nasal tissue (middle panel), and MLN (lower panel) + Typical flow cytometry plots of cells. Data represent 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. As Figure 9A - 9D described, gating was set for alveolar macrophages (AM), interstitial macrophages (IM), CD11b + DC, and CD11b - DC. (D-F) SRB in the 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. For statistical analysis, two-way ANOVA was used for (B-F). *p < 0.05, **p < 0.01, and ***p < 0.001. The experiment was repeated twice with similar results.

[0042] Figure 9A - 9D . Gating strategy for flow cytometry analysis of cells isolated from the designated tissues. (A) NK cells were identified by NK1.1 + and CD3 - in lung cells, and CD3 + cells were divided into CD4 + and CD8 + T cells. Lung CD11c - cells were divided into neutrophils such as CD11b + Ly6C + Ly6G + , whereas inflammatory monocytes were identified as CD11b+ Ly6C hi Ly6G - 。Based on gating on CD11c + cells, four populations were discriminated by CD24 and CD11b markers, where AM was CD24 - CD11b - Siglec F + and IM was CD24 - CD11b + , CD24 + CD11b - DC was CD103 + MHC II + , and tissue-resident CD24 + CD11b + DC also expressed MHCII but not CD103. (B) During influenza virus infection or after PS-GAMP administration, CD11b + DC could be divided into monocyte-derived DC (Mono-DC) or tissue-resident-like DC (tDC). Mono-DC was Ly6C hi , and MHC II expression varied with their activation status. On the other hand, tDC was Ly6C lo MHC II hi . (C and D) Gating strategies for CD11b + DC and CD11b - DC in the MLN (C) or DC in the nasal tissue (D). Gating strategies for T cells, NK cells, neutrophils, and monocytes in the MLN, nasal tissue, and brain were similar to those in the lung (A).

[0043] Figure 10A - 10B . Analysis of cells capturing PS-liposomes in the lung. (A) Mice were intranasally administered the prepared nano4-SRB as shown in Fig. 1A. After 12 hours, CD11c + SRB + cells were mainly characterized as CD24 - CD11b - AM, and CD11c - SRB + cells were mainly EpCAM + CD11b - AEC, which was also positive for MHC II. (B) In mice receiving nano4-SRB, direct nanoparticle uptake (SRB + DC, and CD11b + DC) was analyzed for AM, CD103 + DiD+ )。Approximately half of the AMs that internalized nano4-SRB were SRB + DiD + , whereas DCs rarely captured the nanoparticles. Results are expressed as mean ± SEM. n = 4 mice. The experiment was repeated twice with similar results.

[0044] Figure 11A - 11C . Nano4 delivered cGAMP to AMs. (A) Schematic diagrams of DiD-labeled empty PS-mimicking nanoparticles (DiD-PS) and PS-mimicking nanoparticles encapsulating cGAMP (DiD-PS-GAMP). (B) Intranasal inoculation with DiD-PS or DiD-PS-GAMP (20 μg cGAMP). In mice receiving DiD-PS (red) or DiD-PS-GAMP (blue), DiD was analyzed in lung cells by flow cytometry 12 or 36 hours later + CD11c + cells. CD40 expression was also evaluated in these cells to verify STING activation in the cells. Representative histograms of CD40 expression are given in the middle, and the mean fluorescence intensity (MFI) is summarized in the right panel. n = 4. (C) DiD-expressing CD40 + (blue) or DiD - AMs (red) were analyzed similarly to (B). n = 4. Results are expressed as mean ± SEM. Each symbol represents an individual mouse in the right panels of B and C. For statistical analysis, a t-test was used 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.

[0045] Figure 12 . In in vitro culture, positively charged nano5 was captured by PS. DiD-labeled Nano4 or nano5 was incubated with PS for 30 minutes. Nanoparticle aggregates on PS were visualized by confocal microscopy. The regions outlined in the second column of images are magnified on the right. BF, bright field. Scale bars, 100 μm in the first and second columns of images and 10 μm in the third and fourth columns of images. Data represent 10 similar results from two separate experiments.

[0046] Figure 13A - 13C. AM takes up Nano4 isolated from non - human primates (NHP). AM and PS are isolated from rhesus macaques. (A) DiD - nano4 or DiD - nano5 is cultured with rhesus macaque PS for 30 minutes. Nano5, rather than nano4, aggregates on PS and is visualized by confocal microscopy. The outlined area is magnified in the corresponding figure on the right. Scale bar, 10 μm. Data represent 5 similar results. (B) Monkey AM is isolated and cultured with DiD - nano4 (upper panel) or DiD - nano5 (lower panel) pretreated with or without nanoparticles for 3 hours and imaged by fluorescence microscopy. Live cells are stained with Calcein - AM and nuclei are stained with Hoechst. Scale bar, 50 μm. The DiD fluorescence intensity in cells is quantified by Image J (C). n = 221 - 275. Each symbol represents an individual cell. Results are expressed as mean ± SEM. Statistical analysis, one - way ANOVA for (C). Between the designated groups, ***p < 0.001.

[0047] Figure 14 . AM captures nano4 in the lung. At 12 hours after intranasal administration of DiD - nano4 to mice, lungs are collected and stained with the AM - specific marker Siglec F for cryosections and visualized by fluorescence microscopy. Scale bar, 30 μm. The square in the second figure is magnified in the figure on the right. Data represent 6 similar results in two separate experiments.

[0048] Figure 15 . TEM of nanoparticle distribution in the lung. As Figure 7A shown, nanogold is encapsulated in nano4 (nano4 - gold) and nano5 (nano5 - gold). Mice are administered nanoparticles intranasally. Lungs are collected 6 hours later and prepared for TEM. Note: nano4 - gold is captured in the cytoplasmic vesicles of AM (red arrows), and some nanogold is observed in the cytoplasmic vesicles (hollow red arrows, lower panel). In contrast, nano5 - gold is mainly present on the surface of alveoli (blue arrows). The area outlined in the upper figure is magnified in the corresponding lower figure. Scale bar, 2 μm for the upper figure and 500 nm for the lower figure.

[0049] Figure 16 . In nano4 uptake, from Sftpa1 / Sftpd - / -The AM of mice has a similar ability to WT AM. SP-A / D are large hydrophilic proteins and are established as the first line of innate defense. These two collectins can integrate with PS-coated bacteria, viruses, cell debris, apoptotic cells, and various nanoparticles to facilitate their endocytosis or phagocytosis by AM (20). To test whether this might be the mechanism for nano4 uptake by AM, AM were isolated from WT or Sftpa1 / Sftpd - / - mice and cultured with DiD-nano4 pretreated with WT PS for 30 minutes. DiD fluorescence in the cells was captured by confocal microscopy and quantified by Image J software. In the presence of WT PS, AM from Sftpa1 / Sftpd - / - mice were found to take up nano4 as effectively as WT AM( Figure 16 ), but not in the presence of SP-A / D-deficient PS (Figure 11). n = 25 - 32. Each symbol represents a single cell. Statistical analysis, one-way ANOVA. *p < 0.05 and **p < 0.01 between the designated groups, ns, not significant. The experiment was repeated twice with similar results.

[0050] Figure 17A - 17C .PS-GAMP does not induce significant inflammation in the lungs, nose, and central nervous system (CNS). Swiss Webster mice were administered intranasally with PBS, PS-GAMP, VN04 H5N1 vaccine, or vaccine plus PS-GAMP or CT. (A) Histological examination of the lungs (first and second row figures), nose (third and fourth row figures), and CNS (fifth and sixth row figures) 2 days after immunization. The alveoli and bronchi of the lungs, nasal-associated lymphoid tissue of the nose, and olfactory bulb region of the brain tissue were outlined by dashed rectangles and magnified in the corresponding lower figures. The olfactory bulb region of the brain tissue is directly connected to the olfactory nerves in the nasal cavity. Data represent two separate experiments examined in triplicate each. Scale bars for the lungs and nose, 400 μm in the upper figures and 100 μm in the lower figures. Scale bar for the CNS, 800 μm in the upper figure and 200 μm in the lower figure. (B) Eosinophil infiltrates, epithelial damage, and necrosis of each mouse were analyzed as previously reported (48). Numbers represent the number of mice with (+) or without (-) eosinophil infiltrates, epithelial damage, and necrosis. n = 6 mice. (C) 2 days after immunization, the expression of the designated cytokines and chemokines in the CNS of mice receiving the 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 a single mouse. Results are expressed as mean ± SEM.

[0051] Figure 18A - 18V. Changes in inflammatory 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 infected with 1×LD 50 of CA09 H1N1 influenza virus (red). At the indicated days post-infection or post-immunization (d.p.i.), neutrophils, NK, CD4 + , and CD8 + T cells, monocytes, and CD11b + and CD11b - DC in the lungs (A to G) or MLN (H to N) were analyzed by flow cytometry. Neutrophils, NK, CD4 + , and CD8 + T cells, monocytes, and DC (O to T) in nasal tissues or neutrophils and monocytes (U and V) in the brain were analyzed similarly. n = 4. Results are presented as mean ± SEM. Statistical analysis, one-way ANOVA. Compared with day 0, *p < 0.05, **p < 0.01, and ***p < 0.001. The experiment was repeated twice with similar results.

[0052] Figure 19A - 19C . In contrast to viral infection, PS-GAMP does not induce significant inflammation in the lungs. Mice were intranasally immunized with CA09 H1N1 vaccine plus 20 μg of PS-GAMP (A), or infected with 1×LD 50 of CA09H1N1 influenza virus (B). Lungs were analyzed by H&E staining at the indicated days post-immunization or post-infection. Data represent two separate experiments each tested in triplicate. Scale bar, 400 μm for the upper panels and 100 μm for the lower panels. (C) Lung inflammation was quantified according to the scoring system shown on the right (49). n = 6. Results are presented as mean ± SEM. By non-parametric test, compared with day 0, *p < 0.05 and ***p < 0.001.

[0053] Figure 20 . PS-GAMP induces transient production of immune mediators in the lungs. Mice were intranasally administered with 20 μg of PS-GAMP (blue), or infected with 1×LD 50 of CA09 H1N1 influenza virus (red). The mRNA levels of the indicated mediators were determined by real-time RT-PCR at each time point and normalized to untreated mice. n = 4. Results are presented as mean ± SEM. Statistical analysis, one-way ANOVA. Compared with day 0, *p < 0.05, **p < 0.01, and ***p < 0.001. The experiment was repeated twice with similar results.

[0054] Figure 21A - 21C . PS-GAMP transiently increases IFN-β protein in BALF. Mice were intranasally administered 20 μg of PS-GAMP (blue), or infected with 1×LD 50 CA09 H1N1 influenza virus (red). Protein levels of IFN-β (A), TNF-α (B), and IL-10 (C) in BALF were measured by ELISA at each time point. 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.

[0055] Figure 22A - 22G . PS-GAMP does not systemically induce any inflammation. Mice were intranasally immunized 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.

[0056] Figure 23A - 23D . PS-GAMP increases the number of CD11b + DCs that uptake extracellular Ag in the lung and MLN. (A) Mice were intranasally vaccinated with OVA-AF647 with or without 20 μg of PS-GAMP. Lung CD11c + cells capturing OVA 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 related to cell activation. After 36 hours, OVA uptake was analyzed based on gating of DCs prepared from MLN, revealing that OVA + DCs were mainly CD11b + DCs. (C) As shown by fewer CD11c + DiD + cells when mice were intranasally administered 20 μg of DiD-PS-GAMP and analyzed similarly, DCs do not directly uptake PS-GAMP in the MLN. (D) DiD + cells were also tracked in the MLN from 0 to 60 hours after PS-GAMP administration. n = 4. Results are expressed as mean ± SEM. The experiment was repeated twice with similar results.

[0057] Figure 24A - 24E. PS-GAMP does not enhance in vivo Ag uptake or processing. 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 the BODIPY fluorophore (DQ) and remains self-quenched until the OVA protein is proteolytically processed to generate DQ-green fluorescence, which is commonly used to evaluate Ag processing. For this purpose, mice were intranasally administered PBS (gray), or AF647-OVA and DQ-OVA together, in the presence (red) or absence (blue) of PS-GAMP, and euthanized after 24 h for flow cytometry analysis (A). AF647-OVA was analyzed based on gating of CD11b + DCs, which was further quantified for OVA cleavage based on DQ-green fluorescence. The percentage and number of AF647 + CD11b + DCs are summarized in (B) and (C). The AF647 and DQ-green MFIs in these cells are presented in (D) or (E), respectively. Each symbol represents an individual mouse in B–E. Results are expressed as mean ± SEM. Statistical analysis was one-way ANOVA for (B–E). In the presence or absence of PS-GAMP, *p < 0.05, **p < 0.01, and ***p < 0.001. ns, not significant. The experiment was repeated twice with similar results. Note: There was no difference in the DQ-green fluorescence or OVA MFI in CD11b + CD11b + DCs with or without PS-GAMP (D and E). However, in the presence of PS-GAMP, the percentage and number of CD11b + DCs positive for OVA were significantly increased (B and C), which was mainly attributed to the increased number of CD11b + DCs secondary to the increased immune mediators induced by PS-GAMP.

[0058] Figure 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, carboxyfluorescein succinimidyl ester (CFSE)-labeled OT-I cells were transferred to the vaccinated mice. Three days after cell transfer, the lungs and MLN were harvested. (B) Ag-specific proliferation of OT-I cells was analyzed by the progressive decrease in CFSE fluorescence. Inset in the first two panels (PBS and OVA): a reduced scale of the y-axis to show the CFSE decrease. Cells with high division (≥6, hi) were gated. The numbers of highly divided cells in the lungs (C) and MLN (D) were summarized. n = 4-6. Each symbol represents an individual mouse in (C) and (D). For statistical analysis, one-way ANOVA was used for (C and D). Results are shown as mean ± SEM. In the presence or absence of PS-GAMP, **p < 0.01 and ***p < 0.001. The experiment was repeated twice with similar results.

[0059] Figure 26A - 26C . CD8 + T cell responses in the spleen, lungs, and MLN. C57BL / 6 mice were immunized intranasally with the CA09 H1N1 vaccine plus 20 μg of PS-GAMP. Mice receiving PBS served as controls. (A) Spleen cells were isolated 7 days after immunization and stimulated with the CA09H1N1 vaccine. Representative cell count profiles of CD4 + and CD8 + T cells producing IFN-γ are shown. (B) Four days after immunization, representative cell count profiles of NP 366-374 + CD8 + T cells in the lungs are shown. (C) The percentages of PA + CD8 + T cells were determined based on gating of CD3 224-233 (blue) or PB1 703-711 (red) positive cells. Each panel represents 4 similar results from the same group. n = 4. Data are shown as mean ± SEM. The experiment was repeated twice with similar results.

[0060] Figure 27A - 27C . Early virus-specific GB + CD8 + T cells and BALF antibodies. (A) C57BL / 6 mice were either left unimmunized, or immunized with the CA09 H1N1 vaccine alone or the vaccine plus 20 μg of PS-GAMP, and then challenged with 10 × LD 50 CA09 H1N1 virus 2 days later. Lungs were harvested 4 days after infection. (B) Based on gating of GB + CD8 +Gating of T cells to obtain NPs 366-374 Tetramers (blue), PB1 703-711 (red), or PA 224-233 (green) positive cell percentages. Parallel analysis of CD8 + T cells from unimmunized / unchallenged mice as negative control (gray). n = 3. (C) Six days after immunization, Ag-specific IgA and IgM titers were analyzed in BALF. n = 4. Data are represented as mean ± SEM. The experiments were repeated twice with similar results.

[0061] Figure 28A - 28I . Figure 4A - 4J Supplementary data. (A) Schematic of vaccination and virus challenge schedule. (B to F) Body weight changes (B, C, E, and F) or survival (D) of mice corresponding to those described in Fig. 4A - 4E, respectively. (G and H) Mice were immunized intranasally with either the H7-Re1 H7N9 vaccine alone or in combination with 20 μg of PS-GAMP or poly IC and challenged with a clinically isolated SH13 H7N9 virus 14 days later. n = 10 - 13. (I) Body weight changes of the mice described in Fig. 4F. Statistical analysis was 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.

[0062] Figure 29A - 29B . In vivo SRB + AM to SRB + Inverse correlation of AEC over time, while DiD + AM percentage remained unchanged. SRB-DiD-nano4 was inoculated intranasally into mice. (A) After inoculation, SRB + AM and SRB + Percentage change of AEC relative to the total number of lung cells was tracked over time. n = 4. (B) DiD + AM was analyzed by flow cytometry 12 and 18 hours after nanoparticle administration. n = 4. Results are represented as mean ± SEM. Statistical analysis was a t-test. *p < 0.05 and **p < 0.01 when comparing between 18 and 12 hours. All experiments were repeated twice with similar results.

[0063] Figure 30A - 30D. cGAMP enters AECs from AMs. (A) Mice were intraperitoneally injected with the gap junction inhibitor CBX or PBS for 3 consecutive days, and then 20 μg of PS-GAMP was administered intranasally. After 12 hours, AMs and AECs were sorted and analyzed for Ifnb1 (B) and Gmcsf (C) expression by real-time RT-PCR. The 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–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.

[0064] Figure 31A - 31E . Tissue and cell distribution of poly IC. Mice received 20 μg of rhodamine-labeled poly IC intranasally. (A) After 12 hours, the lungs were dissected and digested for flow cytometry analysis of poly IC uptake by the CD11c + and CD11c - subsets. CD11c + poly IC + cells were further confirmed as CD24 - CD11b - AMs. Then, poly IC uptake was analyzed based on gating for EpCAM + AECs (B) or CD11c + CD24 + DCs (C). MLNs (D) and nasal epithelial and lymphoid tissues (E) were also prepared as single-cell suspensions to determine poly IC uptake. Data represent two separate experiments tested in triplicate each.

[0065] Figure 32A - 32B . Cell reconstitution efficacy after bone marrow (BM) cell transfer. Mice were pretreated with lethal irradiation before infusion with BM cells isolated from mice carrying reciprocal CD45 alleles CD45.1 and CD45.2 (surface biomarkers of all white blood cells). Donor cells were distinguished from recipients by specific antibodies for CD45.1 or CD45.2. After 3 months of infusion, the transfer efficacy was analyzed (A) and summarized in (B) by quantifying CD45.1 or CD45.2 expression on white blood cells in different tissues in the recipients. Each symbol represents an individual mouse in (B). n = 5–7. The experiment was repeated twice with similar results.

[0066] Figure 33A - 33K. Supplementary data for cross - protection studies. (A to K) correspond to the body weight changes of the mice described in Figures 6A - 6K. Results are presented 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.

[0067] Figure 34A - 34B . Vaccination with trivalent seasonal influenza vaccine and PS - GAMP induces cross - protective immunity against mismatched influenza B virus. (A) Schematic of the vaccination / sampling schedule. BALB / c mice were immunized with either the trivalent seasonal influenza vaccine (2018 - 19) (SIV) alone or in combination with 20 μg of PS - GAMP and challenged with 4 × 10 5 TCID 50 mismatched Florida06 B virus 1 month later. (B) Lungs were isolated 4 days after immunization and analyzed for virus titers. Each symbol represents an individual mouse in (B). n = 8 - 9. Results are presented as mean ± SEM. Statistical analysis, one - way ANOVA. **p < 0.01 in the presence or absence of PS - GAMP. ns, not significant. Experiments were repeated twice with similar results.

[0068] Figure 35A - 35E . PS - GAMP / inactivated influenza vaccine induces virus - specific lung CD8 + T RM cells. (A) Schematic of the vaccination / sampling timeline of the OT - I mouse model. Mice were transferred with OT - I cells and 1 hour later, immunized intranasally with OVA in the presence or absence of PS - GAMP. 35 days later, mice were injected intravenously with anti - CD8β antibody to deplete circulating CD8 + T cells and then sacrificed for flow cytometry analysis. (B) Total CD8 + and CD8α + were gated on total CD8 + T cells in the lung (not shown in the figure), and lung OT - I cells were identified as CD45.2 + and CD8β - (after antibody intravenous injection) (first two panels). OT - I cells with a T RM phenotype were identified as CD103 + CD69 + CD49a + . OT - I cells in the spleen were used as controls (gray). Lung OT - I T RMThe number of cells was summarized in (C). n = 6. (D and E) Mice were immunized intranasally with the CA09 H1N1 vaccine in the presence or absence of PS-GAMP. Lungs were isolated 6 months later for flow cytometry. For NP 366-374 + CD8 + T cells were gated and CD103 and CD69 expression was verified (D), and for NP 366-374 + CD8 + T RM cell numbers were summarized in (E). n = 4. NP in the spleen 366-374 + CD8 + T cells were used as controls. Results are expressed as mean ± SEM. For statistical analysis, a t-test was used 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.

[0069] Figure 36A - 36D . FTY720 does not affect cross-protection induced by influenza vaccine / PS-GAMP. (A and B) Mice were immunized intranasally with the CA09 H1N1 vaccine alone or together with 20 μg of PS-GAMP and challenged with 5 × LD 50 GZ89 H3N2 virus 1 month later. (C and D) Mice were immunized and challenged as described in A and B except that from -2 to 14 days post-challenge, the mice additionally received daily injections of FTY720 (1 mg / kg / day). n = 6 - 8. For statistical analysis, two-way ANOVA was used for (B and D) and a time-course test for (A and C). *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the vaccine alone. All experiments were repeated twice with similar results.

[0070] Figure 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 body weight (A) and temperature (B) of the animals were monitored for 6 days. (C) Serum was collected 4 weeks post-immunization and tested for Perth H3N2-specific IgG titers (C). HAI titers were also determined against the Perth H3N2 (D) or Michigan H1N1 (E) virus strains. n = 4. Each symbol represents an individual animal in C to E. Results are expressed as mean ± SEM. For statistical analysis, one-way ANOVA was used for (C and D). **p < 0.01 in the comparison of the presence vs absence of PS-GAMP. Detailed Description

[0071] The cGAS-cGAMP-STING pathway is an important immune surveillance pathway that is activated in the presence of cytoplasmic DNA, for example due to microbial infections or pathophysiological conditions including cancer and autoimmune disorders. Cyclic GMP-AMP synthase (cGAS) belongs to the nucleotidyltransferase family and is a universal DNA sensor that is activated upon binding to cytosolic dsDNA to produce the signaling molecule cyclic GMP-AMP (or 2'-3'-cGAMP or cyclic guanosine monophosphate-adenosine monophosphate). As a second messenger during microbial infections, 2'-3'-cGAMP binds and activates STING, leading to the production of type I interferons (IFNs) and other co-stimulatory molecules that trigger an immune response. In addition to its role in infectious diseases, the cGAS / STING pathway has emerged as a promising new target for autoimmune diseases and cancer immunotherapy. It has been 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 strong and beneficial immune response against cancer cells. In an additional context, dysregulation of the cGAS / STING pathway is involved in self-DNA-induced inflammation and autoimmune disorders such as systemic lupus erythematosus (SLE) and Aicardi-Goutieres syndrome.

[0072] Despite decades of research, effective mucosal adjuvants remain lacking. 2'-3'-cGMP-AMP (cGAMP), a natural agonist of stimulator of interferon genes (STING), is a second messenger produced in response to DNA virus infection or tissue damage (10,11). It stimulates the production of type I interferons (IFN-I), which helps determine the strength of the T-helper 1 (Th1) immune response, particularly those of CD8 + T cells (12,13). STING agonists are potent adjuvants that can trigger a strong anti-tumor immunity and enhance intradermal influenza vaccines (13,14) after intratumoral administration. 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 robust barrier that separates the external air from the inner alveolar epithelium in the alveoli and prevents nanoparticles and hydrophilic molecules from entering AECs (15,16).

[0073] There is a high expectation to develop a "universal" influenza vaccine that confers protection not only against variants within a subtype but also against other subtypes of influenza viruses. However, it remains unclear whether such a universal influenza vaccine is achievable. It has long been recognized in human and animal models that viral infection can stimulate heterosubtypic immunity mediated mainly by CD8 + T cells (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 cells present in the lung. This is mainly due to the fact that the PS-GAMP-adjuvanted influenza vaccine stimulates immunity induced by viral infection, characterized by AEC activation, rapid CD11b + DC recruitment and differentiation, and a robust CD8 + T cell response in the respiratory system. PS-GAMP is a stand-alone adjuvant that is compatible not only with inactivated influenza virus vaccines but also with other vaccines such as vaccines including cocktails of multiple B and T cell epitopes or influenza vaccine subunits. The ability of PS-GAMP to enhance the capacity of non-replicating influenza vaccines for strong heterosubtypic immunity makes it a promising adjuvant for a "universal" influenza vaccine if its efficacy is demonstrated in humans. As such, it can confer significant advantages compared to "replicating" vaccines.

[0074] Unlike conventional vaccine adjuvants that primarily target APCs, PS-GAMP activates both AMs and AECs; without wishing to be bound by theory, AEC activation seems crucial for adjuvanticity, as blockade of gap junctions and STING deficiency in AECs significantly attenuate adjuvanticity, whereas STING deficiency in myeloid cells does not. The key role of AECs in coordinating innate and adaptive immune responses, as compared to AMs, is consistent with that 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 following integration of SP-A and SP-D into the PS-bionic liposomes, which is not feasible in any non-PS-bionic liposomes (39-41). Additionally, the adjuvant can induce robust protection within just 2 days after immunization, in stark contrast to existing influenza vaccines that require at least 10-14 days to become effective. Early cross-protection is highly important for protecting first responders and high-risk individuals, especially when antiviral-resistant viruses or highly pathogenic viruses such as H5N1 and H7N9 viruses emerge as pandemics. Since viral spread can accelerate exponentially after an epidemic expands into a pandemic, early protection during the epidemic will be the most effective means of limiting viral spread and minimizing or preventing the epidemic from becoming a pandemic, saving millions of lives (42).

[0075] Pulmonary surfactant (PS)-bionic nanoparticles

[0076] The present disclosure provides a composition comprising nanoparticles having an average size of 200-400 nm. The nanoparticles comprise a plurality of pulmonary surfactant-bionic molecules, wherein the nanoparticles are negatively charged; and one or more cargo molecules encapsulated by the nanoparticles, wherein the cargo molecules have a molecular weight of 1200 Da or less.

[0077] The present disclosure provides a method of 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 an antigen to the subject.

[0078] The present disclosure provides a method of treating a subject having influenza. The method comprises administering to the subject a therapeutically effective amount of a composition as described herein; and administering an antigen to the subject. In some embodiments, the cargo molecule is cGAMP and the antigen is an influenza vaccine.

[0079] 1. The present disclosure provides methods of treating a subject having an airway disease. The methods include 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); 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)); an agonist or antagonist of a receptor (e.g., chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), chemokine receptor 2 (CCR2)); an agonist or antagonist of an ion channel (e.g., GABA receptor, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or voltage-gated sodium channel); an inducer of IFN-α; a long-acting muscarinic antagonist / anticholinergic (LAMA); an inhibitor against IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; a CXCR2 antagonist; a molecule that blocks a proinflammatory cytokine (e.g., TNF-α, TNF-β, or IL-6); a molecule that blocks IL-17 / T H 17; a macrolide; a molecule that activates HDAC2; a STAT6 inhibitor (e.g., AS1517499); an antiviral small molecule drug (e.g., oseltamivir (Tamiflu), Relenza, or zanamivir); and / or favipiravir (T705).

[0080] The present disclosure provides 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.

[0081] Compared to a similar method without using the PS-bionic nanoparticles, 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 molecule as described herein.

[0082] nanoparticle

[0083] In some embodiments, the nanoparticles are liposomes, vesicles, emulsions, or micelles.

[0084] In some embodiments, the nanoparticles may comprise more than one surfactant, including detergents, wetting agents, emulsifying agents, foaming agents, or dispersing agents. In some embodiments, the surfactant comprises at least one hydrophobic end and / or at least one hydrophilic end. In some embodiments, the surfactant is positively charged, neutral, or negatively charged.

[0085] In some embodiments, the surfactant is a lipid. In some embodiments, the surfactant is a phospholipid. In some embodiments, the nanoparticles may comprise more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 layers of surfactant. In some embodiments, the nanoparticles are water-in-oil-in-water emulsions.

[0086] In some embodiments, the percentage of surfactant in the nanoparticles can range 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%, 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 wt%, about 1 wt%, about 2 wt%, about 3 wt%, about 4 wt%, about 5 wt%, about 10 wt%, about 15 wt%, about 20 wt%, about 25 wt%, about 30 wt%, about 35 wt%, about 40 wt%, about 45 wt%, about 50 wt%, about 55 wt%, about 60 wt%, about 65 wt%, about 70 wt%, about 75 wt%, about 80 wt%, about 85 wt%, about 90 wt%, about 95 wt%, or about 100 wt%.

[0087] In some embodiments, the nanoparticles as described herein can have an average size as follows: from 200 nm to 210 nm, from 210 nm to 220 nm, from 220 nm to 230 nm, from 230 nm to 240 nm, from 240 nm to 250 nm, from 250 nm to 260 nm, from 260 nm to 270 nm, from 270 to 280 nm, from 280 nm to 290 nm, from 290 nm to 300 nm, from 300 nm to 310 nm, from 310 nm to 320 nm, from 320 nm to 330 nm, from 330 nm to 340 nm, from 340 nm to 350 nm, from 350 nm to 360 nm, from 360 nm to 370 nm, from 370 nm to 380 nm, from 380 nm to 390 nm, or from 390 nm to 400 nm.

[0088] Pulmonary surfactant (PS)

[0089] Pulmonary surfactant is a surface-active lipoprotein complex (phospholipoprotein) formed by type II alveolar cells. The proteins and lipids that make up the surfactant have both hydrophilic and hydrophobic regions. By adsorbing to the air-water interface of the alveoli, where the hydrophilic head groups are in water and the hydrophobic tails face the air, the major lipid component of the surfactant, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), reduces surface tension.

[0090] 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 dipalmitoyl phosphatidylcholine (DPPC), up to 8-15% anionic phosphatidylglycerol (DPPG), and a relatively small fraction of other lipids (17).

[0091] Pulmonary surfactant (PS)-biomimetic nanoparticles

[0092] In some embodiments, the PS-bionic nanoparticles can be nanoparticles comprising a variety of PS-bionic molecules, including but not limited to, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate-(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 lysophospholipid.

[0093] In some embodiments, the PS-bionic molecule is a lipid, protein, lipoprotein, phospholipid, or phosphoprotein.

[0094] In some embodiments, the PS-bionic molecule is a domain, moiety, portion, or the entire molecule of pulmonary surfactant. In some embodiments, the PS-bionic molecule is a natural product. In some embodiments, the PS-bionic molecule is synthetic.

[0095] In some embodiments, the PS-bionic molecule is positively charged, neutral, or negatively charged. In some embodiments, the PS-bionic molecule has at least one hydrophobic terminus and / or at least one hydrophilic terminus.

[0096] In some embodiments, the PS-bionic molecule comprises more than one fatty acid group or its salt, and / or more than one head group. In some embodiments, the fatty acid group may comprise a digestible, long-chain (e.g., C8-C50), substituted or unsubstituted hydrocarbon. 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 in the cis conformation. In some embodiments, the double bond of the unsaturated fatty acid may be in the trans conformation. In some embodiments, the fatty acid group is palmitic acid. In some embodiments, the head group is phosphatidylcholine.

[0097] Cargo molecules of the PS-bionic nanoparticles

[0098] Cargo molecules that can be carried in the nanoparticles as described herein may include those having a therapeutic or prophylactic effect on cells of the lung, such as alveolar epithelial cells (AEC) and / or alveolar macrophages (AM). Examples include reagents that enhance the immune response to a co-administered antigen (immunostimulants), such as adjuvants to stimulate the immune response; reagents that block signal pathways associated with inflammation (anti-inflammatory drugs or immunosuppressants), such as particularly for inhibiting inflammation-related lung diseases, including allergy, asthma, and chronic obstructive pulmonary disease (COPD); and anti-cancer drugs, including chemotherapeutic agents. The cargo molecules may be completely encapsulated by the PS (e.g., contained within the PS membrane forming the outer surface of the nanoparticle), may be mixed into the PS (e.g., in solid nanoparticles), or may be on / in / attached to the membrane.

[0099] In some embodiments, cargo molecules can be transferred through gap junctions present between AM and AEC and are limited to those small molecules 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 in the range of from 10 Da to 1200 Da, from 50 Da to 1200 Da, from 100 Da to 1200 Da, from 200 Da to 1200 Da, from 300 Da to 1200 Da, from 400 Da to 1200 Da, from 500 Da to 1200 Da, from 600 Da to 1200 Da, from 700 Da to 1200 Da, from 800 Da to 1200 Da, from 900 Da to 1200 Da, from 1000 Da to 1200 Da, or from 1100 Da to 1200 Da. In some embodiments, cargo molecules can have a molecular weight in the range of 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, cargo molecules can 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.

[0100] In some embodiments, the cargo molecule can be an immunostimulant (used as an adjuvant), e.g., stimulator of interferon genes (STING) agonists (e.g., cGAMP, CDN, MK-1454, ADU-S100, E7766); agonists for intracellular Toll-like receptors including TLR3, TLR7, TLR8, or TLR9 (e.g., imiquimod, resiquimod (R848), imidazoquinoline (IMQ), motolimod, CU-CPT4a, IPH-3102, or ampratide); and / or agonists for Nodinitib (NOD1), NOD2, NLPR3 or NPLRC3 (e.g., muramyl dipeptide (MDP), FK565, or FK156).

[0101] In some embodiments, the cargo molecule can be an anti-inflammatory agent for airway diseases (e.g., asthma, chronic obstructive pulmonary disease (COPD), or allergy), 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)); an agonist or antagonist of a receptor (e.g., chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), chemokine receptor 2 (CCR2)); an agonist or antagonist of an ion channel (e.g., GABA receptor, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or voltage-gated sodium channel); an inducer of IFN-α; a long-acting muscarinic antagonist / anticholinergic (LAMA); an inhibitor against IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; a CXCR2 antagonist; a molecule that blocks a pro-inflammatory cytokine (e.g., TNF-α, TNF-β, or IL-6); a molecule that blocks IL-17 / TH17; a macrolide; a molecule that activates HDAC2; and / or a STAT6 inhibitor (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.

[0102] In some embodiments, the cargo molecule can be a small molecule antiviral agent for treating pulmonary viral infections such as influenza A and B viruses, respiratory syncytial virus (RSV), rhinovirus, parainfluenza virus, or severe acute respiratory syndrome (SARS) coronavirus. Small molecule antiviral agents include oseltamivir (Tamiflu), Relenza, and zanamivir for inhibiting the neuraminidase of influenza virus; and favipiravir (T705) for treating various pulmonary viral infections.

[0103] In some embodiments, the cargo molecule is a chemotherapeutic agent for 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 carcinoma.

[0104] In some embodiments, the cargo molecule is a labeling reagent. For example, the nanoparticle can include more than one detectable moiety. For example, in addition to the cargo molecule, such as a fluorescent dye, such as carbocyanine, indocyanine, oxacarbocyanine, thuicarbocyanine, merocyanine, polymethine, coumarin, rhodamine, sulforhodamine B (SRB), xanthene, fluorescein, boron-dipyrromethene (BODIPY) dye, or derivatives thereof, including but not limited to, BODIPY FL, BODIPY R6G, BODIPY TR, BODIPY TMR, BODIPY581 / 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, HiLyteFluor 647, HiLyte Fluor 680, HiLyte Fluor 750, IR800 (ammonium perchlorate dimethyl{4-[1,5,5-tris(4-dimethylaminophenyl)-2,4-pentadienylidene]-2,5-cyclohexadien-1-yl}), IRDye 800CW, IRDye800RS, IRDye 700DX, ADS780WS, ADS830WS, ADS832WS, 1,1-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 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 the outer surface membrane or attached to the outer surface membrane).

[0105] In some embodiments, the cargo molecule is a small molecule or an antibody fragment, such as an antigen-binding fragment of an antibody.

[0106] STING agonist

[0107] The stimulator of interferon genes (STING) agonist can be any suitable agonist. In some embodiments, the STING agonist is a nucleic acid, protein, peptide, or small molecule.

[0108] In some embodiments, the STING agonist can be a nucleotide STING agonist or a non-nucleotide STING agonist.

[0109] Nucleotide STING agonists include natural cyclic dinucleotides (CDNs), e.g., cGAMP; or synthetic CDNs, e.g., the 'dithio' analogue ADU-S100 (sulfur-modified phosphodiester bond on the c-di[AMP] scaffold), or MK-1454. Non-nucleotide STING agonists include vascular disrupting agents, e.g., 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.

[0110] cGAMP

[0111] As used herein, "cGAMP", or cyclic GMP-AMP, or 2'-3'-cGMP-AMP refers to cyclic guanosine monophosphate-adenosine monophosphate.

[0112] antigen

[0113] In some embodiments, the nanoparticles comprise an antigen or are co-administered with an antigen. In some embodiments, the antigen is a viral antigen.

[0114] In some embodiments, the antigen is a respiratory syncytial virus (RSV) antigen. In some embodiments, the antigen is an 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.

[0115] 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 the proteins.

[0116] Chemotherapeutic agent

[0117] As used herein, a "chemotherapeutic agent" is a cytotoxic drug or a 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 disrupting agents (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; retinoids; 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, endoxan, chlorambucil, melphalan, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, paclitaxel, docetaxel, etoposide, teniposide, talampanel, bleomycin, carboplatin, cisplatin, oxaliplatin, all-trans retinoic acid, vinblastine, vincristine, vindesine, vinorelbine, and bevacizumab (or antigen-binding fragments thereof). Additional examples of chemotherapeutic agents are known in the art.

[0118] In some embodiments, the chemotherapeutic agent 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 carcinoma.

[0119] Method for preparing PS-bionic nanoparticles

[0120] The nanoparticles 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-phosphate-(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, at a mass ratio of about 10:1:1:1, or 5-12:0.5-1.5:0.5-1.5:0.5-1.5.

[0121] In some embodiments, more than one surfactant can be mixed in any mass ratio known in the art.

[0122] The mixture can be dissolved in chloroform, dichloromethane, trichloroethylene, methyl chloroform, or other organic solvents known in the art.

[0123] 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 higher.

[0124] In some embodiments, the concentration of cGAMP in the nanoparticle solution can 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 higher.

[0125] 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%.

[0126] Drug Compositions and Administration Methods

[0127] The methods described herein include using a pharmaceutical composition comprising the nanoparticles described herein as an active ingredient.

[0128] A pharmaceutical composition typically comprises a pharmaceutically acceptable carrier. As used herein, the phrase "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial, antiviral, and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with the administration of the drug. Supplementary active compounds can also be incorporated into the compositions, e.g., additional adjuvants.

[0129] A pharmaceutical composition is generally formulated to be compatible with its intended route of administration. Examples of routes of administration include nasal (e.g., inhalation).

[0130] Methods for formulating 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 spray may include the following components: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, 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 acetate, citrate, or phosphate, and agents for adjusting tonicity such as sodium chloride or glucose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0131] For intranasal administration or administration by inhalation, nanoparticles can be delivered, for example, in the form of a solution, powder, aerosol, or suspension from a pump spray container squeezed or pumped by the subject, or as an aerosol spray from a pressurized container or nebulizer optionally with a suitable propellant. Formulations suitable for intranasal administration can be in the form of dry powder from a dry powder inhaler (alone, or as a mixture, e.g., as a dry blend with a carrier such as lactose, or as mixed component particles, e.g., mixed with a phospholipid such as phosphatidylcholine), or as an aerosol spray from a pressurized container, pump, nebulizer, atomizer (e.g., using electrohydrodynamics to generate 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.

[0132] The pharmaceutical composition can also be prepared in the form of a suppository for nasal delivery (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or as a retention enema.

[0133] The pharmaceutical composition may be contained in a container, package, or dispenser, such as in an inhaler, nebulizer, dropper, optionally with instructions for administration for the methods described herein.

[0134] Method of using PS-bionic nanoparticles

[0135] In some embodiments, the PS-bionic nanoparticles can be used to promote a protective immune response against an antigen, such as as part of a vaccine, for example to treat or reduce the risk of developing a viral or bacterial infection, such as influenza (or flu), for example in the lungs. In some embodiments, the PS-bionic nanoparticles can be used to treat asthma, respiratory allergies, or chronic obstructive pulmonary disease (COPD), or reduce one or more symptoms thereof.

[0136] In some embodiments, the PS-bionic nanoparticles can be administered to mucous membranes (e.g., nasal or lung tissue). In some embodiments, the PS-bionic nanoparticles can be administered intranasally (e.g., via an inhaler, nebulizer).

[0137] In some embodiments, the PS-bionic nanoparticles can be used to increase an immune response (e.g., activate innate immunity in the lungs; initiate a CD8 + T cell response; protection against a virus, e.g., subtype-specific protection against an influenza virus; or heterologous subtype protection against an influenza virus).

[0138] In some embodiments, the PS-bionic nanoparticles can be used as a chemotherapy adjuvant to treat cancers such as lung cancer. In these methods, the cargo is a chemotherapeutic agent, and the method comprises administering a therapeutically effective amount of the nanoparticles, e.g., an amount sufficient to cause a reduction in tumor size, tumor number, tumor growth rate, or metastasis.

[0139] Examples

[0140] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0141] Materials and methods

[0142] PS-GAMP synthesis

[0143] All lipids were purchased from Avanti Polar Lipids and included 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphate-(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 of 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 Na2HPO4, and 0.147 mM KH2PO4). Optionally, cGAMP was replaced with SRB (SigmaAldrich) and / or 0.5 μmol of 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 mixture of lipids and cGAMP was sonicated for 30 min at 50 °C under N2 to obtain a water-in-oil emulsion, followed by gentle removal of the solvent by rotary evaporation at 220 rpm. Excess buffer was added to the mixture and rotary evaporation was continued for an additional 5 min 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). The encapsulation efficiency was determined by UV absorption of cGAMP at 260 nm in Nanodrop (Life Technologies) and confirmed by liquid chromatography-mass spectrometry (LC-MS) (Agilent). Free cGAMP was removed by size-exclusion column G-50 (GE Healthcare). 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 under vacuum at -45 °C by Freezone 4.5 (Labconco). The lyophilized liposomes (PS-GAMP) were stored at -20 °C until use and were used for all in vivo studies unless otherwise specified.

[0144] Animals

[0145] 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 Laboratories or Charles River Laboratories. MHC II-EGFP mice expressing MHC class II molecules fused to enhanced green fluorescent protein (EGFP) were a gift from Dr. H. Ploegh of the Massachusetts Institute of Technology. Four-month-old female ferrets free of influenza were purchased from Marshall BioResources. Healthy naive 6-year-old male rhesus monkeys were purchased from the Beijing Institute of Xieerxin Biology Resource, China. In accordance with institutional, hospital, and NIH guidelines, the animals were housed in pathogen-free animal facilities at Massachusetts General Hospital (MGH) or Fudan University. The study was evaluated and approved by the MGH or the Fudan University Institutional Animal Care and Use Committee.

[0146] Influenza viruses and vaccines

[0147] The SH13 H7N9 virus (A / Shanghai / 4664T / 2013), SH09 H1N1 virus (A / Shanghai / 37T / 2009), and rgGZ89 H3N2 virus, which consists of the H3 and N2 of the A / Guizhou / 54 / 1989 H3N2 virus and the backbone of the A / Puerto Rico / 8 / 1934 (PR8) virus, were obtained from Fudan University. The pandemic CA09 H1N1 virus was requested from the American Type Culture Collection (ATCC, #FR-201). The PR8 (NR-348), A / Aichi / 2 / 68 H3N2 (Aichi, NR-3177), rgPerth H3N2 [A / Perth / 16 / 2009 H3N2 × PR8 (NR-3499)], and B / Florida / 4 / 2006 (Florida06, NR-9696) virus strains were obtained from BEI Resources, NIAID. The reverse genetics (rg)-modified VN04 (rgVN04) H5N1 virus was a gift from Dr. R. Webby of St. Jude Children’s Research Hospital and consists of the H5 and N1 genes from the A / Vietnam / 1203 / 2004 H5N1 virus and the PR8 virus backbone. The A / Michigan / 45 / 2015 H1N1 (Michigan15, FR-1483) and antiviral-resistant A / North Carolina / 39 / 2009 H1N1 virus (NC09, FR-488) were obtained from International Reagent Resources, CDC. Viruses were amplified for 3 days in 10-day-old embryonated chicken eggs (Charles River Laboratories) at 35 °C, harvested, purified by sucrose gradient ultracentrifugation, and frozen at -80 °C. For mouse challenge, the viruses were adapted to mice by 3 cycles of intranasal instillation-lung homogenate preparation and tested for their infectivity in mice according to the standard protocol, by the 50% lethal dose (LD 50 ).

[0148] The monovalent CA09 H1N1 vaccine (NR-20347, Sanofi Pasteur, Inc.) and the whole inactivated H5N1 vaccine (NR-12148, Baxter AG) were obtained from BEI Resources, NIAID. The H7-Re1 H7N9 whole 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. The 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. The Ag concentration was quantified by BCA protein assay and SDS-PAGE based on the HA content.

[0149] Mouse immunization and challenge

[0150] Mice were sedated with ketamine / xylazine and intranasally inoculated with 30 μl (15 μl per nostril) of the designated influenza vaccine or a mixture of vaccine and adjuvant. The VN04 H5N1, SIV 18-19, and CA09 H1N1 SV vaccines were used at the respective doses of 1 μg (HA content), 1 μg, or 0.5 μg per mouse, whereas the H7-Re1 and SH09 H1N1 vaccines were each administered at 0.25 μg or 3 μg per dose. 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, tonabersat, and meclofenamate were obtained from Sigma Aldrich and intraperitoneally injected into individual mice at the respective doses of 25, 10, or 20 mg / kg / day for 4 consecutive days (from 2 days before immunization to 1 day after immunization) (31, 32). To deplete CD8 during vaccination and challenge +T cells were administered anti-CD8α (53-6.7, BioLegend) antibody to mice at a dose of 200 μg / day, 2 days before immunization and at days 0, 2, and 4 after immunization. C57BL / 6 mice were used for challenge studies, and unless otherwise indicated, Swiss Webster mice or BALB / c mice were challenged as an alternative to C57BL / 6 mice for Aichi H3N2 virus, Florida06 B influenza virus, and GZ89 virus because C57BL / 6 mice are relatively resistant to these viruses. To verify antiviral drug resistance of NC09 virus, naïve mice were treated with oseltamivir (20 mg / kg / day) 6 hours before challenge and daily thereafter until the end of the study. At the indicated days after immunization, immunized and control mice were challenged by intranasal instillation of 10×LD 50 mouse-adapted homologous virus, except for H7N9 virus which was 40×LD 50 . However, heterologous virus at 5×LD 50 each was used for challenge, except for Florida06 B influenza virus which was at a dose of 4×10 5 TCID 50 because this virus is non-lethal to mice. After challenge, body weight and survival were monitored daily for 12 days.

[0151] Ferret immunization and challenge

[0152] Four-month-old female ferrets negative for anti-influenza virus antibody were anesthetized with ketamine / xylazine / atropine and immunized intranasally with vehicle, influenza vaccine, or a mixture of vaccine and PS-GAMP. To detect early protection, 2 days after immunization, ferrets receiving 9 μg of either the individual CA09 H1N1 vaccine or with 200 μg of PS-GAMP were challenged with 10 6 TCID 50 CA09 H1N1 virus. To evaluate cross-protection, ferrets were 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, were challenged with 10 6 TCID 50 heterologous subtype Michigan15 H1N1 virus. Body temperature was monitored by two microchips (BioMedicData Systems) implanted in each animal, and clinical symptoms were scored according to a published protocol (Table 1) (44). Two weeks after virus challenge, animals were humanely euthanized by sedation and injection of 0.5 ml of Euthanasia-III into the heart.

[0153] Table 1. Ferret clinical symptom scores (44)

[0154]

[0155] Tissue Processing and Flow Cytometry

[0156] Lungs, nasal tissues, MLNs, and spleens were excised from designated mice and processed into single-cell suspensions for flow cytometry analysis. Specifically, lungs and nasal tissues were minced into 1-mm 2 pieces and digested with 1 ml of collagenase D (2 mg / ml) / DNase I (5 mg / ml) (both from Roche) at 37 °C for 60 minutes and then passed through a 40-μm cell strainer (18). To collect BALF, mice were first perfused completely with ice-cold PBS, followed by intratracheal lavage with 0.5% BSA in PBS. Single-cell suspensions of spleens and MLNs were prepared by passing the tissues 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 antibody (clone 93, 10 μg / ml, BioLegend) for 20 minutes, and stained with fluorescent-conjugated antibodies for 30 minutes on ice, or stained with NP 366-374 PA 224-233 PB1 703-711 MHC I tetramers for 1 hour on ice. After surface staining, activated T cells were fixed and permeabilized, 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 subsets in the murine respiratory system were gated and analyzed as described (18). Information on the various antibodies is shown in Table 2.

[0157] Table 2. Antibodies and Tetramers for Flow Cytometry

[0158]

[0159]

[0160] Cytokine and Chemokine Assays

[0161] C57BL / 6 mice were administered PS-GAMP intranasally at 20 μg, or with 1 × LD 50CA09 H1N1 virus was used to infect C57BL / 6 mice. Lungs were harvested at the indicated times and prepared for total RNA extraction using an RNA purification kit (Roche). To determine cytokines in the brain, mice were administered intranasally either the VN04 H5N1 vaccine alone (1 μg HA) or in combination with PS-GAMP (20 μg) or CT (10 μg), and were sacrificed 48 h later to collect brain tissues 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 using specific ELISA kits.

[0162] Table 3. Primers for real-time PCR

[0163]

[0164] Histology

[0165] Swiss Webster mice were administered intranasally 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 a positive control. At the indicated 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 a NanoZoomer (Hamamatsu).

[0166] Confocal microscopy

[0167] To track DiD-labeled liposomes in the lungs, an equal amount of DiD-nano4 or DiD-nano5 was administered intranasally to C57BL / 6 mice. After 12 hours, the lungs were excised, embedded in optimal cutting temperature (OCT) compound (Sakura Finetek), and cut into 5-μm cryosections. The sections were mounted with ProLong Antifade Mountant containing DAPI (Life Technologies) and imaged by confocal microscopy (Olympus FV1000, UPLSAPO 60XW). To visualize in vitro AM uptake of nanoparticles, the mouse lungs were lavaged 6 times with 1 ml of PBS containing 0.5% BSA and 5 mM EDTA. The lung lavage fluids were pooled and centrifuged at 200×g. The cells were collected, washed thoroughly with PBS, and cultured in RPMI 1640 medium for 45 minutes, followed by removal of non-adherent cells. The adherent cells were collected as AMs, suspended at 2×10 5 cells / ml in the medium, and added to 96-well plates at 200 μl / well. To purify PS, lung lavage fluids were 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 pelletize PS. The supernatant (6 ml) was concentrated to 200 μl through a 3-kDa Amicon ultrafiltration filter unit (Merk Millipore) and mixed with the PS pellet prepared above. Then, the resulting PS (100 μg total protein) was mixed with DiD-nano4 or DiD-nano5 (12 μg lipid content in the nanoparticles) for 30 minutes prior to addition to AM cell cultures with 4×10 4 cells in 200 μl of the medium. After 4-hour incubation at 37°C and 5% CO2, the cells were stained with the live dye calcein-AM (Life Technologies). Uptake of liposomes was quantified by confocal microscopy (Olympus FV1000, UPLSAPO 60XW), followed by ImageJ software analysis.

[0168] Statistical analysis

[0169] The two-tailed Student t-test was used to analyze the differences between two groups. By PRISM software (GraphPad), ANOVA or Kruskal-Wallis test was used to analyze the differences among multiple groups. 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 experiment was conducted under highly standardized and predefined conditions, except for the microscopic images and H&E section examinations, which were evaluated in an investigator-blinded manner.

[0170] Hemagglutination inhibition (HAI) assay

[0171] Serum samples were collected from immunized and control animals at the specified time and treated with receptor-destroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) at 37 °C for 20 h, followed by heat inactivation at 56 °C for 30 min. The resulting serum samples were serially diluted and incubated with 4 hemagglutination units (HAU) of the specified influenza virus at 37 °C for 1 h. The virus treated with the serum was incubated with 0.5% chicken red blood cells (for H1N1 and H7N9) or horse red blood cells (for H5N1) at room temperature for 30 min. The HAI titer was defined as the reciprocal of the highest serum dilution that inhibited 4 HAU of the given virus.

[0172] Enzyme-linked immunosorbent assay (ELISA)

[0173] Influenza-specific IgG, IgG1, IgG2a, IgA, and IgG2c antibody titers were determined by ELISA. Briefly, 1 μg / ml of recombinant HA was coated onto ELISA plates in NaHCO3 buffer, pH 9.6, overnight, and serial dilutions of serum samples were added thereto. Antibody subtypes were quantified by HRP-conjugated goat anti-mouse IgG (NA931V, GE healthcare, diluted 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) antibodies. The titers of specific antibody subtypes were quantified by using SIGMAFASTM OPD as a substrate and reading the reaction at A490 on a plate reader (Molecular Devices).

[0174] Cellular immune response

[0175] Spleen cells were isolated from mice 7 days after immunization by passing the spleen through a 40-μm filter and then lysing red blood cells with ACK (ammonium-chloride-potassium) buffer for 4 minutes on ice. Cells at 1×10 6 / ml were cultured overnight with influenza vaccine (1 μg / ml) and anti-CD28 (clone 37.51, BD Pharmingen) antibody at 4 μg / ml. Golgi-Plug (BD Pharmingen) was added to the cultures and incubated for an additional 5 hours. First, stimulated cells were stained with fluorescent-conjugated antibodies against CD3, CD4, and CD8, followed by intracellular staining with anti-IFN-γ antibody. All antibodies are listed in Table S2. Stained cells were acquired on a FACSAria II (BD) and analyzed using FlowJo software (Tree Star).

[0176] Generation of chimeric mice by bone marrow transplantation

[0177] Chimeric mice were generated by bone marrow (BM) transplantation as described in (33). Briefly, BM cells were harvested from the femurs and tibias of sex- and age-matched donor mice with different CD45 alleles. Recipient mice received lethal irradiation from a 137Cs γ-irradiator (Mark I, 30 J.L. Shepherd) at a dose of 1100 rad, administered in two fractions with a 3-hour interval. 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 into 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 until 14 days after irradiation and were maintained for 3 months to complete full reconstitution of the donor population, which was confirmed by flow cytometry analysis of the lungs, MLN, spleen, and peripheral blood mononuclear cells (PBMC) after staining with anti-CD45.1 (clone A20, BioLegend, 2 μg / ml) or anti-CD45.2 (clone 104, BioLegend, 2.5 μg / ml) antibody.

[0178] BM-derived dendritic cells (BMDCs) and BM-derived macrophages (BMMs)

[0179] BMDCs and BMMs were prepared as previously described (45). Briefly, BM cells were harvested from the tibias and femurs of 4- to 6-week-old C57BL / 6 mice. Cells at 1×10 6Cells were cultured for 7 days at a concentration of cells / ml to generate BMDC or BMM. CD11c+ BMDCs were further purified by high-speed cell sorting in a FACSAria II (BD).

[0180] PS is required for AM uptake of nano4 in non-human primates (NHPs).

[0181] After euthanizing the rhesus monkeys, the lungs were surgically removed, filled with 150 ml of cold RPMI 1649 medium supplemented with antibiotics, immersed in the cold medium, and transported to the laboratory on ice. AM and PS were isolated as described in (46). Briefly, the filled RPMI 1640 medium was collected from the lungs and centrifuged at 200×g to remove cell debris, and then centrifuged at 8000×g for 20 minutes to pelletize the PS. The supernatant (30 ml) was concentrated to 1 ml using a 10 kDa Amicon ultrafiltration filter unit (Merk Millipore) and mixed with the PS pellet prepared above to obtain concentrated PS containing both lipids and surfactant proteins. AM was isolated by washing the lungs 6 times with 100 ml of PBS containing 0.5 mM EDTA. The lung lavage fluids were pooled and centrifuged at 200×g to collect the cells. The cells were washed thoroughly with PBS and cultured in RPMI 1640 for 20 minutes, followed by removal of non-adherent cells. Concentrated PS with a total protein of 2 mg was mixed with DiD-nano4 or DiD-nano5 (48 μg lipid content) for 30 minutes, and then cultured with 1.6×10 5 AMs. The AMs were stained with the live dyes calcein-AM (Life Technologies) and Hoechst (Sigma). AM uptake was evaluated by confocal microscopy (Olympus FV3000, UPLSAPO40×) and analyzed using ImageJ software.

[0182] Transmission electron microscopy (TEM)

[0183] To determine the ultrastructural localization of nano4 and nano5 in alveoli, gold nanoparticles (5 nm, Alfa Aesar) were encapsulated into nano4 or nano5 by the reverse-phase evaporation method as described in (47). Mice were intranasally administered with an equal amount of gold nanoparticle-nano4 or nano5. After 12 hours, the lungs were isolated, fixed overnight in Karnovsky fixative at 4 °C, post-fixed in 1% OsO4 in 0.1 M sodium cacodylate buffer for 1.5 hours, dehydrated in a graded ethanol series, infiltrated with s-propylene oxide / Epon t812 gradient mixture, 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-XR41M4.0 Megapixel Cooled sCMOS camera (Advanced Microscopy Techniques).

[0184] Example 1. PS-GAMP was made from PS components

[0185] Based on the PS component (17), we synthesized a series of liposomes to encapsulate cGAMP ( Figure 7A ). In terms of lipid composition and charge, negatively charged nano4 is close to PS. Compared with the vaccine control alone, when introduced intranasally (i.n.) together 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, with no weight loss at the same time (Figure 7B - 7E). In contrast, neutral liposomes (such as nano1), liposomes with cationic 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) replacing anionic phosphatidylglycerol (DPPG) (such as nano3 or nano5), or liposomes lacking PEG2000 (such as nano2 and nano3) showed significantly less adjuvanticity, resulting in significant weight loss at the same time ( Figure 7A-7E). Although their sizes and encapsulation efficiencies are similar to those of nano4 (Figs. 7F - 7G). Thus, being negatively charged and PEG2000 seem to play important roles in the function and safety of liposomes. Unexpectedly, when bone marrow-derived dendritic cells (BMDCs) were stimulated in vitro with cGAMP encapsulated in positively charged liposomes (nano3 or nano5), higher levels of Ifnb1 were expressed compared to when stimulated with negatively charged liposomes (nano2 and nano4) (Fig. 7H). A similar pattern was observed when bone marrow-derived macrophages (BMMs) were stimulated with positively or negatively charged liposomes encapsulating cGAMP (Fig. 7I). This emphasizes the necessity of in vivo evaluation of the safety and efficacy of nanoparticles. Then trehalose was added to the liposome suspension before lyophilization to increase the stability of nano4 ( Figure 7A ). The resulting nano6 liposomes, which we designated as PS-GAMP, were stable at -20 °C for at least 6 months and exhibited zeta potentials, sizes, functions, and safety similar to freshly prepared nano4 ( Figure 7A , 7F, 7J, and 7B - 7E). In addition, the PS-GAMP-adjuvanted influenza vaccine induced high Ag-specific IgG titers in wild-type (WT) mice but not in STING-deficient mice, confirming that cGAMP, rather than any other component, is responsible for the adjuvanticity of PS-GAMP (Fig. 7K).

[0186] Example 2: Uptake of PS-GAMP by alveolar macrophages requires surfactant proteins A and D

[0187] Next, the cellular targets of nano4 and its cargo were investigated by labeling the membranes of nano4 and nano5 with DiD, a fluorescent lipophilic cyanine, and encapsulating another fluorescent dye (sulfonyl rhodamine B, SRB) with a molecular mass and net negative charge equivalent to cGAMP within the liposomes (Fig. 1A). The liposomes were administered intranasally to mice, and their nasal tissues, brains, mediastinal lymph nodes (MLN), and lungs were analyzed by flow cytometry at various time points. The lung was the only tissue where we found the SRB + signal compared to the control (Figs. 1B and 8A). Here, nano4 was directly taken up by CD11b - CD11c + CD24 - alveolar macrophages (AMs), and indirectly taken up by CD11b - CD11c - EpCAM + MHC II + AECs (Figs. 1B - 1D and 10A) (18). Thus, AECs were the source of SRB + but not 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).

[0188] Surprisingly, AMs isolated from lung lavage fluid did not effectively uptake nano4 ex vivo. 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). Figure 12 ). Under similar conditions, no such aggregates were formed when PS was incubated with nano4 ( Figure 12) Similar results were obtained when AM and PS were isolated from non-human primates (NHP). Figure 13A - 13C ) Thus, PS may play an evolutionarily conserved role in PS-GAMP endocytosis. Consistent with these ex vivo observations, DiD-nano4 was located in single cells positive for the biomarker Siglec F for AM after intranasal administration (Figure 1H and Figure 14 ). In contrast, positively charged nano5 interacted electrostatically and fused with negatively charged PS, presenting diffuse staining along the alveolar surface (Figure 1H). The different localizations of nano4 and nano5 were confirmed by transmission electron microscopy (TEM) using nano5 and nano4 labeled with nanogold. Figure 15 ) In vitro validation of the efficient uptake of nano4 only in the presence of PS suggested that surfactant proteins (SP)-A and -D (termed "collectins") play a role in this uptake. Indeed, PS isolated from Sftpa1 - / - Sftpd - / - mice did not enhance the in vitro nano4 uptake by WT AM, in sharp contrast to PS isolated from WT mice (Figure 1I). Moreover, nano4 uptake was severely impaired in Sftpa1 - / - Sftpd - / - mice (Figure 1J), which was not due to any defect in Sftpa1 - / - Sftpd - / - AM, because after pre-culturing in vitro with WT PS, Sftpa1 - / - Sftpd - / - AM took up an amount of nano4 comparable to that of WT AM. Figure 16 )

[0189] Example 3: PS-GAMP transiently activates innate immunity in the lung

[0190] The dependence on SP-A and SP-D in nano4 uptake suggests the involvement of natural and molecular-specific mechanisms of particle clearance in the lung, 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, the whole inactivated VN04H5N1 vaccine, or a combination of both, the mouse lung, nasal tissue, and brain were histologically indistinguishable from the PBS control tissue (Figure 17A - 17B). There was no cell death, damage to the epithelial barrier, or obvious infiltration of inflammatory cells in these tissues. Figure 17A - 17C)。Moderate and transient infiltration of monocytes was only found in the lungs on day 3, which was significantly less severe compared to monocytes responding to viral infection (Figure 18E). We did not observe any significant cytokine production in the brain compared to the control (Figure 17C). In stark contrast, the VN04H5N1 vaccine formulated with cholera toxin (CT) elicited a large inflammatory cell infiltrate in the lungs and measurable cytokine mRNA expression in the brains of some mice ( Figure 17A - 17C ).

[0191] Despite the lack of obvious lung inflammation histologically over time (Figure 19A), PS-GAMP was found to rapidly and potently, but only transiently, activate innate immunity. Ifnb1, Gmcsf, and Tnf as well as Ccl2, Ccl3, Ccl5, and Cxcl10 mRNA expression peaked at 12 hours post-stimulation and subsided within 48 hours ( Figure 20 ). In contrast, a significantly higher level of these mediators was induced by low-dose infection with the CA09 H1N1 influenza virus ( Figure 20 ), causing worsening lung inflammation during the course of viral infection, despite strong Il10 expression (Figures 19B - 19C and 20). Protein levels in BALF also verified transient IFN-β production, but TNF-α and IL-10 were above the limit of detection ( Figure 21A - 21C ). In stark contrast, these cytokines were produced significantly higher from day 2 to day 6 as the infection progressed ( Figure 21A - 21C ). The transient activation of innate immunity was restricted to the lungs, as serum IFN-β, IFN-γ, IL-6, IL-10, and TNF-α levels were unchanged compared to the control (Figure 22C - 22G). This was consistent with the lack of adjuvant side effects in terms of mouse body weight and temperature (Figures 22A - 22B).

[0192] Example 4: PS-GAMP is a potent adjuvant for both humoral and cellular immune responses

[0193] Although PS-GAMP only transiently activates innate immunity, this effect seems 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 increases serum hemagglutination inhibition (HAI) antibody and BALF IgA titers in a dose-dependent manner (Figures 2A-2B). The adjuvant is potent in both the primary and booster immune responses, increasing Ag-specific IgG1 in serum 10-fold, IgG more than 100-fold, and IgG2c ~1,000-fold compared to the VN04 H5N1 vaccine alone (Figures 2C-2E). In addition to the whole inactivated VN04 H5N1 vaccine, PS-GAMP also exhibits strong adjuvanticity when combined with split virion (SV) vaccines such as the A / California / 7 / 2009 (CA09) H1N1 vaccine. Compared to the SV vaccine alone, the adjuvant enhances the HAI titer 10-fold, BALF IgA 60-fold, and IgG 10,000-fold (Figures 2F-2H). Under similar conditions, poly IC shows 5-fold, 30-fold, and 100-fold lower efficacy in enhancing the HAI titer, BALF IgA, and serum IgG, respectively (Figures 2F-2H). PS-GAMP not only enhances the humoral immune response but also significantly augments the cellular immune response. The PS-GAMP-adjuvanted CA09 H1N1 vaccine increases IFN-γ + CD8 + T cells 24-fold compared to the vaccine alone or increases IFN-γ + CD8 + T cells 8-fold compared to the vaccine formulated with poly IC (Figures 2I and 26A). In all vaccination groups, the duo also induces the highest amount of IFN-γ + CD4 + T cells (Figures 2J and 26A). The strong immune response translates to complete protection against a 10×LD 50 CA09 H1N1 virus challenge, with only moderate weight loss to no weight loss (Figures 2K-2L). In contrast, the poly IC-adjuvanted CA09 H1N1 vaccine only confers partial (33%) protection against virus challenge, with severe weight loss.

[0194] Example 5: PS-GAMP Elicits a Potent CD8 + T Cell Response

[0195] We investigated which DC subset is involved in PS-GAMP-mediated adjuvanticity and found that after intranasal administration of PS-GAMP, CD11b + DCs, rather than CD11b -DCs were increased 14-fold and 36-fold in the lung (upper panel) and MLN (lower panel), respectively, relative to day 0, at day 3 (Figure 3A). Among CD11b + DCs, monocyte-derived CD11b + DCs (Mono-DCs) and tissue-resident CD11b + DCs (tDCs) were distinguished by MHC II and Ly6C expression (Figure 3B) (23, 24). MHC II hi CD11b + tDCs have been shown to be the most capable lung DCs for cross-presentation during influenza virus infection (23). Significantly, after PS-GAMP administration, these cells accumulated massively, similar to those in the early (first 3 days) of viral infection, whereas pro-inflammatory mono-DCs only slightly increased during the same experimental period, although significant relative to day 0 (Figure 3B). Subsequently, CD11b + tDCs were decreased in the lung and MLN receiving PS-GAMP, in sharp contrast to the continuous accumulation of these CD11b + DCs in both the lung and MLN at more than 3 days post-infection (Figure 3B). Changes in other immune cell types in the lung, MLN, nasal tissue, and brain after immunization or infection are detailed in Figure 18A - 18V . In addition to DCs, natural killer (NK) cells and CD4 + T cells were transiently increased in the lung for 1 or 2 days, while other immune cells remained unchanged during the experimental period (Figure 3A).

[0196] These CD11b + DCs appear to be efficient in Ag cross-presentation and can induce robust CD8 + T cell proliferation. 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 10-fold increase in OVA + DCs in the MLN with predominantly CD11b + DCs compared to mice receiving OVA alone (Figure 3C and Figure 23B). These DCs matured and were activated, as indicated by upregulation of CD40 and CD85 (Figure 3D - 3E). This effect is presumably secondary to AEC and AM activation, considering that most MLN DCs were negative for PS-GAME (Figure 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 (Figure 24D-24E). Thus, the robust proliferation of OT-I cells in the presence of PS-GAMP may be due to CD11b + enhanced differentiation and maturation of DCs. In turn, when OVA and PS-GAMP were introduced together, these cells led to a greater than 6-fold increase in highly proliferative OT-I cells in the lung and MLN compared to OVA alone ( Figure 25A - 25D ).

[0197] As early as 4 days after immunization with a PS-GAMP-adjuvanted influenza vaccine, a large number of nucleoprotein (NP) 366-374 -specific CD8 + T cells were observed in the lung and to a lesser extent in the MLN (Figure 3F-3G and Figure 26B). NP 366-374 is the major CD8 + T cell epitope, and CD8 224-233 T cells specific for other epitopes such as PA 703-711 or PB1 + were undetectable in these animals, likely due to the low copy number of these proteins in the inactivated influenza vaccine (Figure 26C)(25). These virus-specific CD8 + T cells expressed the early activation biomarker granzyme B (GB) upon viral challenge (Figure 27A)(26). After receiving a PS-GAMP-adjuvanted CA09 H1N1 vaccine, GB + CD8 + T cells were significantly increased in the BALF at 4 days and in the lung at 6 days (Figure 3H). More than 65% of these GB + CD8 + T cells were positive for NP 366-374 , whereas only a small number of cells were positive for PA 224-233 or PB1 703-711 (Figure 27B). In a similar context, the vaccine alone did not significantly expand GB + CD8 + T cells (Figure 3H). The CD8 + T cell response elicited by PS-GAMP was superior to poly IC or the TLR2 agonist Pam2CSK4 (27,28)(Figure 3I). Although a T cell immune response was induced shortly after immunization, Ag-specific BALF IgA and IgM were undetectable at these early time points (Figure 27C). Thus, PS-GAMP in CD8 +Key events that mimic viral infection in T cell induction without triggering excessive lung inflammation or immunopathology( Figure 17A - 17C , 18A - 18V , 19A - 19C, 20, 21A - 21C, and 22A - 22G).

[0198] Example 6: PS - GAMP provides strong protection as early as 2 days post - immunization

[0199] CD8 + The rapid induction of CD8 T cells prompted us to determine how quickly PS - GAMP could achieve protection. To this end, as shown in Figure 28A, mice were challenged on days 0, 2, 4, 6, 8, or 14 post - immunization. Inclusion of PS - GAMP in the vaccination completely protected mice from homologous virus challenge as early as 2 days post - immunization (Figure 4A). At all early challenge time points (days - 2, - 4, and - 6), mice only experienced a slight weight loss (<10%), and all mice survived (Figure 4A and Figure 28B). When challenged on day 8 post - immunization, mice did not suffer any weight loss and 100% survived (Figure 4A and Figure 28B). This early protection was not directly caused by innate immunity, as administration of PS - GAMP alone at day 0 or 2 days prior did not confer any protection (Figure 4B and Figure 28C). To determine whether CD8 + T cells were responsible for the early protection, CD8 + T cells were depleted by intraperitoneal (i.p.) injection of anti - CD8 antibody every other day starting 2 days prior and ending 4 days post - immunization. Depletion of CD8 + T cells abolished the early protection, as evidenced by similar dramatic weight loss and 100% mortality as those of mice receiving the vaccine alone (Figure 4C and Figure 28D). To rule out that this early protection was unique to the CA09 H1N1 vaccine, we extended the study to the H5N1 vaccine, which is a less immunogenic antibody compared to the CA09 H1N1 vaccine. Again, for mice immunized 2 - 8 days prior in a manner dependent on the adaptive immune response, the presence of PS - GAMP conferred 75% - 100% protection against rgVN04 H5N1 virus challenge (Figure 4D and Figure 28E), as no protection was obtained with PS - GAMP alone (Figure 4E and Figure 28F). Under similar conditions, the vaccine combined with CT did not provide any early protection (Figure 4E and Figure 28F), compellingly suggesting that exacerbated inflammation does not have to be associated with a strong adaptive immune response( Figure 17A - 17C)。In addition to the rgH5N1 virus, 2 or 14 days after immunization with an inactivated H7N9 vaccine (H7-Re1) adjuvanted with PS-GAMP, mice were significantly or completely protected from a lethal challenge with a clinical isolate of the previously pandemic A / Shanghai / 4664T / 2013 (SH13) H7N9 virus (Figure 4F and Figures 28G - 28I). Under similar conditions, the vaccine adjuvanted with poly IC did not confer any benefit compared to the vaccine alone (Figure 4F and Figure 28I).

[0200] The ability of PS-GAMP to rapidly establish protection was also demonstrated in an FDA-approved ferret model. When challenged with the homologous CA09H1N1 virus, ferrets that received the CA09 H1N1 vaccine adjuvanted with PS-GAMP 2 days previously experienced <5% weight loss, along with mild to no clinical symptoms, and only a transient fever on day 2 post-viral challenge (Figure 4G - 4I). Virus shedding was significantly reduced from day 4 onwards (Figure 4J). However, the CA09H1N1 vaccine alone did not protect animals from weight loss, did not improve clinical symptoms or reduce virus shedding after a similar viral challenge compared to controls, although it moderately reduced body temperature (Figure 4G - 4J).

[0201] Example 7: AEC is essential for PS-GAMP-mediated adjuvanticity

[0202] cGAMP has been well documented to be readily transferred through gap junctions that exist between AM and AEC (29, 30). The dynamic flow from AM to AEC was demonstrated by the progressive loss of SRB in AM 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 was not attributable to the loss of liposomes, as DiD +The number of cells remained unchanged until 18 hours later (Figure 29B). The entry of SRB into AEC was blocked by the gap junction blocker (29) carbenoxolone (CBX) (Figure 5A and 5C), which did not affect the SRB uptake by AM (Figure 5A - 5B). In AM and AEC sorted from lungs receiving PS - GAMP, CBX significantly attenuated the transcription of Ifnb1 and Gmcsf in AEC while increasing Ifnb1 transcription in AM (Figure 30B - 30C). This is most likely the result of elevated cGAMP levels in the cells. Thus, there is a gap junction - mediated flow of cGAMP from AM to AEC. On the other hand, poly IC remained predominantly (>97%) in AM after intranasal immunization (Figure 31A). In the lung, only 0.4% of total AEC and 4% of total DC took up poly IC (Figure 31B - 31C). MLN and nasal tissue DC as well as nasal epithelial cells rarely internalized poly IC (Figure 31D - 31E).

[0203] PS - GAMP induced 100 - fold higher IgG2c titers than poly IC (Figure 5D). However, when mice were treated with CBX or two other gap junction inhibitors, tonabersat and meclofenamate, before and during immunization, the adjuvanticity was significantly attenuated (Figure 5D)(31, 32). In contrast, these inhibitors had a minor effect on poly IC - mediated adjuvanticity (Figure 5D), consistent with poly IC, a large molecule, not being able to enter neighboring cells through gap junctions (Figure 31A). Blocking the entry of cGAMP into AEC reduced the recruitment of CD11b + DC by 50% (Figure 5E) and showed a more profound effect on the early CD8 + T cell response in both BALF and lung (Figure 5F - 5G). In addition, in BALF and lung, chimeric mice (ST→WT) including Sting - deficient (Sting - / - or ST) bone marrow (BM) cells and WT AEC had similar levels of CD8 + T cells as WT→WT mice (Figure 5H - 5J and Figure 32A - 32B )(33). In contrast, mice with STING deficiency in AEC, prepared by transferring WT BM cells into Sting - deficient mice (WT→ST mice), produced significantly lower levels of Ag - specific CD8 + T cells (Figure 5I - 5J). These WT→ST mice showed poor protection by 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 (Figure 5K - 5L). We also found GB + 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).

[0204] Example 8: PS-GAMP extends protection against heterologous subtypes of influenza virus

[0205] 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 ( Figure 34A - 34B) breadth of the immune response. These findings suggest that PS-GAMP can enhance multiple influenza vaccines simultaneously and is equally effective for both influenza A and B virus vaccines.

[0206] Long-lived Ag-specific memory CD8 + T cells can be rapidly recalled in the context of viral infection and are required for adequate control of viral replication in the lung (2, 3). In mice receiving OT-I cells, relative to OVA alone, after immunization with OVA combined with PS-GAMP, as measured by CD103 + CD49a + CD69 + the number of labeled lung CD8 + T RM cells increased 20-fold (Figures 35A - 35C). Additionally, 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 correlated with persistent influenza-specific CD8 + T RM cells in the lung, which could be readily detected 6 months after immunization (Figures 35D - 35E). These CD8 + T RM cells, rather than circulating memory CD8 + T cells, contributed to the observed long-term protection, as their function was not affected by the T cell egress inhibitor FTY720 ( Figure 36A - 36D )(3).

[0207] 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 the vaccine alone, the body weight and temperature of the animals were not affected by the immunization, indicating good safety of PS-GAMP in ferrets (Figures 37A-37B). Compared with what the vaccine alone did at 28 days post-immunization, the immunization induced 40-fold higher serum IgG titers and 5-fold higher HAI titers against the homologous Perth H3N2 virus (Figures 37C-37D), but no HAI antibodies were detected against the heterologous subtype A / Michigan / 45 / 2015 H1N1 (Michigan H1N1) virus, as expected (Figure 37E). In the case of challenge with the Michigan H1N1 virus, ferrets receiving the vaccine and PS-GAMP showed significantly less weight loss and milder clinical symptoms compared with animals receiving PBS or the vaccine alone, especially in the late stage of infection (>7 days), and normalized their body temperature faster (Figures 6L-6N). Two days post-infection, the animals also shed significantly less amount of virus (Figure 6O). The ability of vaccination to inhibit virus replication led to a significant improvement in clinical outcomes, and the accelerated weight recovery may be the result of major T cell immunity in the animals.

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[0258] Other embodiments

[0259] It should be understood that although the present invention has been described in connection with the detailed description thereof, the foregoing description is intended to be illustrative and not intended to limit the scope of the present 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 comprising negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-mimetic molecules, wherein said lung surfactant-mimetic molecules comprise polyethylene glycol, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphate-(1'-rac-glycerol), and cholesterol; and more than one cargo molecule, wherein said more than one cargo molecule comprises cyclic guanosine monophosphate-adenosine monophosphate.

2. A composition comprising nanoparticles prepared by a method comprising the steps of: combining a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphate-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], and cholesterol with cyclic guanosine monophosphate-adenosine monophosphate to produce said nanoparticles.

3. A composition formulated for intranasal administration or administration by inhalation, comprising negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-mimetic molecules, wherein said lung surfactant-mimetic molecules comprise polyethylene glycol; and more than one cargo molecule, wherein said more than one cargo molecule comprises a STING agonist.

4. Use of a composition in the manufacture of a medicament for treating or preventing a disease, disorder or condition by a method comprising intranasally or by inhalation administering the composition to a subject, wherein said composition comprises negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-mimetic molecules, wherein said lung surfactant-mimetic molecules comprise polyethylene glycol; and more than one cargo molecule, wherein said more than one cargo molecule comprises a STING agonist.

5. Use of a composition in the manufacture of a medicament for reducing the risk of developing a viral or bacterial infection, wherein said composition comprises negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-mimetic molecules, wherein said lung surfactant-mimetic molecules comprise polyethylene glycol; and more than one cargo molecule, wherein said more than one cargo molecule comprises a STING agonist.

6. Use of a composition in the manufacture of a vaccine adjuvant for protecting against viral or bacterial infection by a method comprising administering the composition and a vaccine to a subject, wherein said composition comprises negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-mimetic molecules, wherein said lung surfactant-mimetic molecules comprise polyethylene glycol; and more than one cargo molecule, wherein said more than one cargo molecule comprises a STING agonist.

7. Use of a composition in the manufacture of a medicament for promoting an immune response to an antigen, wherein said composition comprises negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-mimetic molecules, wherein said lung surfactant-mimetic molecules comprise polyethylene glycol; and One or more cargo molecules, wherein the one or more cargo molecules comprise a STING agonist.

8. A composition comprising negatively charged nanoparticles, said nanoparticles comprising Multiple pulmonary surfactant - biomimetic molecules; and one or more cargo molecules, wherein each of the one or more cargo molecules is selected from alkylating agents, anthracyclines, cytoskeletal disrupting agents (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, retinoids, and / or vinca alkaloids and derivatives.

9. Use of the composition in the preparation of a medicament for treating cancer by a method comprising intranasally or by inhalation administering the composition to a subject, wherein the composition comprises negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-bionic molecules, wherein the lung surfactant-bionic molecules comprise polyethylene glycol; and one or more cargo molecules, wherein each of the one or more cargo molecules has a molecular weight of 1200 Da or less.

10. A composition comprising negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-bionic molecules; and one or more cargo molecules, wherein each of the one or more cargo molecules is selected from agonists for intracellular Toll-like receptors including TLR3, TLR7, TLR8, or TLR9 (e.g., imiquimod, resiquimod (R848), imidazoquinoline (IMQ), motolimod, CU-CPT4a, IPH-3102, or amiprilose), and agonists for Nodinitib (NOD1), NOD2, NLPR3 or NPLRC3 (e.g., muramyl dipeptide (MDP), FK565, or FK156).

11. A composition comprising negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-bionic molecules; and One or more cargo molecules, wherein each of the one or more cargo molecules is selected from 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)); an agonist or antagonist of a receptor (e.g., chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), chemokine receptor 2 (CCR2)); an agonist or antagonist of an ion channel (e.g., a GABA receptor, transient receptor potential cation channel, subfamily A, member 1 (TRPA1), or a voltage-gated sodium channel); an inducer of IFN-α; a long-acting muscarinic antagonist / anticholinergic (LAMA); an inhibitor against IL-5, IL-13, IL-33, or thymic stromal lymphopoietin; a CXCR2 antagonist; a molecule that blocks a pro-inflammatory cytokine (e.g., TNF-α, TNF-β, or IL-6); a molecule that blocks IL-17 / TH17; a macrolide; a molecule that activates HDAC2; and / or a STAT6 inhibitor (e.g., AS1517499).

12. A composition comprising negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-bionic molecules; and One or more cargo molecules, wherein each of the one or more cargo molecules is selected from cyanine, indocyanine, oxacyanine, thiacyanine, merocyanine, polymethine, coumarin, rhodamine, sulforhodamine B (SRB), xanthene, fluorescein, boron-dipyrromethene (BODIPY) dyes such as 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 (ammonium perchlorate dimethyl{4-[1,5,5-tris(4-dimethylaminophenyl)-2,4-pentadienylidene]-2,5-cyclohexadien-1-yl}), IRDye 800CW, IRDye 800RS, IRDye 700DX, ADS780WS, ADS830WS, ADS832WS, 1,1-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate (DiD), 1,1’-dioctadecyl-3,3,3’3’-tetramethylindocyanine and (DiI, also known as DiIC18(3)).

13. A composition comprising negatively charged nanoparticles, said nanoparticles comprising a plurality of lung surfactant-bionic molecules; and one or more cargo molecules, wherein each of the one or more cargo molecules has a molecular weight of 1200 Da or less.

14. Use of the composition according to claim 13 in the preparation of a medicament for preventing or treating a disease, disorder or condition.

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