Invariant natural killer t cell-activating vaccine against bacterial infections

Abstract

Provided are vaccine compositions comprising a liposome, a bacterial polysaccharide antigen, and an invariant natural killer T (iNKT) cell agonist, methods of treating or preventing a bacterial infection using the compositions, and nasal spray devices comprising them.

Description

[0001] INVARIANT NATURAL KILLER T CELL-ACTIVATING VACCINE AGAINST BACTERIAL

[0002] INFECTIONS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to vaccines comprising an invariant natural killer T (iNKT) cell agonist and a bacterial polysaccharide antigen and their use in treating or preventing bacterial infections, and to a nasal spray device for delivering the vaccines.

[0005] BACKGROUND OF THE INVENTION

[0006] Streptococcus pneumoniae, or pneumococcus, causes both non-invasive (pneumonia, otitis media, sinusitis, and conjunctivitis) and invasive diseases (IPDs, such as meningitis, sepsis, and osteomyelitis), which are estimated to kill 1,000,000 children every year. Its capsular polysaccharide is the most virulent component, with over 100 different repeating unit structures, determining its serotype. S. pneumoniae has developed resistance against all antibiotics except vancomycin, and in many countries, macrolide antimicrobials, penicillins, and cephalosporins cannot be assumed effective anymore. Therefore, vaccination remains the most practical and economical strategy against S. pneumoniae for both prophylactic and therapeutic purposes.

[0007] Pneumococcal polysaccharide vaccines (PPSV, Pneumovax® 23) and conjugate vaccines (PCV, Prevnar® 7, 13, 20, & Vaxneuvance™ 15) are the most available licensed vaccines. The PPSV23 was proved to have weak immunogenicity because the classical major histocompatibility complex (MHC) molecule cannot present carbohydrate-based antigens, which is essential for inducing T-dependent adaptive immunity. To break this restriction, the carbohydrate antigens can be conjugated to a carrier protein (e.g., CRM197, a detoxified variant of diphtheria toxin). Based on this strategy, Prevnar®s, Vaxneuvance™ 15, and some preclinical vaccines were developed, whose overall efficacies were significantly improved but still showed limitations in older adults.

[0008] Another approach is to link the carbohydrate antigen to an immunogenic lipid, such as o- galactosylceramide (o-GalCer), which can activate semi-invariant natural killer T (iNKT) cells and further initiate the iNKT-assisted adaptive immune responses. Several semisynthetic vaccines (Bai et al., PNAS, 2013; Deng et al., Chemical Science, 2014; Cavallari, Nature Chemical Biology, 2014; Shute, Journal of Immunology, 2021) using this approach have shown positive results. However, their fabrication processes contain steps of chemical synthesis, which might escalate the cost and make them less accessible to vulnerable populations in low-income areas. Notably, all currently licensed vaccines require to be stored and delivered within a cold chain, which can account for as high as 50 % of the total cost. This further presents an unignorable obstacle to their distribution in developing countries, where most death cases occur.

[0009] SUMMARY OF THE INVENTION

[0010] The present inventors have discovered that a vaccine composition comprising a carbohydrate antigen in combination with an iNKT cell agonist in a liposome formulation induces a potent and effective immune response.

[0011] Compared with the carrier protein-conjugation strategy, which activates normal T cells, the designed vaccine targeting iNKT cells holds several advantages.

[0012] First, the iNKT cell-mediated adaptive immune responses are universal among individuals. The classical polymorphic MHC (human leukocyte antigen, HLA in humans) has many alleles, and their frequency distributions vary significantly among different populations. In contrast, the ligand of TCRs on iNKT cells, the CDld molecule in mice (CDla-e in humans), is highly structurally conserved, which helps bring a more predictable and consistent immunization efficacy.

[0013] Second, iNKT cells respond faster after stimulation. Upon activation, iNKT cells immediately proliferate and differentiate into functional phenotypes within hours, while it takes days for conventional T cells. Moreover, iNKTFH cells could help the germinal centers' formation by day 3, which generally takes 10 days for conventional TFH cells.

[0014] Third, iNKT cells can both activate innate immunities by the "pathogen-associated molecular patterns (PAMPs) alike" functions and differentiate into "T-helper" phenotypes to contribute to adaptive immune responses. The bridging capability between innate and adaptive immunity can result in a synergistic effect and improve bacterial clearance efficacy.

[0015] In preferred embodiments, the capsular polysaccharide is extracted from bacterial culture instead of being produced by chemical synthesis. This makes the vaccine cost relatively low, making it more accessible to risky populations in low-income areas. Furthermore, the liposome can be fabricated by simple extrusion. The whole formulation can be stored safely at room temperature for at least 3 weeks; as a result, the cold chain can be avoided during transportation. Moreover, different from existing licensed Pneumovax® and Prevnar®s, which are administered systemically (intramuscularly or subcutaneously), the vaccine according to the present invention can be intranasally applied with the help of a cationic liposome formulation. This immunization route is more patient-compliant and effective in stimulating the local respiratory mucosal immune systems, which are proximal to infection sites.

[0016] So, in a first aspect the present invention relates to a composition, comprising : a) a liposome, b) at least one bacterial polysaccharide antigen, and c) at least one invariant natural killer T (iNKT) cell agonist, wherein the iNKT cell agonist is integrated in a lipid bilayer of the liposome, and wherein the at least one bacterial polysaccharide antigen is within the lumen of the liposome and / or attached to a lipid bilayer of the liposome.

[0017] In some embodiments, the at least one iNKT cell agonist is selected from any one of: o- galactosyl ceramide (o-GalCer), o-galactosyldiacylglycerol (o-GalDag), o- glucosyldiacylglycerol (o-GIcDag), o-glucuronsylceramide (o-GIcACer), o- galacturonosylceramide (o-GalACer), phosphatidyl-myo-inositol mannoside (PIM2), and any derivative of any one of these agonists, preferably o-GalCer, or any combination thereof.

[0018] In some embodiments, the at least one bacterial polysaccharide antigen is within the lumen of the liposome.

[0019] In some embodiments, the liposome is cationic at physiological pH.

[0020] In some embodiments, the at least one bacterial polysaccharide antigen is a non-modified native polysaccharide.

[0021] In some embodiments, the bacterial polysaccharide antigen is a capsular polysaccharide derived from a bacterium selected from: Bordetella pertussis, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, and Streptococcus pneumoniae, preferably S. pneumoniae.

[0022] In some embodiments, the composition comprises at least two different capsular polysaccharides, which are derived from at least two different serotypes of S. pneumoniae, optionally selected from the following: 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, and In some embodiments, the at least one bacterial polysaccharide antigen is a capsular polysaccharide derived from S. pneumoniae, and the iNKT cell agonist is o-GalCer.

[0023] In a second aspect, the invention relates to a pharmaceutical composition comprising the composition according to embodiments of the first aspect of the invention and a pharmaceutically acceptable vehicle and / or diluent and / or immunological adjuvant.

[0024] In a third aspect, the invention relates to a method of treating or preventing a bacterial infection in a mammal, such as a human being, said method comprising administering a pharmaceutically effective amount of the composition according to embodiments of the first aspect of the invention or of the pharmaceutical composition according to the second aspect of the invention to said mammal.

[0025] In some embodiments, the bacterial infection is an airway infection, such as an upper airway and / or lung infection.

[0026] In some embodiments, the composition or the pharmaceutical composition is administered intranasally, subcutaneously, orally or intramuscularly, preferably intranasally, or any combination thereof.

[0027] In a fourth aspect, the composition according to embodiments of the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention is for use in a method according to embodiments of the third aspect of the invention.

[0028] In a fourth aspect, the invention relates to a nasal spray device, comprising : a pressurised aerosol canister including a vial containing the pharmaceutical composition according to the second aspect of the invention, an actuator, and a delivery outlet for the aerosol plume.

[0029] In some embodiments, the nasal spray device is configured to deliver metered doses of the pharmaceutical composition to the nasal cavity of a mammal, such as a human being.

[0030] LEGENDS TO THE FIGURES

[0031] Fig. 1 : Schematic illustration of an exemplary composition according to the invention, the Lipo+CPS12F&oGC vaccine, comprising a cationic liposome containing the capsular polysaccharide CPS12F from S. pneumoniae in its lumen and the iNKT cell agonist o- galactosylceramide (o-GalCer) integrated in its lipid bilayer. Fig. 2: Size distribution and stability of the liposomal vaccine Lipo+CPS12F&oGC. The liposomal vaccine was stored at 2-8 °C (in a fridge) or 18-24 °C (at room temperature). The hydrodynamic size was measured weekly for 2 months with a zetasizer.

[0032] Fig. 3: Lipo+CPS12F&oGC stimulated elevated proinflammatory cytokines secretion compared with other formulations. Concentrations of a) IL-12p70, b) IFN-y, c) IL-4, and d) IL-17A in serum and saliva. BALB / cJRj mice were immunized with Lipo+(group 1, blank cationic liposomes as placebo), Lipo+oGC (group 2), or 3 nmol antigen (repeat units of the polysaccharides) in Lipo+CPS12F (group 3), LipoCPS12F&oGC (group 4), or Lipo+CPS12F&oGC (group 5 & 6) via intranasal instillation (i.n., group 1-5) or subcutaneous injection (s.c., group 6). Serum and saliva were collected 18 hours after the priming vaccination, and IL-12p70, IFN-y, IL-4, and IL-17A inside were quantified via cytometric bead array flow cytometry. Data were plotted as mean ± SEM (n=8). Welch's ANOVA tests and multiple comparisons were executed to assess statistical significance. *, **, ***, **** represent P<0.1, P<0.01, P<0.001, P<0.0001.

[0033] Fig. 4: Lipo+CPS12F&oGC induced superior high-affinity CPS12F-specific antibody production compared with other formulations. Levels of CPS12F-specific a) IgM, b) IgGpOiy, and c) IgA in serum and saliva 2 weeks after the final vaccination. BALB / cJRj mice were immunized with Lipo+(group 1, blank cationic liposomes as placebo), Lipo+oGC (group 2), or 3 nmol antigen (repeat units of the polysaccharides) in Lipo+CPS12F (group 3), LipoCPS12F&oGC (group 4), or Lipo+CPS12F&oGC (group 5 & 6) via intranasal instillation (i.n., group 1-5) or subcutaneous injection (s.c., group 6) for 3 times at 2 weeks intervals. Serum and saliva were collected at the start of the study and 2 weeks after each vaccination. Antibodies were quantified via indirect ELISA. Data were plotted as mean ± SEM (n = 8). Dunnett's T3 multiple comparisons tests were done to assess statistical significance. *, **, ***, **** represent P<0.1, P<0.01, P<0.001, P<0.0001.

[0034] Fig. 5: Lipo+CPS12F&oGC induced superior high-affinity CPS12F-specific antibody production compared with other formulations. Dissociation constants (KD) calculated for antibodies in serum and saliva. BALB / cJRj mice were immunized with Lipo+(group 1, blank cationic liposomes as placebo), Lipo+oGC (group 2), or 3 nmol antigen (repeat units of the polysaccharides) in Lipo+CPS12F (group 3), LipoCPS12F&oGC (group 4), or Lipo+CPS12F&oGC (group 5 & 6) via intranasal instillation (i.n., group 1-5) or subcutaneous injection (s.c., group 6) for 3 times at 2 weeks intervals. Serum and saliva were collected at the start of the study and 2 weeks after each vaccination. Their affinities were evaluated via competitive ELISA, in which the binding rate refers to the ratio of [Ab bound with suspension CPS12F] / [Ab bound with bottom CPS12F], and the KDequals the half-binding concentration of CPS12F. Rabbit antiserum against CPS12F from SSI Diagnostica was used as an external control. Data were plotted as mean ± SEM (DF = 29). Curves were fitted using the one-site total binding model. Dunnett's T3 multiple comparisons tests were done to assess statistical significance. *, **, ***, **** represent P<0.1, P<0.01, P<0.001, P<0.0001.

[0035] Fig. 6: Lipo+CPS12F&oGC generated antibodies with improved anti-S. pneumoniae 12F activity compared with other formulations. Half-killing dilution folds of the antibodies in serum and saliva samples. BALB / cJRj mice were immunized with Lipo+(group 1, blank cationic liposomes as placebo), Lipo+oGC (group 2), or 3 nmol antigen (repeat units of the polysaccharides) in Lipo+CPS12F (group 3), LipoCPS12F&oGC (group 4), or Lipo+CPS12F&oGC (group 5 & 6) via intranasal instillation (i.n., group 1-5) or subcutaneous injection (s.c., group 6) for 3 times at 2 weeks intervals. Serum and saliva collected 2 weeks after the final vaccination were pooled-assessed via OPKA. Data were fitted in the nonlinear dose-response model. Half-killing dilution folds were interpolated and plotted as mean ± SEM (DF>38). Dunnett's T3 multiple comparisons tests were done to assess statistical significance. *, **, ***, **** represent P<0.1, P<0.01, P<0.001, P<0.0001.

[0036] Fig. 7: Lipo+CPS12F&oGC vaccination protected mice from S. pneumoniae 12F challenge. Survival curves of vaccinated mice after bacteria challenge. BALB / cJRj mice were immunized with Lipo+(group 1, blank cationic liposomes as placebo), Lipo+oGC (group 2), or 3 nmol antigen (repeat units of the polysaccharides) in Lipo+CPS12F (group 3), LipoCPS12F&oGC (group 4), or Lipo+CPS12F&oGC (group 5 & 6) via intranasal instillation (i.n., group 1-5) or subcutaneous injection (s.c., group 6) for 3 times at 2 weeks intervals. Three weeks after the final vaccination, mice were challenged with 1 x 105CFU S. pneumoniae 12F via i.n. instillation and carefully monitored for the next 14 days. Results were plotted as survival curves. Gehan-Breslow-Wilcoxon tests were done to assess statistical significance. *, **, ***, **** represent P<0.1, P<0.01, P<0.001, P<0.0001.

[0037] DETAILED DISCLOSURE OF THE INVENTION

[0038] Definitions

[0039] By "liposome", as used herein, is meant an artificial vesicle comprising a lipid bilayer, which lipid bilayer encapsulates a lumen. A liposome may comprise phospholipids and / or other types of lipids. By "integrated in a lipid bilayer of the liposome", as used herein to describe the structural arrangement of the iNKT cell agonist, is meant at least partially integrated in the lipid bilayer. By "attached", as used herein to describe that the at least one bacterial polysaccharide may be attached to a lipid bilayer of the liposome, is meant directly connected or coupled, in a covalent or non-covalent manner, or connected through a linker or anchored, such as via a lipid.

[0040] By "bacterial polysaccharide antigen", as used herein, is meant a polysaccharide derived from a bacterium, such as, e.g., a bacterial capsular polysaccharide antigen, which is derived from the capsule of an encapsulated bacterium. The term "polysaccharide" has its normal meaning in the art, i.e. a saccharide chain comprising a certain number of linked monosaccharide units, such as at least 10 or at least 13 monosaccharide units. In contrast, a saccharide chain comprising a lower number of linked monosaccharide units, such as between 3 and 9 or between 3 and 12 monosaccharide units, is normally termed an "oligosaccharide" (see, e.g., Prestegard et al., Essentials of Glycobiology, 2015). The term "antigen" also has its normal meaning in the art, i.e. a substance that can be recognized specifically by the immune system (e.g. when bound by an antibody). Thus, as used herein, a "bacterial polysaccharide antigen" is a polysaccharide as defined above, derived from a bacterium, which has antigenic properties.

[0041] By "invariant natural killer T (iNKT) cell agonist", as used herein, is meant any molecule which can be presented to and is recognized by iNKT cells via their T cell receptor, and which activates them. An example of such a molecule is o-galactosylceramide (o-GalCer), which can be presented to iNKT cells by antigen-presenting cells presenting o-GalCer bound to a CDld molecule. The terms "derivative" and "analog" of an iNKT cell agonist are used interchangeably herein. The terms mean a variant iNKT cell agonist which is different from the iNKT cell agonist from which it is derived, or which it is an analog of, but which is still similar in chemical structure and / or function to that iNKT cell agonist. Often a derivative / analog will be a variant of the relevant molecule in which one or a few chemical groups have been changed, exchanged or removed.

[0042] By "immunological adjuvant", as used herein, is meant any molecule or substance which is capable of enhancing the immune response against an immunogen. In the context of the present invention, the invariant natural killer T (iNKT) cell ligand is itself an immunological adjuvant since it enhances the immune response against the bacterial polysaccharide antigen. As used herein, when it is described that the pharmaceutical composition comprises the inventive composition (comprising an iNKT cell agonist) and an immunological adjuvant, it is meant that the pharmaceutical composition comprises another immunological adjuvant in addition to the iNKT cell agonist. The immunological adjuvant may form part of the liposome composition.

[0043] By "non-modified native polysaccharide", as used herein, is meant a polysaccharide chain in its natural form, i.e. a non-chemically modified polysaccharide chain, meaning that it is not chemically different from the polysaccharide in its natural form. The polysaccharide chain may be directly obtained by extraction from bacterial culture, or may be synthesized, but preferably obtained by extraction. The process of obtaining the polysaccharide may comprise physical processing steps, such as ultrasonication to reduce polymerization degree.

[0044] Specific embodiments of the invention

[0045] Vaccine

[0046] In a first aspect, the invention relates to a composition, comprising : a) a liposome, b) at least one bacterial polysaccharide antigen, and c) at least one invariant natural killer T (iNKT) cell agonist, wherein the iNKT cell agonist is integrated in a lipid bilayer of the liposome, and wherein the at least one bacterial polysaccharide antigen is within the lumen of the liposome and / or attached to a lipid bilayer of the liposome.

[0047] Liposome

[0048] In some embodiments, the liposome is cationic at physiological pH.

[0049] In some embodiments, the liposome is anionic (negative surface charge) or neutral (no surface charge) at physiological pH, whereas in other, preferred, embodiments, it is cationic (positive surface charge) at physiological pH. By physiological pH is meant the physiological pH of the relevant organ, and it may be, but is not limited to, a pH of 7-8, such as 7.0, such as 7.2, such as 7.4, such as 7.6, such as 7.8, and such as 8.0.

[0050] A neutral liposome may, e.g., comprise a phospholipid selected from C12-C18 acyl saturated and unsaturated phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylglycerol phospholipids such as DLPC, DLPE, DLPG, DMPC, DMPE, DMPG, DPPC, DPPE, DPPG, DSPC, DSPE, DSPG, DOPC, DOPE, and DOPG, or PEGylated lipids, such as those selected from DLPE, DMPE, DPPE, DSPE, DOPE phospholipids PEGylated with either PEG350, PEG500, PEG750, PEGlk, PEG2k, PEG3k, and PEG5k, and any derivative thereof, or any combination thereof. Additionally, it may comprise a sterol, such as cholesterol, cholesterol hemisuccinate, lanosterol and ergosterol, any derivative thereof, such as PEGylated derivatives, with either PEG350, PEG500, PEG600, PEG750, PEGlk, PEG2k, PEG3k, PEG5k, l,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k) or Distearoyl- rac-glycerol-PEG2K (DSG-PEG2k), or any combination thereof. A cationic liposome may, e.g., comprise any combination of the components described above for the neutral liposome, and in addition it may comprise a cationic lipid selected from trimethylammonium propane (TAP), ethylphosphocholine (EPC), DC-chol, DOTMA, DOSPA, DDAB, DMDAP, DPDAP, DSDAP, DODAP, DODMA, DOBAQ, DLin-DMA, DLin-KC2-DMA, DLin- MC3-DMA, C12-200, A6, OF-02, A18-Iso5-2DC18, YSK05, 7C1, G0-C14, L319, OF-Deg-Lin, 306-O12B, 3060110, FTT5, 9A1P9, 98N12-5, 304013, CKK-E12, Spermine-chol, and MVL5, or any combination thereof.

[0051] In preferred embodiments, a cationic liposome according to the invention comprises PC, cholesterol and TAP, such as PC in the form of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and TAP in the form of l,2-distearoyl-3-trimethylammonium-propane (DSTAP).In some embodiments, the composition according to the first aspect of the invention comprises:

[0052] - at least one neutral phospholipid, such as PC, such as DSPC;

[0053] - optionally at least one cationic phospholipid, such as TAP, such as DSTAP;

[0054] - at least one sterol, such as cholesterol;

[0055] - at least one iNKT cell agonist, such as o-GalCer; and

[0056] - at least one bacterial polysaccharide antigen; in a molar ratio of: (neutral phospholipid) 3-6, such as 3.5, such as 4.0, such as 4.5, such as 5.0, and such as 5.5: (cationic phospholipid) 0.1-3, such as 0.15, such as 2.0, and such as 2.5: (sterol) 2-4, such as 2.5, such as 3.0, and such as 3.5: (iNKT cell agonist) 0.01-2, such as 0.015: (bacterial polysaccharide antigen) 0.01-2, such as 0.015.

[0057] In some embodiments, the composition according to the first aspect of the invention comprises: DSPC; DSTAP; cholesterol; o-GalCer; and at least one bacterial polysaccharide antigen; in a molar ratio of: (DSPC) 3-6, such as 3.5, such as 4.0, such as 4.5, such as 5.0, and such as 5.5: (DSTAP) 0.1-3, such as 0.15, such as 2.0, and such as 2.5: (cholesterol) 2- 4, such as 2.5, such as 3.0, and such as 3.5: (o-GalCer) 0.01-2, such as 0.015: (bacterial polysaccharide antigen) 0.01-2, such as 0.015.

[0058] In specific embodiments, the molar ratio is: (DSPC) 5.4: (DSTAP) 1.5: (cholesterol) 3.0: (o- GalCer) 0.015: (bacterial polysaccharide antigen) 0.015.

[0059] In some embodiments, the liposome has a hydrodynamic size distribution of 1 to 1000 nm, preferably 100 to 200 nm, as measured by dynamic light scattering (DLS).

[0060] Liposomes belong to the larger group of nanoparticles. The present invention also enables compositions comprising other types of nanoparticles than a liposome.

[0061] Thus, in one aspect, the invention relates to a composition, comprising: a) a nanoparticle, b) at least bacterial polysaccharide antigen, and c) at least one invariant natural killer T (iNKT) cell agonist, wherein the iNKT cell agonist and the bacterial polysaccharide antigen is integrated in, present within, and / or attached to the nanoparticle.

[0062] Bacterial polysaccharide antigen

[0063] In some embodiments, the at least one bacterial polysaccharide antigen is a non-modified native polysaccharide.

[0064] As defined above, by non-modified native polysaccharide is meant that the polysaccharide chain is in its natural form, i.e. it is non-chemically modified, meaning that it is not chemically different from the polysaccharide in its natural form. The polysaccharide chain may be directly obtained by extraction from bacterial culture, or may be synthesized, but preferably obtained by extraction. The process of obtaining the polysaccharide may comprise physical processing steps, such as ultrasonication to reduce polymerization degree.

[0065] In some embodiments, the at least one bacterial polysaccharide antigen comprises a saccharide chain of at least 10, such as at least 11, such as at least 12, and such as at least 13, linked monosaccharide units.

[0066] In some embodiments, the at least one bacterial polysaccharide antigen is non-conjugated, meaning that it is not conjugated to any other molecule.

[0067] In some embodiments, the at least one bacterial polysaccharide antigen is not conjugated to a peptide or protein.

[0068] In some embodiments, the at least one bacterial polysaccharide antigen is not conjugated to the iNKT cell agonist.

[0069] In some embodiments, the at least one bacterial polysaccharide antigen is not attached to a lipid bilayer of the liposome.

[0070] By "attached" or "conjugated" as used in this context is meant directly connected or coupled, in a covalent or non-covalent manner, connected through a linker or anchored, such as via a lipid. In some embodiments, the bacterial polysaccharide antigen is a capsular polysaccharide.

[0071] In further embodiments, the capsular polysaccharide is derived from a bacterium selected from: Bordetella pertussis, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, and Streptococcus pneumoniae.

[0072] In preferred embodiments, the capsular polysaccharide is derived from S. pneumoniae.

[0073] In preferred embodiments, the capsular polysaccharide is derived from S. pneumoniae serotype 12F.

[0074] A vaccine should preferably target as many different serotypes of a bacterial species as possible. In the case of S. pneumoniae, there are currently more than 100 serotypes documented (see URL: https: / / www.pneumogen.net / gps / serotypes.html, accessed in Oct 2023).

[0075] Thus, in some embodiments, the composition comprises at least two, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, such as at least 11, such as at least 12, such as at least 13, such as at least 14, such as at least 15, such as at least 16, such as at least 17, such as at least 18, such as at least 19, such as at least 20, such as at least 21, such as at least 22, such as at least 23, such as at least 24, such as at least 25, such as at least 26, such as at least 27, such as at least 28, such as at least 29, such as at least 30, such as at least 40, and such as at least 50, different capsular polysaccharides, which are derived from the same number of different serotypes of S. pneumoniae.

[0076] In further embodiments, the at least two different serotypes are selected from the following : 1, 2, 3, 4, 5, 6A, 6E(6A), 6B, 6E(6B), 6C, 6D, 6H, 7F, 7A, 7B, 7C, 7D, 8, 9A, 9L, 9N, 9V, 10F, 10A, 10B, IOC, 10D, 10F, 11F, 11A, 11B, 11C, 11D, 11E, llAv, 11F, 12F, 12A, 12B, 13, 14, 15F, 15A, 15B, 15C, 16F, 16A, 17F, 17A, 18F, 18A, 18B, 18C, 19F, 19A, 19B, 19C, 20, 20A, 20B, 21, 22F, 22A, 23F, 23A, 23B, 24F, 24A, 24B, 24C, 25F, 25A, 7 , 28F, 28A, 29, 31, 32F, 32A, 33F, 33A, 33B, 33C, 33D, 34, 35F, 35A, 35B, 35C, 35D, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48, 9X, 11X, 16X, 18X1, 18X2, 18X3, 29X, 33X, and 36X.

[0077] More preferably, the at least two different serotypes are selected from the following : 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, and 35B. The present invention also enables compositions comprising other types of antigens than a bacterial polysaccharide antigen.

[0078] Thus, in one aspect, the invention relates to a composition, comprising : a) a liposome, b) at least one antigen, and c) at least one invariant natural killer T (iNKT) cell agonist, wherein the iNKT cell agonist is integrated in a lipid bilayer of the liposome, and wherein the at least one antigen is within the lumen of the liposome and / or attached to a lipid bilayer of the liposome.

[0079] The at least one antigen according to this aspect may, e.g., be selected from: a protein, a peptide, a polysaccharide, and a lipid. iNKT cell agonist

[0080] The iNKT cell agonist may be any molecule which activates an iNKT cell through its T cell receptor, preferably wherein the molecule is presented to the iNKT cell bound to a CDlx, such as a CDld, receptor. The iNKT cell agonist may be either naturally occurring or synthetic.

[0081] In some embodiments, the at least one iNKT cell agonist may be at least two, such as 2, such as 3, such as 4, such as 5, such as 6, such as 7, such as 8, such as 9, and such as 10, different iNKT cell agonists.

[0082] In some embodiments, the at least one iNKT cell agonist is selected from any one of: o- galactosylceramide (o-GalCer), o-galactosyldiacylglycerol (o-GalDag), o- glucosyldiacylglycerol (o-GIcDag), o-glucuronsylceramide (o-GIcACer), o- galacturonosylceramide (o-GalACer), phosphatidyl-myo-inositol mannoside (PIM2), and any derivative of any one of these ligands, or any combination thereof. o-GalCer, o-GalDag, o-GIcDag, o-GIcACer, o-GalACer, and PIM2 have all been shown to act as iNKT cell agonists, which is why they are classified as such herein. However, they may have additional modes of action, which do not necessarily go through iNKT cells, e.g., they may activate other cell types. Regardless of the specific mode of action, and the cell type activated (or inhibited), in a given vaccine and under given circumstances, the listed molecules and their analogues described herein are still considered "iNKT cell agonists" for the purposes of describing them as components of a composition according to the invention. In preferred embodiments, the at least one iNKT cell agonist is o-GalCer.

[0083] • a-GalCer o-galactosylceramide (o-GalCer), also known as KRN7000, is a synthetic glycolipid consisting of a D-galactose moiety linked to a phytosphingosine (reviewed in Romano and Clausen, Eur. J. Org. Chem., 2022, hereby incorporated by reference herein in its entirety). o-GalCer acts as a potent iNKT cell agonist. o-GalCer is commercially available (as of October 2023, see Example 1, Materials and methods, "Liposomal vaccines preparation"), and it can also be synthesized from D-galactose by different routes, such as the synthesis routes described in Romano and Clausen (see Scheme 1).

[0084] In some embodiments, the at least one iNKT cell agonist is a derivative or analogue of o- GalCer.

[0085] Examples of analogs of o-GalCer include those shown in Figure 2 in Romano and Clausen, i.e. : OCH, PBS-25, C20 :2, 24, 25, AGP, 7DW8-5, C34, DB06-1, and AH03-1.

[0086] Especially preferred analogues of o-GalCer are analogues containing C-6" modifications. Examples of such analogues include those shown in Figure 3 in Romano and Clausen, i.e. : NU-o-GalCer, PyrC-o-GalCer, AH10-7, 27, 28, 29, 30, and 31.

[0087] Structural arrangement and preferred combinations of the vaccine composition

[0088] In preferred embodiments, the at least one bacterial polysaccharide antigen is within the lumen of the liposome. In such embodiments, the antigen may either be in "free form", such as present in soluble or dispersed form (i.e., in solution, in suspension or in colloidal suspension), within the lumen, i.e. unattached to a lipid bilayer of the liposome, or it may be attached, such as covalently conjugated or such as non-covalently anchored, to a lipid bilayer of the liposome, preferably in a manner wherein the polysaccharide protrudes into or is otherwise in direct contact with the aqueous phase within the lumen. Preferably, the antigen is within the lumen of the liposome without being attached to a lipid bilayer of the liposome.

[0089] In other embodiments, the antigen is not within the lumen of the liposome but attached to a lipid bilayer of the liposome, such as wherein the antigen is attached, such as covalently conjugated or such as non-covalently anchored, to the outer surface of the liposome. In preferred embodiments, the at least one bacterial polysaccharide antigen is a capsular polysaccharide derived from S. pneumoniae, and the iNKT cell agonist is o-GalCer.

[0090] Production of the vaccine composition

[0091] The vaccine composition according to the present invention may be prepared by the extrusion method as described in Example 1 (see under Materials and methods, "Liposomal vaccines preparation"), or it may be prepared using any one of the methods summarized in Lombardo et al., Pharmaceutics, 2022, hereby incorporated by reference herein in its entirety. These methods include methods for liposome fabrication, such as thin-film hydration, self-assembly, injection, and reverse-phase evaporation, and methods for liposome downsizing, such as extrusion, sonication, and high-pressure homogenization. They also include methods using some newer technologies, such as lyophilization, dense gas methods, microfluidic methods, and membrane contactor methods.

[0092] Pharmaceutical composition

[0093] In a second aspect, the invention relates to a pharmaceutical composition comprising the composition according to embodiments of the first aspect of the invention and a pharmaceutically acceptable vehicle and / or diluent and / or immunological adjuvant.

[0094] Preferred vehicles include diluents such as water, saline, glycerol, and ethanol, and substances such as pH buffering substances.

[0095] In some embodiments, the pharmaceutical composition comprises the composition according to embodiments of the first aspect of the invention and an immunological adjuvant.

[0096] The person skilled in the art will know how to select a suitable immunological adjuvant for the present purpose. Such adjuvant may, e.g., be an adjuvant described in: Guerrero Manriquez and Tuero, Human Vaccines & Immunotherapeutics, 2021 (hereby incorporated by reference herein in its entirety).

[0097] Preferred adjuvants include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate; (2) adjuvants containing one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS); (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; and (5) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

[0098] The pharmaceutical composition may be prepared as an injectable solution, in solid form suitable for suspension in liquid vehicles prior to injection, or, preferably, as a solution or in solid form suitable for producing an aerosol for inhalation. In some embodiments, the composition in solid form may be a freeze-dried composition, which may provide for an increased shelf-life prior to resuspension than a composition in solution.

[0099] In some embodiments, the pharmaceutical composition may form part of a kit-of-parts with a device for vaccine delivery, such as the nasal spray device according to the fifth aspect of the invention, as described below.

[0100] Vaccination method

[0101] In a third aspect, the invention relates to a method of treating or preventing a bacterial infection in a mammal, such as a human being, said method comprising administering a pharmaceutically effective amount of the composition according to embodiments of the first aspect of the invention or of the pharmaceutical composition according to embodiments of the second aspect of the invention to said mammal.

[0102] In some embodiments, the bacterial infection is an airway infection, such as an upper airway and / or lung infection.

[0103] However, the infection may also be any other type of bacterial infection, such as an infection of the ear canal, brain, muscle, skin, eye or bone marrow.

[0104] In some embodiments, the composition according to embodiments of the first aspect of the invention or the pharmaceutical composition according to embodiments of the second aspect of the invention is administered intranasally, subcutaneously, orally or intramuscularly, preferably intranasally, or any combination thereof.

[0105] In some embodiments, the immunization scheme includes that the mammal (e.g. the human) receives a priming administration and one or more booster administrations. Further according to the invention, the composition according to embodiments of the first aspect of the invention or the pharmaceutical composition according to embodiments of the second aspect of the invention is for use in a method of medical treatment.

[0106] In a fourth aspect of the invention, the composition according to embodiments of the first aspect of the invention or the pharmaceutical composition according to embodiments of the second aspect of the invention is for use in a method of treating or preventing a bacterial infection in a mammal, such as a human being. As such, the composition or the pharmaceutical composition is for use in a method according to embodiments of the third aspect of the invention.

[0107] Furthermore, the invention provides for use of the composition according to embodiments of the first aspect of the invention or the pharmaceutical composition according to embodiments of the second aspect of the invention for treating or preventing a bacterial infection in a mammal, such as a human being.

[0108] Finally, the invention provides for use of the composition according to embodiments of the first aspect of the invention or the pharmaceutical composition according to embodiments of the second aspect of the invention in the manufacture of a medicament for treating or preventing a bacterial infection in a mammal, such as a human being.

[0109] Nasal spray device

[0110] In a fifth aspect, the invention provides a nasal spray device, comprising : a pressurised aerosol canister including a vial containing the pharmaceutical composition according to embodiments of the second aspect of the invention, an actuator, and a delivery outlet for the aerosol plume.

[0111] In some embodiments, the nasal spray device is configured to deliver metered doses of the pharmaceutical composition to the nasal cavity of a mammal, such as a human being. EXAMPLE 1

[0112] Materials and methods

[0113] Liposomal vaccines preparation

[0114] The liposomal vaccines were prepared by the extrusion method. The protocols were modified from the manufacturer's instructions (Avanti Polar Lipids, "Mini-extruder extrusion technique" and "Liposome preparation", accessed on URL: https: / / avantilipids.com / divisions / equipment- products / mini-extruder-extrusion-technique and https: / / www.sigmaaldrich.com / DK / en / technical-documents / protocol / cell-culture-and-cell- culture-analysis / transfection-and-gene-editing / liposome-preparation, respectively, in October 2023).

[0115] Briefly, 53.5 % of DSPC (Avanti Polar Lipids® 850365P), 15 % of DSTAP (Avanti Polar Lipids® 890880P), 30 % of cholesterol (Sigma-Aldrich® C8667), and 1.5 % of o-GalCer (Biosynth® MG15978) at molar ratio were weighed, dissolved in t-BuOH / H2O (9 / 1, v / v) mixture at 70 °C (above the phase transition temperatures, Tm, DSPC = 55 °C, Tm, DSTAP < 5 °C), mixed and stirred at 70 °C in a heating block for 30 minutes, frozen with liquid nitrogen, and lyophilized overnight on a freeze dryer (LaboGene™ ScanVac CoolSafe) to remove the solvent. On the next day, the lyophilized lipid membrane was rehydrated in HEPES-buffered saline (10 mM HEPES, 150 mM NaCI, pH adjusted to 7.4, and filtered through 0.22 pm membrane), mixed with 1.5 % hydrophilic CPS12F (SSI Diagnostica 76939) at the molar ratio, diluted to a lipid concentration of 40 mM, stirred at 70 °C for 1 hour, and finally extruded through a 0.2 pm hydrophilic membrane (Nuclepore™ 10417004) 21 times in an extruder (Avanti Polar Lipids) at 70 °C.

[0116] Size distribution and stability evaluation

[0117] Hydrodynamic size distribution was assessed by dynamic light scattering (DLS) in a zetasizer (MalvernNanoZS). The samples were separately stored at room temperature (18-24 °C) or in the fridge (2-8 °C) and repeatedly characterized by the zetasizer for stability evaluation. Mouse strain, vaccination route, and sa method

[0118] Animal experiments in this study were licensed (2020-15-0201-00482-C1 and 2022-15- 0201-01249) by the Danish Animal Testing Authority (Dyreforsogstilsynet) and approved (AEHAN_001 and PW_002) by the Bio Facility at the Technical University of Denmark. 6-8 weeks old BALB / cJRj female mice were purchased from JANVIER LABS®; after 1 week of acclimation, they were randomized with a block size of 4 and bred in IVC cages at DTU Bio Facility. The vaccinations were carried out via subcutaneous (s.c.) injections at 50 pL or intranasal (i.n.) installations (UCSF IACUC, "IACUC standard procedure: Intranasal instillation in rodents", accessed on URL: https : / / iacuc.ucsf. edu / sites / g / files / tkssra751 / f / wysiwyg / STDPROCEDU RE- MiscRodentProcedures-IntranasalInstillationinRodents.pdf, in October 2023) at 25 pL for each nostril, which would give enough exposure to NALT and iBALT.

[0119] For cytokine and antibody quantification, at defined time points, blood samples were collected by sublingual bleeding and processed into the serum to evaluate the systemic immune responses, and saliva samples were collected with sampling sponges (Nasco® B01245WA) after pilocarpine injection (Medchem Express HY-B0726, saliva secretion stimulator, i.p., 0.36 pg / g of body weight) to evaluate mucosal immune responses (Zubeidat, STAR Protocols, 2022). Samples were stored at 2-8 °C for the short term and -80 °C for the long term.

[0120] For germinal center examination and cell phenotyping, 3 days after the final vaccination, half of the mice were sacrificed by cervical dislocation, and their lung and spleen were cryopreserved with OCT (CellPath KMA-0100-00A) for immunohistochemistry analysis or prepared into single-cell suspensions for multi-color flow cytometry analysis (data not shown). Another half of the mice were challenged with bacteria 1 week after the final serum and saliva sampling to evaluate the efficacy of protection in vivo.

[0121] At the end of the study, all remaining mice were euthanized by cervical dislocation.

[0122] Cytokines (IL-12, IL-4, IFN-y, and IL-17) were quantified with BD™ cytometric bead array (CBA) assay kits (BD Biosciences 562264, 562272, 562233, 562261) via flow cytometry according to the manufacturer's protocol ("BD cytometric bead array (CBA) mouse enhanced sensitivity master buffer kit instruction manual", accessed on URL: https: / / www.bdbiosciences.com / content / dam / bdb / marketing-documents / Mouse-Enhanced- Sensitivity-Master-Buffer-Kit.pdf, in October 2023).

[0123] Briefly, serum and saliva samples were collected 18 hours after the priming vaccination, diluted at 5 folds for serum and 10 folds for saliva, captured by antibodies conjugated with beads with specific fluorescent profiles by incubating for 2 hours, further loaded with PE- labelled antibodies for concentration evaluation by incubating for 2 hours. Samples were prepared in 96-well round-bottom plates (Corning® 3365). Unbound components were washed away by centrifugation (400 x g, 5 min) and discarding the supernatant. Then, samples and standards were acquired on a flow cytometer (BD© LSRFortessa™) through FSC-A, FSC-W, SSC-A, SSC-W, PE-A, APC-A, and APC-Cy7-A channels, and their median fluorescence intensities of PE channel (MFIPE) were calculated in FlowJo 10. Finally, in GraphPad Prism 9, the standard curves were fitted, the concentrations of the cytokines were interpolated, and Dunnett's T3 multiple comparisons tests were executed to assess statistical significance.

[0124] Antibody quantification

[0125] At the start of the study and 2 weeks after each vaccination, serum and saliva were collected from the mice and stored at -80 °C. CPS12F-specific antibodies in the samples were measured by indirect ELISA.

[0126] Briefly, 384-well plates (Nunc™ MaxiSorp™ 464718) were coated with 100 ng CPS12F (SSI Diagnostica 76939) per well, blocked with BSA (Invitrogen™ DS98200); then, diluted testing samples, HRP-conjugated goat anti-mouse IgM (abeam ab97230), IgA (abeam ab97235), IgGpoiy (abeam ab6789), and TMB chromogen solution (Invitrogen™ SB02) were sequentially added and incubated in the plate. Unbound components in the well were washed away with PBS + 0.05 % (w / v) Tween 20 buffer (PBST, Thermo Scientific™ Pierce™ 28352) between each step. After that, 0.16 M sulfuric acid (Thermo Scientific™ SS04) was added to stop the reaction, and OD45o in each well was acquired in a microplate reader (TECAN Spark® Cyto) referenced by OD5so. Finally, the data were analyzed in GraphPad Prism 9, and Dunnett's T3 multiple comparisons tests were done to assess statistical significance.

[0127] Antibody affinity measurement

[0128] The affinities of antibodies in the samples to CPS12F were determined by competitive ELISA, in which the binding kinetics K equals the half-binding concentration of CPS12F (Martineau, Springer Protocols Handbooks, 2010). Briefly, diluted testing samples were incubated with serially diluted CPS12F (SSI Diagnostica 76939) and transferred to 384-well plates (Nunc™ MaxiSorp™ 464718, coated with 2 ng CPS12F per well and blocked with BSA, Invitrogen™ DS98200, in advance); then, HRP- conjugated goat anti-mouse IgGpOiy(abeam ab6789) and TMB chromogen solution (Invitrogen™ SB02) were sequentially added and incubated in the plate. Unbound components in the well were washed away with PBS + 0.05 % (w / v) Tween 20 buffer (PBST, Thermo Scientific™ Pierce™ 28352) between each step. After that, 0.16 M sulfuric acid (Thermo Scientific™ SS04) was added to stop the reaction, and OD45o in each well was acquired in a microplate reader (TECAN Spark® Cyto) referenced by OD5so. Finally, in GraphPad Prism 9, the data were fitted in sigmoidal & 4 parameters logistical curves, the half-binding concentration was interpolated, and the statistical significance were assessed by Dunnett's T3 multiple comparisons tests.

[0129] Protection efficacy evaluation in vitro

[0130] The vaccine's protective efficacy was evaluated in vitro via the opsonophagocytic killing assay (OPKA). The protocol was modified from the published protocol (Nahm and Burton, "Protocol for multiplexed opsonophagocytic killing assay (UAB-MOPA) for antibodies against Streptococcus pneumoniae", accessed on URL: https: / / www.vaccine.uab.edu / uab-mopa.pdf, in October 2023).

[0131] Briefly, BALB / cJRj mice were immunized with vaccines 3 times at 2 weeks intervals; 2 weeks after the final vaccination, serum and saliva samples were collected and cryopreserved at - 80°C until use. S. pneumoniae 12F (SSI Diagnostica 82201) bacteria was cultured in THY broth (Todd-Hewitt broth (OXOID™ CM0189B) with 0.5 % (w / v) yeast extract (BioReagents™ BP1422)) with 5 % (v / v) CO2at 37 °C until its ODSoo reaches 0.8; then, it was added with 15 % (v / v) glycerol (Invitrogen™ 15514011, cryoprotectant), aliquoted at 0.5 mL, cryopreserved at -80 °C, and quantified by serial dilution culture on THYA plates (THY broth with 1.5 % (w / v) agar (BD BACTO™ 214010)) and CFU counting. HL-60 cells (ATCC® CCL- 240™) were differentiated into phagocytes with 0.8 % (v / v) of DMF (Thermo Scientific™ 210585000) added into the medium for 3-5 days culture. On the day of assay, cryopreserved bacteria were thawed, diluted to 5xl04CFU / mL with freshly prepared opsonization buffer (5 % (v / v) heat-inactivated FBS (Gibco™ A3160802) and 0.1 % (w / v) gelatin (Thermo Scientific™ 410875000) in HBSS (Gibco™ 24020091)), and aliquoted at 10 pL (500 CFU) into 96-well conical-bottom plates. Serum and saliva samples were treated at 56 °C for 30 minutes to deactivate endogenous complement, serially diluted by 3 folds at 30 pL (20 pL left) and mixed with bacteria suspension in the plates for 30 minutes of incubation at room temperature. Then, differentiated HL-60 cells were resuspended in HBSS at 1 x 107cells / mL, mixed with baby rabbit complement (MP Biomedicals 08642961) at 4 / 1 (v / v), and aliquoted at 50 pL into the plates for 45 minutes of coculture (cells / bacteria=800 / l) with 5 % (v / v) CO2at 37 °C. After that, 10 pL of each mixture was spotted on the THYA plates, overlayed with THYA supplemented with T.T.C. (OXOID™ SR0148A), cultured at 37 °C for 18 hours; then, red colonies formed by un-phagocytosed bacteria were counted with software NICE (NIST's Integrated Colony Enumerator, accessed on URL: https: / / doi.org / 10.18434 / M32073, in October 2023). Finally, in GraphPad Prism 9, the data were fitted in sigmoidal & 4 parameters logistical curves, the half-killing dilution folds were interpolated, and Dunnett's T3 multiple comparisons tests were done to assess statistical significance.

[0132] Protection efficacy evaluation in vivo

[0133] The vaccine's protective efficacy was evaluated in vivo through the bacteria challenge study.

[0134] Briefly, mice were immunized with vaccines 3 times at 2 weeks intervals; 3 weeks after the final vaccination, each mouse was challenged with 1 x 105CFU of S. pneumoniae 12F via i.n. instillation; then, mice were closely monitored for the following 2 weeks; finally, in GraphPad Prism 9, survival curves were plotted, and Gehan-Breslow-Wilcoxon tests were executed to assess statistical significance.

[0135] Results

[0136] Preparation and characterization of cationic liposomal vaccines

[0137] The complete formulation was cationic liposomal CPS12F with o-GalCer, abbreviated as Lipo+CPS12F&oGC (Figure 1, Table 1). CPS12F was the naturally extracted antigen from S. pneumoniae 12F, and o-GalCer was co-formulated to induce iNKT-mediated immune responses. In the liposome recipe, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) was used as the backbone lipid, cholesterol was added to improve the stability, and 1,2- distearoyl-3-trimethylammonium-propane (DSTAP) was included to introduce positive charges on the liposome surface. The vaccine was designed to be preferably administered through the i.n. route. Table 1 : List of vaccine formulations tested in the mouse study

[0138] After static placement, the neutral liposomes were deposited to the bottom; in contrast, the cationic liposomes remained stable in the suspension presumably because their positive surface charge prevented aggregation due to electrostatic repulsion. However, this did not prove the cationic liposome was more stable than the neutral liposome, as the homogeneity could be restored to the neutral liposome suspension by simple vortexing or pipetting.

[0139] Liposomal vaccines of all groups displayed homogeneous hydrodynamic size distribution ranging from 150 to 200 nm. The hydrodynamic size of all liposomal vaccines did not undergo changes after storage at either room temperature (18-24 °C) or in the fridge (2-8 °C). Figure 2 shows hydrodynamic size stability data for the Lipo+CPS12F&oGC formulation of groups 5 and 6 (see Table 1), data is not shown for the other formulations. The storage length was superior to both the widely applied liposomal vaccine (12 hours at room temperature) for COVID-19 and the non-liposomal vaccine PCV20 (96 hours at room temperature) for pneumonia by Pfizer ("Pfizer-Biontech COVID-19 vaccine: Highlights of emergency use authorization", accessed on URL: https: / / labeling. pfizer. com / ShowLabeling.aspx?id = 19542, in October 2023, and "Prevnar 20

[0140] - highlights of prescribing information", accessed on URL: https: / / labeling. pfizer. com / ShowLabeling.aspx?id = 15428, in October 2023). Lipo+CPS12F&oGC stimulated elevated pro-inflammatory cytokines secretion

[0141] It was first investigated whether Lipo+CPS12F&oGC can invoke cells to secrete pro- inflammatory cytokines, including IL-12, IFN-y, IL-4, and IL-17 by cytometric bead array assay. Antigen-presenting cells (e.g., dendritic cells) secrete IL-12, which helps iNKT cells activate and proliferate; iNKT cells further secrete IFN-y, IL-4, and IL-17, which separately promote their differentiation into NKT1, NKT2, and NKT17. Combinations of the cytokines will create different cytokine milieus, which will steer the final immune responses.

[0142] Lipo+CPS12F&oGC, when delivered intranasally (group 5) or subcutaneously (group 6), both induced the elevation of IL-12, IFN-y, IL-4, and IL-17 levels (Figure 3). Moreover, it was observed that the s.c. route resulted in higher levels of cytokines in serum, indicating a stronger response from the systemic immune system, as compared to the i.n. route. However, this difference was not found to be statistically significant in saliva, representing the respiratory mucosal immune system (nasal- and inducible bronchus-associated lymphoid tissue, NALT and iBALT). The cationic formulation (group 5) presented more potential in cytokine stimulation than the neutral formulation (group 4), although the differences were slight. The secretion of IL-12 and IFN-y displayed a strong correlation with iNKT agonist o- GalCer, as the cationic liposomal o-GalCer (group 2) independently induced the production of IL-12 and IFN-y.

[0143] Overall, these results showed that Lipo+CPS12F&oGC successfully stimulated the secretion of pro-inflammatory cytokines. The liposome surface charge, o-GalCer, and immunization route all played a role in this effect. The combination of these cytokines could further activate immune cells and enhance both innate and adaptive immune responses.

[0144] Lipo+CPS12F&oGC induced isotype-switched high-affinity antibodies against CPS12F

[0145] Then, the change in antibody levels after priming-booster vaccinations and their isotype composition 2 weeks after being fully immunized was tested by indirect ELISA.

[0146] All antigen-containing formulations (group 3-6) provoked the secretion of IgM (Figure 4a) and IgGpoiy both in the systemic and mucosal immune systems. Different from IgM, which peaked 2 weeks after the 1stbooster vaccination, IgGpOiylevel continued to rise following the priming and booster vaccinations (data not shown). It validated that IgM is the first responded antibody after stimulation, which can switch into more matured IgG or IgA after B cells go through somatic hypermutation in germinal centers. IgA is better than IgG in clearing pneumococcus in the respiratory mucosal system because IgG mainly functions via complement fixation, opsonization, and their mediated phagocytosis and cytotoxicity (CDC, CDCP, ADCC, and ADCP), during which toxins, such as pneumolysin, might be released and cause inflammation. In contrast, IgA is safer as it acts by neutralizing and agglutinating the bacteria, which will finally be removed by ciliary motion.

[0147] Vaccination with Lipo+CPS12F&oGC (group 5 & 6) effectively helped mice produce IgGpOiy(Figure 4b) and IgA (Figure 4c). While the local-distant immune response effect appeared again in the result of IgGpOiy, it is remarkable that mice vaccinated intranasally displayed higher IgA levels in both serum and saliva. Although the differences were not statistically significant, the p-value at 0.14 in saliva suggested the trend. Both results proved that the i.n. route was more promising since it produced more IgG and IgA in the mucosal system. Similar to the results above, the positive surface charge also increased the secretion of IgGpOiyand IgA, further confirming the cationic liposome platform's capability to deliver mucosal vaccine components.

[0148] Further, the affinity of the antibody in collected samples against CPS12F was measured by competitive ELISA. Lipo+CPS12F&oGC provoked the production of antibodies with higher affinities (Figure 5: i.n. vaccination: KD_serum = 66.9 ± 4.7 x 10“9M, KD_saiiva = 259.5 ± 19.4 x IO"9M; s.c. vaccination: KD_serUm = 29.3 ± 2.3 x IO"9M, KD_saiiva = 530.9 ± 49.2 x IO"9M) than the commercial high-affinity rabbit antiserum (KD= 132.5 ± 6.4 x 10-7M) from SSI Diagnostica. The results indirectly proved that the designed carbohydrate vaccine successfully activated the adaptive immune responses, in which the B cells went through the somatic hypermutation and finally secreted the mature high-affinity antibodies. Similar to the results in the experiments above, iNKT agonist o-GalCer and the cationic liposome both played roles in generating high-affinity antibodies. Furthermore, while s.c. injection generated higher affinity antibodies in serum, i.n. instillation produced higher affinity antibodies in saliva. This feature indicated the superiority of i.n. vaccination in combating respiratory pathogens.

[0149] To sum up, Lipo+CPS12F&oGC efficiently induced the secretion of isotype-switched and affinity-matured CPS12F-specific IgG and IgA both in systemic and mucosal immune systems, which was enhanced by the iNKT agonist o-GalCer and cationic liposome design. The results also suggested that i.n. vaccination was more capable of eliciting local airway immune responses. Generated CPS-specific antibodies efficiently killed S. pneumoniae in vitro

[0150] The protective efficacy of the generated antibodies against S. pneumoniae was then tested in vitro through the opsonophagocytic killing assay (OPKA). The antibodies contributed to bacteria clearance mainly by attaching to the pneumococcus surface and mediating phagocytosis by Fc and FcyR recognition (antibody-dependent cellular phagocytosis, ADCP) or contributing to the formation of complement C3b and mediating phagocytosis by C3b and C3bR recognition (complement-dependent cellular phagocytosis, CDCP). Their efficiencies were quantified as their half-killing dilution folds (Figure 6).

[0151] The serum and saliva induced by Lipo+CPS12F&oGC (group 5 & 6) both displayed elevated anti-S. pneumoniae 12F activity compared with the o-GalCer-lacking (group 3) and neutral liposomal (group 4) formulation, suggesting the significance of iNKT agonist o-GalCer and the cationic liposome in improving the immune responses. It is remarkable that, with the same formulation of Lipo+CPS12F&oGC, while the s.c. route (group 6) produced more effective serum, the i.n. administration (group 5) tended to produce more effective saliva. However, the difference in saliva tests was not statistically significant.

[0152] The result verified the anti-S. pneumoniae efficiency of the serum and saliva collected from mice immunized with Lipo+CPS12F&oGC. The i.n. route was more promising, as it mainly stimulated the respiratory mucosal immune system, which was proximal to the infection and disease site.

[0153] Lipo+CPS12F&oGC vaccination protected mice from S. pneumoniae challenge

[0154] Finally, a bacteria challenge study was performed to evaluate the protective potential of Lipo+CPS12F&oGC. BALB / cJRj mice were fully vaccinated with different vaccines intranasally (group 1-5) or subcutaneously (group 6) and then challenged with S. pneumoniae 12F via i.n. instillation. The treated mice were carefully followed for the next 14 days, and their mortality data were recorded and plotted as the survival curve.

[0155] In Figure 7, it is notable that mice intranasally vaccinated with Lipo+CPS12F&oGC (group 5) all survived the 2 weeks after infection; on the contrary, mice vaccinated with blank cationic liposome (group 1) or cationic liposomal o-GalCer (group 2) were all sacrificed within the first week. It demonstrated the significant (P<0.0001) protection rendered by Lipo+CPS12F&oGC when applied intranasally. Meanwhile, only 2 of 8 (25 %) mice survived after receiving the formulation without o-GalCer (group 3), and 5 of 8 (62.5 %) mice survived after receiving the neutral surface charge formulation (group 4), validating the important roles of the iNKT agonist o-GalCer and nasal mucosa attachment facilitating positive surface charge.

[0156] Notably, 6 of 8 (75 %) mice survived after receiving the whole formulation via s.c. injection (group 6); compared with group 5, although the difference was not statistically significant, the P value of 0.14 indicated the route of vaccination played a role in the final protection strength. S. pneumoniae 12F mainly colonizes and causes disease in the respiratory system. Applying the vaccines intranasally can activate the NALT and iBALT, which is proximal to the infection site and more robust than the remote immune responses elicited by systemic s.c. vaccination.

[0157] These results confirmed that Lipo+CPS12F&oGC, when immunized intranasally, can efficiently protect mice from S. pneumoniae 12F challenge.

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Claims

CLAIMS1. A composition, comprising : a) a liposome, b) at least one bacterial polysaccharide antigen, and c) at least one invariant natural killer T (iNKT) cell agonist, wherein the iNKT cell agonist is integrated in a lipid bilayer of the liposome, and wherein the at least one bacterial polysaccharide antigen is within the lumen of the liposome and / or attached to a lipid bilayer of the liposome.

2. The composition according to claim 1, wherein the at least one iNKT cell agonist is selected from any one of: o-galactosyl ceramide (o-GalCer), o-galactosyldiacylglycerol (o-GalDag), o- glucosyldiacylglycerol (o-GIcDag), o-glucuronsylceramide (o-GIcACer), o- galacturonosylceramide (o-GalACer), phosphatidyl-myo-inositol mannoside (PIM2), and any derivative of any one of these agonists, preferably o-GalCer, or any combination thereof.

3. The composition according to claim 1 or 2, wherein the at least one bacterial polysaccharide antigen is within the lumen of the liposome.

4. The composition according to any one of the preceding claims, wherein the liposome is cationic at physiological pH.

5. The composition according to any one of the preceding claims, wherein the at least one bacterial polysaccharide antigen is a non-modified native polysaccharide.

6. The composition according to any one of the preceding claims, wherein the bacterial polysaccharide antigen is a capsular polysaccharide derived from a bacterium selected from: Bordetella pertussis, Burkholderia pseudomallei, Chlamydophila pneumoniae, Corynebacterium diphtheriae, Haemophilus influenzae, Mycoplasma pneumoniae, and Streptococcus pneumoniae, preferably S. pneumoniae.

7. The composition according to claim 6, wherein the composition comprises at least two different capsular polysaccharides, which are derived from at least two different serotypes of S. pneumoniae, optionally selected from the following : 1, 2, 3, 4, 5, 6A, 6B, 6C, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15A, 15B, 16F, 17F, 18C, 19A, 19F, 20, 20B, 22F, 23A, 23B, 23F, 24F, 31, 33F, and 35B.

8. The composition according to any one of the preceding claims, wherein the at least one bacterial polysaccharide antigen is a capsular polysaccharide derived from S. pneumoniae, and wherein the iNKT cell agonist is o-GalCer.

9. A pharmaceutical composition comprising the composition according to any one of the preceding claims and a pharmaceutically acceptable vehicle and / or diluent and / or immunological adjuvant.

10. A method of treating or preventing a bacterial infection in a mammal, such as a human being, said method comprising administering a pharmaceutically effective amount of the composition according to any one of claims 1-8 or of the pharmaceutical composition according to claim 9 to said mammal.

11. The method according to claim 10, wherein the bacterial infection is an airway infection, such as an upper airway and / or lung infection.

12. The method according to claim 10 or 11, wherein the composition or the pharmaceutical composition is administered intranasally, subcutaneously, orally or intramuscularly, preferably intranasally, or any combination thereof.

13. The composition according to any one of claims 1-8 or the pharmaceutical composition according to claim 9 for use in a method according to any one of claims 10-12.

14. A nasal spray device, comprising : a pressurised aerosol canister including a vial containing the pharmaceutical composition according to claim 9, an actuator, and a delivery outlet for the aerosol plume.

15. The nasal spray device according to claim 14, configured to deliver metered doses of the pharmaceutical composition to the nasal cavity of a mammal, such as a human being.

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