Mucosal vaccine adjuvant

Abstract

This invention relates to a mucosal vaccine composition and to an adjuvant comprising highly deacetylated chitosan for use in a method of immunotherapy, wherein the adjuvant is administered by a mucosal administration.

Description

[0001] Mucosal vaccine adjuvant

[0002] This invention relates to a mucosal vaccine adjuvant and to a composition comprising highly deacetylated chitosan for use in a method of immunotherapy, wherein the adjuvant is administered by a mucosal administration.

[0003] Vaccines are a key tool in increasing public health quality by reducing the incidence and severity of preventable diseases. However, many available vaccines provide a suboptimal immune response and there are many diseases for which no effective vaccine has been developed. Infectious diseases caused by mucosal pathogens are associated with a significant global burden of mortality and morbidity, with lower respiratory tract infections representing the fourth leading cause of death worldwide. A key issue is that many vaccines are formulated for administration by the intramuscular or subcutaneous route, and so the immune response induced by such vaccines is not targeted at the site of infection.

[0004] One method of increasing vaccine potency is the inclusion of an adjuvant. Adjuvants are substances which enhance the immune response induced by an antigen, thereby providing increased protection against a particular disease. However, many approved adjuvants have a weak ability to enhance longterm effective immunity as they are only capable of inducing antibody response and have a limited ability to induce CD8+T cell-mediated immunity, which is critical in the immune defence against intracellular pathogens.

[0005] Chitosan is a cationic polysaccharide that has been evaluated as a potential vaccine adjuvant due to its biocompatible and biodegradable nature. In particular, highly deacetylated chitosan has been shown to be a promising as an immunostimulatory compound when administered intramuscularly or subcutaneously, through its role as an activator of the cGAS-STING pathway and NLRP3 inflammasome (J.L Turley, et al., Biomaterials, 2021 , 275, 120961).

[0006] However, there remains a need in the art for adjuvants and vaccine compositions which can enhance antigen-specific immune response within target tissues, particularly for the prevention of respiratory infection.

[0007] Accordingly, the present invention provides a mucosal vaccine composition comprising an antigen and an adjuvant, wherein the adjuvant is chitosan that is at least 99% deacetylated.

[0008] The present invention further provides an adjuvant comprising chitosan that is at least 99% deacetylated for use in a method of immunotherapy, wherein the adjuvant is administered by mucosal administration in combination with an antigen.

[0009] The inventors of the present invention have surprisingly found that the intranasal administration of vaccine compositions comprising highly deacetylated chitosan as an adjuvant drives the production of antigen-specific CD8+T cells in the lungs of immunised mice, an effect that had not been previously observed by administration through subcutaneous or intramuscular injection. In particular, completely deacetylated chitosan (C100) was also the only highly deacetylated chitosan preparation tested which consistently enhanced all antigen-specific IgG isotypes and nasal wash IgA.

[0010] Since antigen-specific CD8+T cells were only detected after administration directly to the respiratory mucosa, it is apparent that this route of administration provides an additional immunisation-route specific effect that has not been previously observed.

[0011] The present invention will now be described in detail with reference to the accompanying drawings, in which:

[0012] Fig. 1 shows a standard intranasal immunisation protocol;

[0013] Fig. 2 shows an antigen-specific CD8+ T cell gating strategy and FMO controls;

[0014] Fig. 3A and D show pseudo colour dot plots indicating the percentage of antigen-specific CD44hi Tetramer+ CD8+ T cells quantified in the lungs and spleen, respectively, of C57BL / 6J mice intranasally immunised with vaccine compositions and Fig. 3B, C, E, F show the percentage of antigen specific CD8+ T cells in the lungs and spleen, respectively;

[0015] Fig. 4A and B show the IFN-y concentration from lung and spleen re-stimulation with SARS-CoV-2 spike, respectively, of C57BL / 6J mice intranasally immunised with vaccine compositions and Fig. 40 shows the IL-17 concentration from lung re-stimulation with SARS-CoV-2 spike;

[0016] Fig. 5A-D show nasal wash antigen-specific IgA (A), and serum antigen-specific IgG (B), lgG2c (C), and lgG1 (D) titres determined by ELISA, of C57BL / 6J mice intranasally immunised with vaccine compositions;

[0017] Fig. 6 shows the percentage of antigen-specific CD8+ T cells in the lungs of C57BL / 6J mice intranasally immunised with vaccine compositions;

[0018] Fig. 7A shows pseudo colour dot plots indicating the percentage of antigen-specific CD44hi Tetramer+ CD8+ T cells quantified in the lungs of C57BL / 6J mice intranasally immunised with vaccine compositions;

[0019] Fig. 8 shows nasal wash antigen-specific IgA (A), and serum antigen-specific IgG (B), lgG2c (0), and lgG1 (D) titres determined by ELISA, of C57BL / 6J mice intranasally immunised with vaccine compositions; and

[0020] Fig. 9A and B show the IFN-y concentration from spleen and lung re-stimulation with SARS-CoV-2 spike, respectively, of C57BL / 6J mice intranasally immunised with vaccine compositions.

[0021] Fig. 10A-B show the IFN-y concentration from lung (A) and spleen (B) re-stimulation with Influvac Tetra, respectively, and Fig. 10C-E show nasal wash antigen-specific IgA (C) and serum antigen specific IG2c (D) and IgG (E) titres determined by ELISA, of C57BL / 6J mice intranasally immunised with vaccine compositions in a two-dose regimen.

[0022] Fig. 11A shows the IFN-y concentration from lung re-stimulation with Influvac Tetra, and Fig. 11 B-D show nasal wash antigen-specific IgA (B) and serum antigen specific IG2c (C) and IgG (D) titres determined by ELISA, of C57BL / 6J mice intranasally immunised with vaccine compositions in a three- dose regimen.

[0023] Fig. 12 shows a simultaneous intramuscular / intranasal immunisation protocol.

[0024] Fig 13A and B show the IFN-y concentration from lung (A) and spleen (B) re-stimulation with Influvac Tetra, and Fig 13C-D show nasal wash antigen-specific IgA (C) and serum antigen specific IG2c (D) titres determined by ELISA, of C57BL / 6J mice simultaneously immunised intranasally and intramuscularly with vaccine compositions.

[0025] Mucosal vaccine composition

[0026] The vaccine composition of the present invention is a mucosal vaccine. Administration of vaccines directly to the mucosa allows for the induction of protective immune responses at the predominant sites of pathogen infection, thereby preventing an infection from becoming established. Mucosal routes of administration include intranasal and pulmonal administration, as well as oral, sublingual, rectal and vaginal administration.

[0027] The inventors of the present invention have found that administration of a vaccine composition according to the invention to the respiratory mucosa, results in a robust antigen-specific immune response. Accordingly, it is preferred that the mucosal vaccine of the present invention is a pulmonal or intranasal vaccine, more preferably an intranasal vaccine.

[0028] The mucosal vaccine composition according to the invention may further comprise a pharmaceutically acceptable carrier. The carrier may be purified water, sterile saline or phosphate buffered saline (PBS). The composition may be sterile.

[0029] Deacetylated chitosan

[0030] The mucosal vaccine composition according to the present invention comprises chitosan that is at least 99% deacetylated.

[0031] Chitosan is a linear polysaccharide composed of randomly distributed p-( 1 — >4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). Chitosan is typically produced commercially by deacetylation of chitin, which is the structural element in the exoskeleton of crustaceans (such as crabs and shrimp) and cell walls of fungi. Chitin has at least 90% acetylation. The deacetylated chitosan according to the present invention may be derived form a synthetic source, such as linking monomeric glucosamines and / or monomeric N-acetyl-D-glucosamines.

[0032] The degree of deacetylation (%DD) of chitosan refers to the percentage of deacetylated units (p-(1— >4)- linked D-glucosamine) in the molecule. As such, in chitosan which is at least 99% deacetylated, no more than 1% of the monomeric units are N-acetyl-D-glucosamine groups. The degree of deacetylation can be determined by NMR spectroscopy.

[0033] Chitosan may also be referred to as acetylated polyglucosamine. Therefore, 100% deacetylated chitosan (C100), which is a polymer of p-(1— >4)-linked D-glucosamine that does not comprise N-acetyl- D-glucosamine, may also be referred to as polyglucosamine.

[0034] It is preferred that the chitosan is 100% deacetylated.

[0035] In its base form chitosan is not water soluble. As such, when used in vaccine compositions chitosan is often used in the form of a water-soluble salt such as chitosan hydrochloride, aspartate or glutamate. It is also often formulated as nanoparticles and / or cross-linked or covalently attached to another molecule such as an antigen or other ligand.

[0036] However, the inventors of the present invention have found that the enhanced immune response induced by the compositions of the present invention is achieved when the deacetylated chitosan is in the form of a free chain. Such free chains will have an overall net positive charge when in solution, whereas linking of the chitosan can neutralise the net positive charge and cause unwanted aggregation of the chitosan molecules. Accordingly, the deacetylated chitosan is preferably in free-chain form.

[0037] It is also preferred that the deacetylated chitosan is used in a form that is non-water soluble. Accordingly, it is preferred that the chitosan is not used in the form of its hydrochloride, aspartate or glutamate salts. Preferably, the chitosan is used in the form of its free base. It may be necessary to first dissolve the chitosan in an acetic acid solution in order to prepare the vaccine composition.

[0038] The molecular weight of the deacetylated chitosan is another aspect which may also affect its immunostimulatory properties. It is preferred that the molecular weight of the deacetylated chitosan is 100 to 1 ,000 kDa, more preferably 115 to 500 kDa.

[0039] It is further preferred that the deacetylated chitosan is not hydrolysed.

[0040] The deacetylated chitosan should be comprised in the vaccine composition in a sufficient amount to induce an immunostimulatory response. Accordingly, it is preferred that the deacetylated chitosan is present at a concentration of 1-20 mg / mL.

[0041] In a preferred embodiment, the composition is an intranasal vaccine composition comprising an antigen and an adjuvant, wherein the adjuvant is 100% deacetylated chitosan in free chain form. Antigen

[0042] The mucosal vaccine composition according to the invention comprises an antigen. An antigen is a substance that can bind to an antibody or T cell receptor, thereby triggering an immune response. Accordingly, the antigen should be selected in order to provide an immune response against a particular disease target.

[0043] It is preferred that the antigen is selected from a protein or peptide (e.g. a recombinant protein or a glycoprotein), a polysaccharide (e.g. a lipopolysaccharide), a polynucleotide, a cell, or a virus. If the antigen is a virus, then it may be a live attenuated virus, an inactivated virus or a split virus. If the antigen is a cell, then it is preferably a bacterial cell. One or more antigens may be comprised within a bacterial cell lysate.

[0044] The inventors of the present invention have found that the administration of a vaccine composition according to the invention provides an effective immune response against infection. Accordingly, it is preferred that the antigen is derived from a pathogenic species, more preferably from a bacteria or virus. It is further preferred that the pathogenic species from which the antigen is derived is one which infects the respiratory system.

[0045] In a preferred embodiment, the antigen is SARS-CoV-2 spike protein. In another preferred embodiment, the antigen is influenza hemagglutinin.

[0046] The antigen should be comprised in the vaccine composition in a sufficient amount to induce an immunostimulatory response. Accordingly, it is preferred that the antigen is present at a concentration of 1-2,000 pg / mL

[0047] Further immunomodulatory compound

[0048] It has been found by the inventors of the present invention that further immunomodulatory compounds can be incorporated into the compositions of the present invention in order to provide an enhanced immune response. For example, the combination of deacetylated chitosan with a-galactosylceramide results in a significant increase in the production of IFN-y in the re-stimulated lung cells of inoculated mice, compared to the administration of either compound alone. Further increases in antigen-specific immune response were observed when combining deacetylated chitosan with toll-like receptor agonists, such as CpG ODN 1826 and monophosphoryl lipid A, and also cyclic diAMP (CDA) with significantly enhanced production of antigen-specific CD8+T cells and IgG isotypes

[0049] Accordingly, in an embodiment of the present invention, the intranasal vaccine composition comprises a further immunomodulatory compound. Such further immunomodulatory compounds include NKT cell agonists (e.g. a-galactosylceramide), toll-like receptor (TLR) agonists, nod-like receptor agonists, C- type lectin receptor agonists, Al M2-like receptor agonists, RIG-l-like receptor agonists, other pathogen recognition receptor agonists and combinations of pathogen recognition receptor agonists. Toll-like receptor agonists include imiquimod, polyinosinic: polycytidylic acid, CpG oligodeoxynucleotides (CpG ODN), which are short-single stranded synthetic DNA molecules (e.g. CpG ODN 1826) and monophosphoryl lipids (MPL).

[0050] It is preferred that the further immunomodulatory compound is selected from the group consisting of a- galactosylceramide, imiquimod, polyinosinic: polycytidylic acid, CpG ODN 1826, cyclic di-AMP (CDA) or monophosphoryl lipid A. More preferably the further immunomodulatory compound is selected from the group consisting of imiquimod, poly inosinic: polycytidylic acid, CpG ODN 1826, cyclic di-AMP (CDA) or monophosphoryl lipid A. Most preferably the further immunomodulatory compound is CpG ODN 1826, cyclic di-AMP (CDA) or monophosphoryl lipid A.

[0051] The further immunomodulatory compound should be comprised in the vaccine composition in a sufficient amount to induce an immunostimulatory response. It is preferred that the further immunomodulatory compound is present at a concentration of 100-2,000 g / mL

[0052] Method of immunotherapy

[0053] Immunotherapy relates to the prevention or treatment of disease with substances that stimulate the immune response.

[0054] The present invention further provides an adjuvant comprising chitosan that is at least 99% deacetylated for use in a method of immunotherapy, wherein the adjuvant is administered by mucosal administration in combination with an antigen.

[0055] The adjuvant and the antigen may be formulated together or separately in a pharmaceutically acceptable carrier. It is preferred that the adjuvant and antigen are comprised within a mucosal vaccine composition.

[0056] The adjuvant is mixed with the antigen prior to administration in order to form the vaccine composition. Mixing of the adjuvant and the antigen may take place extemporaneously, or during the manufacture of the vaccine.

[0057] In an embodiment, the adjuvant is combined with a vaccine composition that is already known in the art, thereby enhancing the immune response provided by the vaccine composition. The known vaccine composition may be a commercially available vaccine composition. The known vaccine composition may have been formulated for administration by an alternative route e.g. subcutaneous or intramuscular administration. The inventors of the present invention have found that the administration of an adjuvant according to the invention provides an effective immune response against infection by a pathogenic species, in particular where such infection targets the respiratory system.

[0058] Accordingly, it is preferred that the adjuvant is administered by intranasal or pulmonal administration. More preferably, the adjuvant is administered by intranasal administration.

[0059] It is additionally preferred that, the method of immunotherapy is for the prevention of a pathogenic disease, preferably a respiratory disease. More preferably, the pathogenic respiratory disease is caused by a viral or bacterial infection.

[0060] It is further preferred that the respiratory disease is caused by a viral infection selected from a human coronavirus, SARS-CoV, SARS-CoV-2, MERS-CoV, influenza, parainfluenza, or respiratory syncytial virus (RSV), or a bacterial infection selected from the group consisting of Bordetella pertussis, Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae, Mycobacterium tuberculosis and Streptococcus pneumoniae. More preferably, the respiratory disease is SARS-CoV-2. In another more preferred embodiment, the respiratory disease is influenza.

[0061] In a preferred embodiment, the present invention provides an adjuvant comprising chitosan that is 100% deacetylated in free chain form for use in a method of immunotherapy for the treatment of a pathogenic respiratory disease, wherein the adjuvant is administered intranasally in combination with an antigen.

[0062] The adjuvant or vaccine composition may be administered multiple times to ensure that a sufficient immune response is observed. For example, the administration may comprise a prime-boost regime (i.e. a first dose followed by one or more further doses). The time between doses may be at least 1 week, preferably at least 2 weeks. The total number of doses in the prime-boost regimen will be at least 2 doses (1 prime and at least 1 boost). The total number of doses in the prime-boost regimen is selected to provide sufficient immunity against the target disease whilst minimising the number of doses required, thereby providing an efficient vaccination schedule with a high degree of patient compliance. Preferably, the number of doses in the prime-boost regimen is 2 (1 prime and 1 boost) or 3 doses (1 prime and 2 boost).

[0063] In a preferred embodiment, the adjuvant or vaccine composition is for use in the prevention of SARS- CoV-2 and is administered as a 3 dose prime-boost regimen. In another preferred embodiment, the adjuvant or vaccine composition is for use in the prevention of influenza and is administered as a 2 dose prime-boost regimen.

[0064] The adjuvant or vaccine composition may be administered as one or more boosting doses following previous administration with an alternative priming vaccine composition, such as an mRNA or adenoviral vector vaccine. It is preferred that the previously administered vaccine composition is administered by intramuscular injection. The time between the dose of the priming vaccine and the first dose of the boosting vaccine may be at least 1 week, preferably at least 2 weeks.

[0065] In an embodiment, the mucosal vaccine composition is a first vaccine composition administered in combination with the intramuscular or subcutaneous administration of a second vaccine composition comprising an antigen which provides a response against the same disease target. Preferably the first vaccine composition is administered intranasally, and the second vaccine composition is administered intramuscularly. The first vaccine composition and the second vaccine composition may be administered simultaneously or sequentially, preferably simultaneously. It is preferred that the antigen in the first vaccine composition is the same as the antigen in the second vaccine composition.

[0066] The subject administered with the adjuvant or vaccine composition may be mammalian. In one embodiment, the subject is human. In another embodiment, the subject may be a domestic or livestock animal.

[0067] The present invention will now be described with reference to the following examples which are not intended to be limiting.

[0068] Examples

[0069] Materials

[0070] Mice

[0071] C57BL / 6J mice aged 8-15 weeks were obtained from Charles River, UK or bred in-house by the Comparative Medicine Unit of the Trinity Biomedical Sciences Institute (TBSI, TCD).

[0072] General cell culture materials

[0073] Complete RPMI 1640 Medium (cRPMI) - Roswell Park Memorial Institute (RPMI) 1640 medium with GlutaMAX (Gibco) pH 7.1 was supplemented with 50 U / mL penicillin and 50 pg / mL streptomycin (Gibco), and 10% (v / v) ultra-low endotoxin heat-inactivated filter-sterilised FBS of Brazil origin (Gibco).

[0074] Complete RPMI 1640 T cell Medium - RPMI 1640 medium with GlutaMAX (Gibco) pH 7.1 was supplemented with 45.5 pM p-mercaptoethanol (Gibco), 0.88 mM sodium pyruvate (Gibco), 0.88% (v / v) 100X Minimum Essential Medium (MEM) Non-Essential Amino Acids (Gibco), 0.35% (v / v) 100X MEM Vitamins (Gibco), 4.4 U / mL penicillin and 4.4 pg / mL streptomycin (Gibco), and 8% (v / v) ultra-low endotoxin heat-inactivated filter-sterilised FBS of Brazil origin (Gibco).

[0075] Phosphate Buffered Saline (PBS)(Gibco) - purchased from BioSciences Ltd. Trypsin-EDTA (Gibco) - purchased from BioSciences Ltd.

[0076] Ammonium Chloride (ACL) Red Blood Cell Lysis Buffer - 8.29 g (155 mM) of ammonium chloride and

[0077] 1 g (10 mM) of potassium bicarbonate were dissolved in 1 L of endotoxin-free water (HyClone Cytiva).

[0078] 2 mL (0.1 mM) of UltraPure 0.5M EDTA was added and the solution filter-sterilised using a 0.22 pm syringe-driven filter (Millipore).

[0079] Cell counting

[0080] Cell viability was assessed via the Trypan Blue (Sigma) exclusion method using KOVA Glasstic haemocytometer slides (VWR), or via the ChemoMetec NucleoCounter NC-202 using Via-2 cassettes, as indicated.

[0081] In vitro and in vivo treatment

[0082] Table 1

[0083] Antigens for in vivo studies Table 2

[0084] Reagents used for ex vivo restimulations

[0085] Table 3

[0086] Additional reagents used for in vivo studies

[0087] Table 4

[0088] Lung / cervical lymph node digestion reagents

[0089] Digestion Media - per sample: 1 ml_ RPMI 1640 with GlutaMAX with 20 mg / mL Collagenase type I (Gibco) and 10 g / ml_ DNase I (Sigma-Aldrich).

[0090] 5mM PBS-Ethylenediaminetetraacetic acid (EDTA) - 500 ml_ 1X PBS supplemented with 5 ml_ of 0.5M UltraPure EDTA (Gibco).

[0091] Flow cytometry reagents

[0092] Fluorescence activated cell sorting (FACS) buffer - 1X PBS supplemented with 2% (v / v) FBS.

[0093] OneComp eBeads Compensation Beads - used for single stain controls and purchased from BioSciences Ltd.

[0094] Brilliant Stain Buffer - purchased from BD Biosciences.

[0095] 4% Paraformaldehyde (PFA) - purchased from Santa Cruz Biotechnology. Panel used for identification of antigen-specific CD8+T cells after immunisation

[0096] Table 5

[0097] Anti-CD16 / CD32 monoclonal antibody (FcBlock) - purchased from BD Biosciences

[0098] Enzyme-linked immunosorbent assay (ELISA) reagents

[0099] Sodium carbonate coating buffer - 4.2g NaHCOs AND 1.78g Na2COs were dissolved in 1L of reverse osmosis (RO) water (RiOs Essential) and pH adjusted to pH 9.5.

[0100] Phosphate citrate buffer - 10.19g anhydrous citric acid and 14.6g Na2HPO4 were dissolved in 1 L RO water (RiOs Essential), and pH adjusted to pH 5.5.

[0101] 10X PBS - 800g NaCI, 116g Na2HPO4, 20g KH2PO4, and 20g KOI were dissolved in 10L RO water (RiOs Essential), and pH adjusted to pH 7.2.

[0102] Wash buffer - 1X PBS supplemented with 0.05% (v / v) Tween-20

[0103] Antibody ELISA substrate - O-Phenylenediamine (dihydrochloride) (OPD) chromogenic substrate was prepared in phosphate citrate buffer at a concentration of 0.4 mg / mL. Immediately prior to addition to ELISA plate, 7 L of H2O2 was added per 25mL of substrate solution.

[0104] Cytokine ELISA substrate - 3, 3', 5, 5' Tetramethylbenzidine (TMB) substrate kit was purchased from Thermo Fisher and prepared at a 1 :1 ratio of reagent A and reagent B prior to addition to ELISA plate.

[0105] Stop solution - 1 M H2SO4. Reagent diluent for IFN-y ELISA kit - 0.1% bovine serum albumin (BSA) (w / v) and 0.05% Tween 20 (v / v) in Tris-buffered Saline (20 mM Trizma base, 150 mM NaCI), pH adjusted to pH 7.2-7.4.

[0106] Horseradish peroxidase (HRP)-conjugated Streptavidin for non-commercial ELISA kits - Streptavidin- HRP was purchased from BD Pharmingen and used at a dilution of 1 / 2000 in all non-commercial ELISAs where a non-HRP conjugated detection antibody was used.

[0107] Commercial ELISA kits used for quantification of IFN-y, CXCL10, IL-17, and Flt3L.

[0108] Table 6

[0109] Antibodies for quantification of serum and mucosal antibody titres.

[0110] Table 7

[0111] *non-HRP conjugated antibodies. Streptavidin-HRP step is required

[0112] Methods

[0113] Isoflurane-induced anaesthesia and intranasal (IN) instillation of formulations

[0114] Mice were anaesthetised using 3% isoflurane with 1.5% O2 for 5 minutes prior to immunisation. Formulations were administered dropwise directly into the nares in a total volume not exceeding 25 pL. Mice were placed in an inclined recovery position when returned to their cage and monitored for 30 minutes, and again 12-24 hours after immunisation.

[0115] Analysis of immune responses to intranasal (IN) immunisation

[0116] Mice were immunised intranasally by direct instillation of formulation into the nares under anaesthesia as described hereinabove with the following formulations, depending on the individual experiment:

[0117] PBS / vehicle control;

[0118] SARS-CoV-2 spike antigen only control (2 pg); Chitin-derived polymers (Table 1) with SARS-CoV-2 spike (2 pg); a-GalCer (10 pg) with SARS-CoV-2 spike (2 pg);

[0119] C100 (100 pg), a-GalCer (10 pg), with SARS-CoV-2 spike (2 pg); or

[0120] TLR ligands (Table 1 ) with SARS-CoV-2 spike (2 pg).

[0121] Mice were immunised according to a standard intranasal vaccination regimen (Fig. 1 ), with a priming immunisation on day 0, followed by two boosting immunisations on day 14 and day 28. On day 35 mice were euthanised through controlled CO2 exposure and cervical dislocation. Prior to organ collection, mice were perfused with 5 ml_ PBS by direct injection into the right ventricle. Spleens, lungs, cervical lymph nodes, blood, and nasal wash were collected. To collect nasal wash, a lateral incision was made through the mandible and nasal turbinates perfused using a BD tuberculin syringe with a 27G needle filled with 300 pL of nasal wash buffer. Spleens and lungs were homogenised (with lungs requiring prior collagenase digestion) and cells isolated for flow cytometry and ex vivo antigen restimulation assays. Cervical lymph nodes were collagenase digested, homogenised, and cells isolated for ex vivo restimulation assays. Cytokine concentrations from supernatants and antibody titres from sera and nasal wash were quantified by ELISA.

[0122] Cell culture

[0123] All cells were cultured in a cell culture incubator at 37°C with 95% humidity and 5% CO2.

[0124] Cell counting

[0125] Unless specified, live cells used in all in vitro and in vivo experiments were counted using the trypan blue exclusion method in disposable KOVA Glasstic haemocytometer slides under a light microscope. Cell suspensions were diluted appropriately in trypan blue, ranging from 1 / 2-1 / 50, dependent on cell density. 10 pL of the trypan blue-diluted cell suspension was added to the counting chamber. Cells were counted in three diagonal squares to obtain mean square count, with cells on the border excluded. Cell concentration was determined using the following formula:

[0126] Cells / mL = (mean cell number) ( 104) (dilution factor)

[0127] Alternatively, cells were counted using the NucleoCounter NC-202 provided by ChemoMetec in Via-2 cassettes. Cell suspensions were left undiluted, or diluted 1 / 10 in cRPMI depending on density. Cells were aspirated using the Via-2 cassette automated loading system and counted automatically after insertion of the cassette into the NucleoCounter. Cell concentration calculations were performed by the accompanying software. Isolation of cells from spleen

[0128] Spleens were collected from euthanised mice and stored in cRPMI until the next step. Cell suspensions were obtained by passing spleens through a 40 pm cell strainer with intermittent washing with cRPMI. Cells were centrifugated at 400 x g for 5 minutes at 4°C, supernatants discarded, and cells resuspended in 1 ml_ of ACL RBC lysis buffer for 2 minutes to lyse red blood cells. The lytic reaction was quenched with 10 mL of cRPMI, and centrifugation repeated. Cells were resuspended and counted as outlined previously at a 1 / 50 dilution. For ex vivo antigen restimulation assays, cells were seeded at a density of 2x106cells / mL in sterile U-bottom 96-well plates in 200 pL of T cell media. Cells were then restimulated with appropriate antigen and control concentration. Then 2x107cells were removed into FACS tubes for tetramer staining and analysis by flow cytometry. Care was taken to ensure that samples destined for tetramer staining were not placed on ice at any stage during the isolation process.

[0129] Digestion of lungs and isolation of cells

[0130] Lungs were collected from euthanised mice and stored in cRPMI until the next step. Prior to collagenase digestion, lungs were transferred into a sterile F-bottom 24-well-plate and cRPMI was discarded. Lungs were minced with a sterile pair of scissors until approx. 2 mm size pieces were obtained. 1 mL of digestion media containing 20 mg / mL collagenase type I and 10 pg / mL DNase I was added to each well and lungs were placed in a cell culture incubator for 1 hour on shaker at 300 rpm. At 1 hour, 5mM PBS-EDTA was added to inhibit the collagenase and single cell suspensions were obtained by passing through a 40 pm cell strainer with intermittent washing with cRPMI. Cells were centrifugated at 400 x g for 5 minutes at 4°C, supernatants discarded, and cells resuspended in 1 mL of ACL RBC lysis buffer for 3 minutes to lyse red blood cells. The lytic reaction was quenched with 10 mL of cRPMI, and centrifugation repeated. Cells were resuspended in T cell media and counted as outlined previously at a 1 / 50 dilution. For ex vivo antigen restimulation assays, cells were seeded at a density of 2x106cells / mL in sterile U-bottom 96-well plates in 200 pL of T cell media. Cells were then restimulated with appropriate antigen and control concentrations. 1x107cells were removed into FACS tubes for tetramer staining and analysis by flow cytometry. Care was taken to ensure that samples destined for tetramer staining were not placed on ice at any stage during the isolation process.

[0131] Isolation of serum from whole blood

[0132] After euthanasia, blood was collected either from the heart if no perfusion was required, or from the hepatic artery. Blood was allowed to coagulate overnight at 4°C. Samples were centrifugated at 11,000 x g for 11 minutes at 4°C and sera carefully aspirated and frozen until use at -20°C.

[0133] Flow cytometry

[0134] OneComp eBeads and cells were utilised as single stain controls for compensation or unmixing, depending on the cytometer used. Auto-fluorescence was accounted and corrected for by using unstained samples as negative fluorescence and fluorescence-minus-one (FMO) controls, where samples are stained for all fluorochromes except one. FMO and unstained controls were repeated and matched per tissue analysed. Samples were acquired using Cytek SpectroFlo software on a fullspectrum Cytek Aurora flow cytometer with 4L V / B / YG / R configuration or using BD FACSDiva software on a BD LSRFortessa flow cytometer with 4L V / B / YG / R configuration, as indicated in each individual experiment. Subsequent analyses were performed on FlowJo software.

[0135] Analysis of antigen-specific CD8+T cell induction following intramuscular or intranasal vaccination through Class-1 tetramer staining

[0136] Splenocytes and cell suspensions obtained from lung digestion were obtained as outlined previously. Cells were stained with PE-labelled H-2kb VNFNFNGL tetramer (15 pg / mL) in 100 pL of T cell media for 90 minutes on an orbital shaker in a cell culture incubator. After 50 minutes, and during the incubation, anti-CD16 / CD32 monoclonal antibody (mAb) (FcBlock) was added to all samples, except OneComp eBeads single stain controls, to block FcyRII / lll and prevent background staining (0.5 pL / sample in 50 pL T cell medium). For the final 30 minutes of staining, fluorochrome-labelled cell surface antibodies targeting F4 / 80, B220 (CD45R), CD11c, CD3s, CD4, CD8 a, and CD44 were added to all samples. Fig. 2. A,B describes the gating strategy in detail along with indicated FMO controls. Samples were then washed with PBS and centrifugated at 400 x g for 5 minutes. Supernatants were discarded, and samples resuspended in live / dead FVS510 stain diluted 1 / 500 in PBS and incubated for 30 minutes in the dark at 4°C. Cells were washed as before and resuspended in 2% paraformaldehyde in PBS and incubated for 15 minutes in the dark at RT to fix samples. Cells were washed as before and resuspended in 300 pL of FACS buffer and stored in the dark at 4°C until acquisition on a flow cytometer.

[0137] ELISA

[0138] Measuring cytokine and other analytes in cell culture supernatants

[0139] Bio-Techne DuoSet ELISAs for IFN-y, CXCL10, IL-17, and Flt3L were performed according to the manufacturer’s guidelines. Briefly, F-bottom high-binding 96-well plates were coated with 50 pL / well capture antibody at RT overnight. Plates were washed three times with wash buffer. Plates were blocked with 200 pL PBS-1 % BSA for 2 hours at RT. Plates were washed as before and incubated with 50 pL / well of undiluted or diluted sample, and serially diluted recombinant standard for 2 hours at RT. Plates were washed as before and incubated with 50 pL / well of detection antibody. After another wash step, plates were incubated with 50 pL / well of streptavidin-HRP in the dark at RT. Plates were washed again, with an additional wash step, and incubated with 50 pL / well of TMB substrate reagent for 20 minutes at RT in the dark. The reaction was stopped by addition of 20 pL 1 M H2SO4 and optical density (CD) values were determined by measuring absorbance at 492nm using a spectrophotometer. Cytokine concentrations were determined by fitting a non-linear regression curve of the recombinant standard and extrapolating unknown values.

[0140] Measuring antigen-specific serum and nasal wash antibodies (IgG, lgG1, lgG2c, lgG2b, IgA)

[0141] F-bottom medium-binding 96-well plates were coated with 2 pg / mL of SARS-CoV-2 spike (50 pL / well) diluted in sodium carbonate coating buffer and incubated at 4°C overnight. Plates were washed three times with wash buffer and blocked with 200 pL of PBS-1% skimmed milk powder for 2 hours at RT. Plates were washed as before and incubated with 50 pL / well of samples serially diluted (1 :2) in PBS- 1% skimmed milk powder for 2 hours at RT.

[0142] Plates were washed again and incubated with 50 pL / well detection antibody (Table 8) for 1 hour at RT. Plates were washed again, and in the case of non-HRP conjugated antibodies (lgG1 and lgG2b), 50 pL / well 1 / 2000-diluted streptavidin-HRP was added and incubated for 30 minutes at RT in the dark. HRP-conjugated antibodies proceeded directly to the substrate step. Plates were washed as described, with an additional wash step, and 50 pL / well OPD substrate supplemented with 7 pL per 25mL solution was added and incubated at RT in the dark. The reaction was stopped by addition of 20 pL 1 M H2SO4 and optical density (OD) values were determined by measuring absorbance at 492 nm using a spectrophotometer.

[0143] Detection antibody dilutions

[0144] Table 8 a-GalCer reconstitution and antigen mixing

[0145] Lyophilised a-GalCer was dissolved in sterile nonpyrogenic dimethyl sulfoxide (DMSO) to create a 5mg / mL stock solution. To ensure complete dissolution, was briefly vortexed, heated at 80°C for 2 minutes, and sonicated in a water bath sonicator for 5 minutes. The solution was aliquoted and stored at -20°C until use. Prior to antigen mixing, frozen a-GalCer was thawed at RT, briefly vortexed, heated at 80°C for 2 minutes, and sonicated for 10 minutes in a water bath sonicator. a-GalCer was diluted to the required concentration in PBS / vehicle and mixed with antigen 40 minutes before immunisation. Immediately prior to immunisation, the solution was briefly vortexed. Example 1

[0146] C1OO, a-GalCer, and C1OO-a-GalCer enhance pulmonary and splenic antigen-specific CD8+ T cell responses

[0147] Five C57BL / 6J mice per group aged 8-12 weeks-old were intranasally immunised with:

[0148] PBS;

[0149] SARS-CoV-2 spike antigen (Ag) (2 pg);

[0150] C100 (100 pg) + Ag (2 pg) , C90 (100 pg) + Ag (2 pg);

[0151] Protasan (100 pg) + Ag (2 pg);

[0152] 072 (100 pg) + Ag (2 pg);

[0153] 050 (100 pg) + Ag (2 pg); a-GalCer (10 pg) + Ag (2 pg); or

[0154] C100 (100 pg) + a-GalCer (10 pg) + Ag (2 pg) (C100-a-GalCer), according to Fig. 1.

[0155] On day 35, antigen-specific CD8+T cell responses were quantified in the lungs and spleen using H- 2Kb SARS-CoV-2 spike (VNFNFNGL) MHO Class I Tetramer according to the gating strategy detailed in Fig. 2. PBS or SARS-CoV-2 spike alone were used as vehicle or antigen only controls, respectively. The results are presented in Fig. 3.

[0156] Fig. 3A and D show pseudo colour dot plots indicating the percentage of antigen-specific CD44hi Tetramer+ CD8+ T cells quantified in the lungs and spleen, respectively, of C57BL / 6J mice intranasally immunised with vaccine compositions comprising SARS-CoV-2 spike antigen (Ag) and various chitosan preparations (C50, C72, Protasan, C90 and C100), and of compositions comprising SARS-CoV-2 spike antigen and a-galactosylceramide (a-GalCer), with and without C100. Fig. 3B, C, E, F show the percentage of antigen specific CD8+ T cells in the lungs and spleen, respectively.

[0157] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01 , *** p < 0.001.

[0158] The results show that the use of C100 provides a significant increase in the number of antigen-specific CD8+T cells in the lungs (Fig 3. A and B), which is greater than that observed for chitosan preparations with a lower deacetylation percentage. Example 2

[0159] C1OO-a-GalCer and a-GalCer are promising drivers of antigen-specific IFN-y while concomitantly suppressing IL-17

[0160] Five C57BL / 6J mice per group aged 8-12 weeks-old were intranasally immunised with:

[0161] PBS;

[0162] SARS-CoV-2 spike antigen (Ag) (2 pg);

[0163] C100 (100 pg) + Ag (2 pg);

[0164] C90 (100 pg) + Ag (2 pg);

[0165] Protasan (100 pg) + Ag (2 pg);

[0166] C72 (100 pg) + Ag (2 pg);

[0167] C50 (100 pg) + Ag (2 pg); a-GalCer (10 pg) + Ag (2 pg); or

[0168] C100 (100 pg) + a-GalCer (10 pg) + Ag (2 pg) (C100-a-GalCer), according to Fig. 1. PBS or SARS-CoV-2 spike alone were used as vehicle or antigen only controls, respectively.

[0169] On day 35, mice were euthanised, and spleen and lung single-cell suspensions were obtained. Cells were plated and re-stimulated ex vivo with cRPMI media or SARS-CoV-2 spike (2 pg / mL) for 72 hours. Supernatants were collected and cytokine levels determined by ELISA. The results are presented in Fig. 4.

[0170] Fig. 4A and B show the IFN-y concentration from lung and spleen re-stimulation with SARS-CoV-2 spike, respectively, obtained from C57BL / 6J mice intranasally immunised with vaccine compositions comprising SARS-CoV-2 spike antigen (Ag) and various chitosan preparations (C50, C72, Protasan (P113), C90 and C100), and of compositions comprising SARS-CoV-2 spike antigen and a- galactosylceramide (a-GalCer), with and without C100. Fig. 4C shows the IL-17 concentration from lung re-stimulation with SARS-CoV-2 spike.

[0171] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01 , *** p < 0.001.

[0172] The results show that the combination of C100 with a-galactosylceramide enhances its adjuvanticity, through a significant increase in the production of antigen-specific IFN-y (Fig. 4 A). Example 3

[0173] Highly deacetylated chitosan preparations are effective drivers of humoral immunity but only C1OO and C1OO-a-GalCer consistently enhance humoral immunity across all IgG isotypes.

[0174] Five C57BL / 6J mice per group aged 8-12 weeks-old were intranasally immunised with:

[0175] PBS;

[0176] SARS-CoV-2 spike antigen (Ag) (2 pg);

[0177] 0100 (100 pg) + Ag (2 pg);

[0178] 090 (100 pg) + Ag (2 pg);

[0179] Protasan (100 pg) + Ag (2 pg);

[0180] 072 (100 pg) + Ag (2 pg);

[0181] 050 (100 pg) + Ag (2 pg); a-GalCer (10 pg) + Ag (2 pg);or

[0182] C100 (100 pg) + a-GalCer (10 pg) + Ag (2 pg) (C100-a-GalCer), according to Fig. 1. PBS or SARS-CoV-2 spike alone were used as vehicle or antigen only controls, respectively.

[0183] On day 35, mice were euthanised, and blood and nasal wash collected. Fig. 5A-D show nasal wash antigen-specific IgA (A), and serum antigen-specific IgG (B), lgG2c (C), and lgG1 (D) titres determined by ELISA, of C57BL / 6J mice intranasally immunised with vaccine compositions comprising SARS-CoV- 2 spike antigen (Ag) and various chitosan preparations (C50, C72, Protasan (P113), C90 and C100), and of compositions comprising SARS-CoV-2 spike antigen and a-galactosylceramide (a-GalCer), with and without C100. The endpoint-titre cutoff point was defined as the mean of the vehicle control group ± 3 SD.

[0184] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 .

[0185] The results show that C100 is the only chitosan preparation to consistently enhance humoral immunity across all IgG isotypes. In particular, it is the only chitosan preparation to produce a significant increase in the production of lgG1 (Fig. 5 D). Furthermore, the combination of C100 with a-galactosylceramide provides an improvement over the use of a-galactosylceramide alone, with a-galactosylceramide not producing a significant increase in IgA. Example 4

[0186] C1OO enhances pulmonary and splenic antigen-specific CD8+ T cell responses.

[0187] Fifteen C57BL / 6J mice (5 per experiment) per group aged 8-12 weeks-old were intranasally immunised with:

[0188] PBS;

[0189] SARS-CoV-2 spike antigen (Ag) (2 pg); or

[0190] C100 (100 pg) + Ag (2 pg).

[0191] On day 35, antigen-specific CD8+ T cell responses were quantified in the lungs and spleen using H- 2Kb SARS-CoV-2 spike (VNFNFNGL) MHO Class I Tetramer. PBS or SARS-CoV-2 spike alone were used as vehicle or antigen only controls, respectively.

[0192] Fig. 6 shows the percentage of antigen-specific CD8+ T cells in the lungs of C57BL / 6J mice intranasally immunised with vaccine compositions comprising SARS-CoV-2 spike antigen and C100.

[0193] Data are expressed as the mean ± SD of fifteen biological replicates pooled from three independent experiments and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. *** p < 0.001.

[0194] The results further support that C100 enhances immune response through the production of antigen specific CD8+ T cells.

[0195] Example 5

[0196] C100, C100-Polyl:C, C100-CpG, and C100-MPLA-SM enhance pulmonary antigen-specific CD8+ T cell responses but none are superior to C 100 alone

[0197] Female C57BL / 6J mice (n = 5) aged 8-12 weeks-old were intranasally immunised with:

[0198] PBS,

[0199] SARS-CoV-2 spike antigen (Ag) (2 pg);

[0200] C100 (100 pg) + Ag (2 pg);

[0201] C100 (100 pg) + a-GalCer (10 pg) + Ag (2 pg);

[0202] C100 (100 pg) + Imiquimod (10 pg) + Ag (2 pg);

[0203] C100 (100 pg) + Poly l:C (10 pg) + Ag (2 pg);

[0204] C100 (100 pg) + CpG ODN 1826 (CpG) (10 pg) + Ag (2 pg);

[0205] C100 (100 pg) + CDA (10 pg) + Ag (2 pg);or C100 (100 pg) + MPLA-SM (10 pg) + Ag (2 pg), according to Fig. 1. PBS or SARS-CoV-2 spike alone were used as vehicle or antigen only controls, respectively.

[0206] On day 35, antigen-specific CD8+ T cell responses were quantified in the lungs using H-2Kb SARS- CoV-2 spike (VNFNFNGL) MHO Class I Tetramer according to the gating strategy detailed in Fig. 2. The results are presented in Fig. 7.

[0207] Fig. 7A shows pseudo colour dot plots indicating the percentage of antigen-specific CD44hi Tetramer+ CD8+ T cells quantified in the lungs of C57BL / 6J mice intranasally immunised with vaccine compositions comprising SARS-CoV-2 spike antigen (Ag) and C100, in combination with a- galactosylceramide (a-GalCer), imiquimod, polyinosinic:polycytidylic acid (Poly l:C), CpG ODN 1826, (CpG) cyclic di-AMP (CDA) or monophosphoryl lipid A (MPLA-SM).

[0208] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01.

[0209] The results show that the combination of C100 with further immunomodulatory compounds also results in the production of antigen-specific CD8+T cells. In particular, significant responses are observed for the combination of C100 with Poly l:C, CpG and MPLA-SM (Fig. 7 B).

[0210] Example 6

[0211] Combination of C100 with other immunomodulators can increase antigen-specific antibody responses in serum and nasal wash

[0212] Five C57BL / 6J mice per group aged 8-12 weeks-old were intranasally immunised with:

[0213] PBS;

[0214] SARS-CoV-2 spike antigen (Ag) (2 pg);

[0215] C100 (100 pg) + Ag (2 pg);

[0216] C100 (100 pg) + a-GalCer (10 pg) + Ag (2 pg);

[0217] C100 (100 pg) + Imiquimod (10 pg) + Ag (2 pg);

[0218] C100 (100 pg) + Poly l:C (10 pg) + Ag (2 pg);

[0219] C100 (100 pg) + CpG ODN 1826 (CpG) (10 pg) + Ag (2 pg);

[0220] C100 (100 pg) + CDA (10 pg) + Ag (2 pg); or or C100 (100 pg) + MPLA-SM (10 pg) + Ag (2 pg), according to Fig. 1. PBS or SARS-CoV-2 spike alone were used as vehicle or antigen only controls, respectively.

[0221] On day 35, mice were euthanised, and blood and nasal wash collected. Fig. 8 shows nasal wash antigen-specific IgA (A), and serum antigen-specific IgG (B), lgG2c (C), and lgG1 (D) titres determined by ELISA, of C57BL / 6J mice intranasally immunised with vaccine compositions comprising SARS-CoV- 2 spike antigen (Ag) and C100, in combination with a-galactosylceramide (a-GalCer), imiquimod, poly inosinic: polycytidy lie acid (Poly l:C), CpG ODN 1826, (CpG) cyclic di-AMP (CDA) or monophosphoryl lipid A (MPLA-SM). The endpoint-titre cutoff point was defined as the mean of the vehicle control group ± 3 SD.

[0222] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01 , *** p < 0.001 , **** p < 0.0001 .

[0223] The results show that the production of IgG isotypes is enhanced when C100 is combined with further immunomodulatory compounds. In particular, the combination of C100 with CpG, CDA and MPLA-SM was observed to result in consistently high IgG production, with significant increases observed across all IgG isotypes.

[0224] Example 7

[0225] Combination of C100 with other immunomodulators can enhance antigen-specific IFN-y in spleen and lung

[0226] Five C57BL / 6J mice per group aged 8-12 weeks-old were intranasally immunised with:

[0227] PBS;

[0228] SARS-CoV-2 spike antigen (Ag) (2 pg);

[0229] C100 (100 pg) + Ag (2 pg);

[0230] C100 (100 pg) + a-GalCer (10 pg) + Ag (2 pg);

[0231] C100 (100 pg) + Imiquimod (10 pg) + Ag (2 pg);

[0232] C100 (100 pg) + Poly l:C (10 pg) + Ag (2 pg);

[0233] C100 (100 pg) + CpG ODN 1826 (CpG) (10 pg) + Ag (2 pg);

[0234] C100 (100 pg) + CDA (10 pg) + Ag (2 pg); or

[0235] C100 (100 pg) + MPLA-SM (10 pg) + Ag (2 pg), according to Fig. 1. PBS or SARS-CoV-2 spike alone were used as vehicle or antigen only controls, respectively. On day 35, mice were euthanised, and spleen and lung single-cell suspensions were obtained. Cells were plated and re-stimulated ex vivo with cRPMI media or SARS-CoV-2 spike (2 pg / mL) for 72 hours. Supernatants were collected and cytokine levels determined by ELISA. The results are presented in Fig. 9.

[0236] Fig. 9A and B show the IFN-y concentration from spleen and lung re-stimulation with SARS-CoV-2 spike, respectively, obtained from C57BL / 6J mice intranasally immunised with vaccine compositions comprising SARS-CoV-2 spike antigen (Ag) and C100, in combination with a-galactosylceramide (a- GalCer), imiquimod, polyinosinic: polycytidy lie acid (Poly l:C), CpG ODN 1826, (CpG) cyclic di-AMP (CDA) or monophosphoryl lipid A (MPLA-SM).

[0237] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01 , *** p < 0.001.

[0238] The results show that that the combination of C100 with either CpG, CDA or MPLA-SM is particularly effective at enhancing adjuvanticity through an increase in the production of antigen-specific IFN-y in restimulated lung cells, with a large effect also observed in the spleen with CDA and MPLA-SM.

[0239] Example 8

[0240] Combination of C100 with a commercial influenza vaccine in a two-dose regimen can enhance antigenspecific IFN-y in lung and antigen-specific nasal-wash IgA, serum lgG2c and IgG

[0241] Five C57BL / 6J mice per group aged 8-12 weeks-old were intranasally immunised two times, two weeks apart, with:

[0242] PBS;

[0243] Influvac Tetra (1 pg hemagglutinin); or

[0244] C100 (100 pg) + Influvac Tetra (1 pg hemagglutinin).

[0245] Influvac Tetra is a commercially available quadrivalent influenza vaccine composition comprising purified, inactivated influenza surface antigen (hemagglutinin) from 4 different influenza strains (2 x A and 2 x B), at a concentration of 120 pg / mL (4 x 30 pg / mL), intended for intramuscular or deep subcutaneous injection at a dose of 0.5 mL.

[0246] PBS or Influvac Tetra alone were used as vehicle or antigen only controls, respectively.

[0247] On day 21, mice were euthanised, and spleen and lung single-cell suspensions and blood and nasal wash were obtained. Cells were plated and re-stimulated ex vivo with cRPMI media or Influvac Tetra (1 pg / mL hemagglutinin) for 72 hours. Supernatants were collected and cytokine levels determined by ELISA. The results are presented in Fig. 10.

[0248] Fig. 10A and B show IFN-y concentration (pg / mL) from lung and spleen re-stimulation with Influvac Tetra (1 pg / mL hemagglutinin), respectively, obtained from C57BL / 6J mice intranasally immunised twice with vaccine compositions comprising influenza hemagglutinin (Influvac) and C100. Fig. 10C-D show nasal wash antigen-specific IgA (A), and serum antigen-specific lgG2c (D), and IgG (E) titres determined by ELISA for the same group of mice. The endpoint-titre cutoff point was defined as the mean of the vehicle control group ± 3 SD.

[0249] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01 , *** p < 0.001.

[0250] The results show that the combination of C100 with a commercially available influenza vaccine (Influvac Tetra) in a two-dose regimen significantly enhances antigen-specific mucosal immune responses.

[0251] Example 9

[0252] Combination of C100 with a commercial influenza vaccine in a three-dose regimen can enhance antigen-specific nasal-wash IgA, serum lgG2c and IgG

[0253] Five C57BL / 6J mice per group aged 8-12 weeks-old were intranasally immunised three times, two weeks apart with:

[0254] PBS;

[0255] Influvac Tetra (1 pg hemagglutinin); or

[0256] C100 (100 pg) + Influvac Tetra (1 pg hemagglutinin).

[0257] PBS and Influvac Tetra alone were used as vehicle or antigen only controls, respectively.

[0258] On day 28, mice were euthanised, and spleen and lung single-cell suspensions and blood and nasal wash were obtained. Cells were plated and re-stimulated ex vivo with cRPMI media or Influvac Tetra (1 pg / mL hemagglutinin) for 72 hours. Supernatants were collected and cytokine levels determined by ELISA.

[0259] Fig. 11A and B show IFN-y concentration (pg / mL) from lung and spleen re-stimulation with Influvac Tetra (1 pg / mL hemagglutinin), respectively, obtained from C57BL / 6J mice intranasally immunised three times with vaccine compositions comprising influenza hemagglutinin (Influvac) and C100. Fig. 11C-D show nasal wash antigen-specific IgA (A), and serum antigen-specific lgG2c (D), and IgG (E) titres determined by ELISA for the same group of mice. The endpoint-titre cutoff point was defined as the mean of the vehicle control group ± 3 SD.

[0260] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against antigen only. * p < 0.05, ** p < 0.01 , *** p < 0.001.

[0261] The results show that the combination of C100 with a commercially available influenza vaccine (Influvac Tetra) in a three-dose regimen significantly enhanced antigen-specific mucosal immune responses.

[0262] Example 10

[0263] Intranasal administration of C100 during simultaneous intramuscular and intranasal vaccination enhances systemic and mucosal immune responses to influenza

[0264] Five C57BL / 6J mice per group aged 8-12 weeks-old were simultaneously immunised with combinations of intramuscular (IM) and intranasal (IN) treatments, to evaluate the individual and combined effects of adjuvant and antigen delivered via the IN route in the context of systemic Influvac Tetra vaccination.

[0265] Mice received either

[0266] PBS IM + PBS IN;

[0267] Influvac Tetra IM only (1 pg hemagglutinin);

[0268] Influvac Tetra IM (1 pg hemagglutinin) + C100 IN (100 pg);

[0269] Influvac Tetra IM (1 pg hemagglutinin) + C100-lnfluvac IN (100 pg C100, 1 pg hemagglutinin); or

[0270] Influvac Tetra IM (1 pg hemagglutinin) + Influvac Tetra IN (1 pg hemagglutinin), according to Fig. 12. PBS or Influvac Tetra IM only were used as vehicle or antigen only controls, respectively.

[0271] On day 21, mice were euthanised, and spleen and lung single-cell suspensions and blood and nasal wash were obtained. Cells were plated and re-stimulated ex vivo with cRPMI media or Influvac Tetra (1 pg / mL hemagglutinin) for 72 hours. Supernatants were collected and cytokine levels determined by ELISA.

[0272] Fig. 13A and B show IFN-y concentration (pg / mL) from lung and spleen re-stimulation with Influvac Tetra (1 pg / mL hemagglutinin), respectively, obtained from C57BL / 6J mice intranasally immunised with vaccine compositions comprising influenza hemagglutinin (Influvac) and C100, in combination with intramuscular immunisation with Influvac. Fig. 13C and show nasal wash antigen-specific IgA and serum antigen-specific lgG2c titres, respectively, determined by ELISA for the same group of mice. The endpoint-titre cutoff point was defined as the mean of the vehicle control group ± 3 SD.

[0273] Data are expressed as the mean ± SD of five biological replicates from one independent experiment and were analysed by one-way ANOVA with Dunnett’s multiple comparison test against mRNA IM only.

[0274] * p < 0.05, ** p < 0.01, *** p < 0.001.

[0275] The results show that modifying traditional vaccination schedules to a simultaneous intramuscular and intranasal strategy significantly improves the efficacy of a commercially available influenza vaccine (Influvac Tetra) at promoting mucosal immunity. The enhancement is only observed when the intranasal component includes both the cognate antigen and C100, not with the antigen alone.

Claims

Claims1. A mucosal vaccine composition comprising an antigen and an adjuvant, wherein the adjuvant is chitosan that is at least 99% deacetylated.

2. The mucosal vaccine composition as claimed in claim 1, wherein the chitosan is 100% deacetylated.

3. The mucosal vaccine composition as claimed in claims 1 or 2, wherein the vaccine is a pulmonal or intranasal vaccine.

4. The mucosal vaccine composition as claimed in any of claims 1-3, wherein the chitosan is present at a concentration of 1-20 mg / mL.

5. The mucosal vaccine composition as claimed in any of claims 1-4, wherein the antigen is selected from a protein or peptide (e.g. a recombinant protein or a glycoprotein), a polysaccharide (e.g. a lipopolysaccharide), a polynucleotide, a cell or a virus.

6. The mucosal vaccine composition as claimed in any of claims 1-5, wherein the antigen is present at a concentration of 1-2,000 pg / mL.

7. The mucosal vaccine composition as claimed in any of claims 1-6, wherein the composition comprises a further immunomodulatory compound.

8. The mucosal vaccine composition as claimed in claim 7, wherein the further immunomodulatory compound is selected from a-galactosylceramide, imiquimod, polyinosinic: polycytidylic acid, CpG ODN 1826, cyclic di-AMP (CDA) or monophosphoryl lipid A.

9. The mucosal vaccine composition as claimed in claim 7 or 8, wherein the further immunomodulatory compound is present at a concentration of 100-2,000 pg / mL.

10. An adjuvant comprising chitosan that is at least 99% deacetylated chitosan for use in a method of immunotherapy, wherein the adjuvant is administered by mucosal administration in combination with an antigen.

11. The adjuvant for use as claimed in claim 10, wherein the chitosan is 100% deacetylated.

12. The adjuvant for use as claimed in claim 10 or 11, wherein the mucosal administration is pulmonal or intranasal administration.

13. The adjuvant for use as claimed in any of claims 10-12, wherein the method of immunotherapy is for the prevention of a pathogenic disease.

14. The adjuvant for use as claimed in claim 13, wherein the pathogenic disease is a respiratory disease.

15. The adjuvant for use as claimed in claim 14, wherein the respiratory disease is caused by a viral infection selected from a human coronavirus (e.g. SARS-CoV, SARS-CoV-2 or MERS-CoV), influenza, parainfluenza or respiratory syncytial virus (RSV), or a bacterial infection selected from Bordetella pertussis, Corynebacterium diphtheriae, Clostridium tetani, Haemophilus influenzae, Mycobacterium tuberculosis and Streptococcus pneumoniae.

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