Polymeric-carbohydrate conjugates or nanoparticle vaccines, methods and uses thereof

Abstract

The present disclosure relates to a polymeric–carbohydrate conjugate or nanoparticle comprising a chitosan derivative and the use of said conjugate or nanoparticle as a vaccine delivery system. Moreover, it relates to the use of said conjugate or nanoparticle in immunization or gene therapy, in particular, for the treatment of infectious diseases.

Description

D E S C R I P T I O NPOLYMERIC-CARBOHYDRATE CONJUGATES OR NANOPARTICLE VACCINES, METHODS ANDUSES THEREOFTECH N ICAL FI ELD

[0001] The present disclosure relates to biomedical materials and immunology. More specifically to polymeric-carbohydrate conjugates or nanoparticles as vaccine delivery systems for prevention and / or treatment of infectious diseases, particularly those affecting the respiratory tract.BACKG ROU N D

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has triggered one of the most impactful pandemics in human history. As of November 2024, the virus has been linked to over 777 million infections and 7.1 million fatalities globally. Beyond the staggering health consequences, the pandemic has significantly disrupted economies and placed immense tension on healthcare systems worldwide, disproportionately affecting vulnerable populations due to physical distancing measures and overburdened medical infrastructure. SARS-CoV-2 is a positive-sense, singlestranded RNA virus belonging to the Betacoronavirus genus, featuring one of the largest known viral RNA genomes (~30 kb) [1], Its genetic material encodes structural proteins essential for viral replication and dissemination, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins. The S protein is particularly crucial, mediating viral entry into host cells by binding the angiotensin-converting enzyme 2 (ACE2) receptor via its receptor-binding domain (RBD) and facilitating membrane fusion with the help of the transmembrane serine protease TMPRSS2 [2] [3], This protein is also the primary immunogenic target for neutralizing antibodies and forms the basis for the design of all COVID-19 vaccines approved by the World Health Organization to date. COVID-19 vaccines, developed with unprecedented speed, are based on diverse platforms such as mRNA, viral vectors, and inactivated viruses [4], While these vaccines have significantly mitigated the pandemic's impact, the emergence of SARS-CoV-2 variants of concern, including Beta (B.1.351), Alpha (B.l.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529), has raised new challenges. Mutations within the S protein not only influence infectivity and severity but also reduce vaccine efficacy by altering the binding affinity of neutralizing antibodies [5], underlining the necessity for next -generation vaccines.

[0003] To address the challenges posed by emerging SARS-CoV-2 variants, vaccine strategies should prioritize durable and broad-spectrum protection, leveraging insights into the virus's molecular biology and immune evasion mechanisms. DNA vaccines are a promising solution due to their flexibility in adapting to new variants by modifying antigenic sequences, as well as their inherent stability, whichsimplifies storage and transport, even in resource-limited settings. Traditional DNA vaccines targeting muscle cells may lead to weaker immune responses due to insufficient co-stimulation. However, directing DNA delivery to professional antigen-presenting cells (APCs), such as dendritic cells (DCs), significantly enhances immune activation. DNA vaccines activate the immune system by delivering plasmid DNA that encodes viral antigens, such as the SARS-CoV-2 S protein. Once inside host cells, the DNA is transcribed into mRNA, and subsequently translated into viral proteins. These endogenous proteins are then processed and presented on MHC class I molecules, triggering the activation and differentiation of CD8+ cytotoxic T cells. These cytotoxic T cells release cytolytic granules containing perforins and granzymes, as well as pro-inflammatory cytokines like TNF-a and IFN-y, to eliminate infected cells . Meanwhile, DCs mature, upregulating co-stimulatory molecules and cytokines, and migrating to lymph nodes, where they present antigens to naive CD4+ T cells through MHC class II molecules, thereby priming the T cells. Upon activation, the CD4+ T cells differentiate into various effector subsets, including Thl, Th2, Thl7, and follicular helper T (Tfh) cells .

[0004] DCs and other APCs express a variety of pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), which are activated not only by pathogen-associated molecular patterns during infection but also by specific ligands.

[0005] The intranasal FluMist™ vaccine showcases the potential of mucosal immunization to elicit a more complete immune response against respiratory pathogens compared to traditional parenteral methods [6], Intranasal vaccine administration capitalizes on the distinctive immunological properties of the nasal mucosa, including its expansive absorptive surface, limited enzymatic degradation, and the presence of nasal-associated lymphoid tissue (NALT). This highly organized lymphoid structure plays a pivotal role in antigen sampling and subsequent immune activation. Microfold cells, specialized for capturing antigens and delivering them to underlying APCs, are central to NALT's function. This process elicits both localized mucosal immunity at the infection site and across distal mucosae, such as the respiratory and genital tracts, as well as systemic immune responses. A hallmark of mucosal immunization is the production of secretory immunoglobulin A (slgA), the predominant antibody in mucosal secretions. slgA not only inhibits pathogen attachment to the nasal epithelium but also displays increased cross-reactivity compared to systemic IgG, offering potential protection against a broad range of viral variants. This capability is exemplified by live-attenuated intranasal influenza vaccines, which have demonstrated cross-protective immunity against diverse strains, likely mediated by slgA's unique properties [7], Beyond immunological benefits, intranasal vaccines are needle-free, simplifying administration, which is critical for large-scale immunization campaigns. Furthermore, NALT's slower aging compared to other lymphoid sites makes intranasal delivery particularly advantageous for elderly populations [8],

[0006] Chitosan, a versatile biopolymer, has garnered substantial research interest in gene delivery and vaccine applications. Its cationic characteristics enable the formation of stable complexes with DNA,protecting the genetic material from enzymatic degradation and ensuring efficient antigen delivery to target cells. Moreover, chitosan's mucoadhesive properties contribute to prolonged antigen retention at mucosal surfaces and enhanced penetration across epithelial barriers. Not only that, but this polymer has also been shown to enhance macrophage activation, induce pro-inflammatory cytokine production, and elicit cytotoxic T cell responses, further enhancing its immunostimulatory potential [9],

[0007] US20230293666A1 discloses a nanoparticle comprising mannose conjugated chitosan and an inactivated influenza A virus antigen.

[0008] EP4319726A1 and WO2022215079A1 describe polymeric nanoparticles for treating COVID- 19.

[0009] EP3902530A4 discloses a polymeric nanoparticle for treating diseases associated with abnormal cell growth or an infection.

[0010] CN107583045B, CN117618389A and CN117467127A disclose chitosan nanoparticles for mucosal vaccination.

[0011] CA3182756A1 discloses an immunogenic construct using a nanoparticle platform containing chitosan and an antigen or antigen producing agent for treating an infectious disease.

[0012] WO2023037387A2 discloses a viral vaccine composition comprising multiple virus types.

[0013] Effective vaccines, particularly mucosal or subcutaneous vaccines, are needed for the prevention and treatment of infectious diseases, with a special emphasis on respiratory infections, such as COVID-19. Since many respiratory pathogens, including SARS-CoV-2, initiate infection through mucosal surfaces, vaccine formulations capable of stimulating both local mucosal immune defenses and systemic protection are highly required.

[0014] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.G EN ERAL DESCRI PTI ON

[0015] The present disclosure relates to a polymeric-carbohydrate conjugate or nanoparticle, so-called functionalized chitosan formulations, and use as a vaccine delivery system for the prevention and / or treatment of infectious diseases, particularly those affecting the respiratory tract, such as COVID-19.

[0016] Surprisingly, the present disclosure has a good transfection ability, enhances the levels of antibodies in the mucosa evidencing targeted delivery and local immune response activation. Besides, the conjugate or nanoparticle of the present disclosure elicits a systemic immune response as well as the release of neutralizing antibodies. In fact, the conjugate or nanoparticle of the present disclosure promote effector memory T-cell development, enhance cytotoxic activity, and stimulate a balanced Thl / Th2 / Thl7 immune response. The simultaneous transfection, targeting, maturation, and balanced multi-armimmune activation through mucosal delivery is unexpected establishing a non-obvious inventive advance over prior nanoparticle vaccine systems.

[0017] An aspect of the present disclosure relates to a polymeric-carbohydrate conjugate or a nanoparticle, wherein said polymeric-carbohydrate conjugate or nanoparticle, comprises chitosan conjugated, i.e., covalently conjugated, with a carbohydrate residue selected from a list consisting of: mannose, gluconic acid, lactobionic acid, or mixtures thereof in other to form a modified chitosan; and a genetic material or an immunogen; with the proviso that, if the carbohydrate residue is mannose, the chitosan is additionally conjugated with at least one further carbohydrate residue selected from a list consisting of: gluconic acid, lactobionic acid, or mixtures thereof.

[0018] In an embodiment for better results, the conjugate or nanoparticle of the present disclosure comprises a mixture of two or more modified chitosan; preferably with a mass ratio ranging from 1:5 to 20:1; more preferably with a mass ratio ranging from 1:5 to 15:1.

[0019] In an embodiment for better results, the mixture of the two or more modified chitosan is chitosan-mannose and chitosan-gluconic acid.

[0020] In an embodiment for better results, the mass ratio between chitosan-mannose and chitosangluconic acid ranges from 1:3 to 10:3; preferably the mass ratio is 7:3.

[0021] In an embodiment for better results, the mixture of the two or more modified chitosan is chitosan-lactobionic acid and chitosan-mannose.

[0022] In an embodiment for better results, the mass ratio between chitosan-lactobionic acid and chitosan-mannose ranges from 1:1 to 10:1; preferably the mass ratio is 9:1.

[0023] In an embodiment for better results, the mixture of the two or more modified chitosan is chitosan-lactobionic acid and chitosan-gluconic acid.

[0024] In an embodiment for better results, the chitosan has a molecular weight ranging from 100 to 200 kDa; preferably 150 to 180 kDa; more preferably 160 to 170 kDa.

[0025] In an embodiment for better results, the degree of deacetylation of the chitosan is at least 50%; preferably ranging from 60 to 95%; more preferably 65 to 94%. The degree of deacetylation was measured using nuclear magnetic resonance (1H NMR).

[0026] In an embodiment for better results, the degree of substitution of mannose, gluconic acid, or lactobionic acid ranges from 10 to 50% measured by nuclear magnetic resonance; preferably ranges from 12 to 30%; more preferably ranges from 15 to 28%.

[0027] In an embodiment for better results, the substitution occurs via / V-linkage.

[0028] In an embodiment for better results, the nanoparticle further comprises a crosslinker.

[0029] In an embodiment for better results, the crosslinker is selected from: sodium tripolyphosphate, sodium sulfate, sodium phosphate, aluminum sulfate, or mixtures thereof.

[0028] In an embodiment for better results, the conjugate or nanoparticle of the present disclosure further comprises laminarin or human serum albumin.

[0030] In an embodiment for better results, the conjugate or nanoparticle of the present disclosure exhibits a particle size ranging from 200 nm to 8 pm.

[0031] In an embodiment for better results, the nanoparticle exhibits a particle size ranging from 100 nm to 500 nm; preferably from 200 to 450 nm.

[0032] In the present application, particle size of the conjugate or nanoparticle may be measured by different techniques, including but not limited to dynamic light scattering (DLS).

[0033] In an embodiment for better results, the conjugate or nanoparticle of the present disclosure exhibits a zeta potential of -10 to +60 mV.; preferably the nanoparticle exhibits a zeta potential ranging from -6 to +57 mV.

[0034] In the present disclosure, the zeta potential of the nanoparticle may be determined by conventional electrophoretic mobility techniques, such as electrophoretic light scattering (ELS), using instruments like a Zetasizer, thereby providing information on the surface charge and colloidal stability of the formulation.

[0035] In an embodiment for better results, the conjugate or nanoparticle of the present disclosure exhibits a polydispersity index ranging from 0.1 to 0.35.

[0036] In the present disclosure the polydispersity index of the nanoparticle may be determined by DLS, providing a dimensionless measure of particle size distribution.

[0037] In an embodiment for better results, the encapsulation / loading efficiency of the genetic material or immunogen is at least 24%.

[0038] In an embodiment for better results, the genetic material is a plasmid DNA; preferably a plasmid DNA encoding a SARS-CoV-2 protein; more preferably a plasmid DNA encoding the SARS-CoV-2 protein.

[0039] In an embodiment for better results, the genetic material is a plasmid DNA encoding the SARS- CoV-2 spike protein.

[0040] In an embodiment for better results, the mass ratio of modified chitosan to genetic material ranges from 4:1 to 10:1.

[0041] In an embodiment for better results, the immunogen is selected from a list consisting of: toll-like receptor agonists, STING agonists, RIG-l-like receptor agonists, NOD-like receptor agonists, or combinations thereof.

[0042] In an embodiment for better results, the immunogen may be combined with an immunopotentiator selected from a list consisting of: toll-like receptor agonists, STING agonists, RIG-l-like receptor agonists, NOD-like receptor agonists, or combinations thereof.

[0043] In an embodiment for better results, said conjugate or nanoparticle is administered intranasally, intramuscularly, orally, or subcutaneously; preferably intranasally or subcutaneously.

[0044] Another aspect of the present disclosure relates to the use of the conjugate or nanoparticle of the present disclosure as a vaccine delivery system; preferably as a DNA vaccine.

[0045] Even another aspect of the present disclosure relates to the use of the conjugate or nanoparticle of the present disclosure in gene therapy or immunization.

[0046] Another aspect of the present disclosure relates to the use of the of the conjugate or nanoparticle of the present disclosure in the treatment of infectious diseases; preferably respiratory infectious diseases; more preferably COVID-19.

[0047] An aspect of the present disclosure relates to a method for producing said nanoparticle comprising the steps of: providing a solution comprising chitosan, modified chitosan, or a mixture of two or more modified chitosan; adding a crosslinker solution to induce coacervation or precipitation; obtaining said nanoparticle; optionally isolating said nanoparticle by centrifugation or filtration.

[0048] In an embodiment for better results, said method further comprises the step of adding the immunogen with the crosslinker solution to encapsulate the immunogen.

[0049] In an embodiment for better results, said method further comprises the step of adsorbing the immunogen to the nanoparticles.

[0050] Even another aspect relates to the use of the conjugate or nanoparticle described herein for the manufacture of a medicament for the treatment of infectious diseases; preferably respiratory diseases; more preferably COVID-19.

[0051] Another aspect of the present disclosure relates to a method for treating or preventing infectious diseases in a subject, the method comprising administering the conjugate or nanoparticle disclosed herein.BRI EF DESCRI PTI ON OF TH E DRAWI N GS

[0052] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0053] Figure 1: Illustration of the results of the physicochemical profile and morphology of functionalized chitosan formulations complexed with luciferase-encoding DNA for vaccine delivery. (A) The size and surface charge (ZP) of the particles were characterized in the sodium acetate buffer, which was the medium used for the polyplex assembly, as well as after being resuspended in cell culture media (RPMI and DMEM, both supplemented with necessary additives). Results are expressed as mean ± SEM, with a minimum of three independent replicates (n > 3). (B) TEM images were collected, providing representative visual characterization of chitosan-based polyplexes suspended within an acetate buffer medium. The figure includes two images of polyplexes formed from the mixture of lactobionic acid- modified chitosan and mannose-modified chitosan (CL-MAN), and one image of polyplexes from gluconic acid-modified chitosan and mannose-modified chitosan (CGA-MAN). These formulations were selected due to their superior transfection efficiency. The scale bar indicates a length of 1000 nm. (C) Gel electrophoresis was employed to assess the ability of chitosan-based polyplexes, formed from two distinct combinations of functionalized polymers, to bind and immobilize pDNA. The analysis was conducted at polymer-to-pDNA ratios of 6:1, 7.5:1, and 9.5:1. (D) Evaluation of the ability of chitosan-functionalized polyplexes to protect DNA from degradation by DNase I. Polyplexes were exposed to either active or inactive DNase I at a concentration of 1 unit per pg of DNA. The gel electrophoresis data demonstrated the capacity of the polyplexes to shield the DNA from enzymatic degradation. The first lane in the current and previous electrophoresis experiments served as a control, containing unbound plasmid DNA.

[0054] Figure 2: Illustration of the results of the physicochemical and morphological characterization of chitosan-based polyplexes. (A) The average size and zeta potential (ZP) of the particles were assessed in both sodium acetate buffer (pH 5.7) as a stock suspension, and after resuspension in cell culture media (supplemented DMEM and RPMI). The data are presented as mean ± SEM, with a minimum sample size of three (n > 3). (B) Representative TEM images of LA-functionalized Chitosan polyplexes dispersed inAcB; Length of scale bar: 1000 nm. (C) One-month storage stability assessed at 4 °C for four lead polyplex formulations, selected to represent distinct compositional features (including LA-modified chitosan, two different laminarin concentrations, and HSA-containing formulation). Size (DLS) and zeta potential (ELS) were measured on day 0 and day 30. Data are presented as mean ± SEM of three independent particle batches.

[0055] Figure 3: Illustration of the results of the impact of chitosan-functionalized polyplexes on transfection efficiency and cell viability was investigated in A549 cells. (A) Transfection efficiency wasmeasured by incubating 1 pg of plasmid DNA (pDNA) with chitosan derivatives modified with lactobionic acid and mannose (CL-MAN) at three different polymer-to-DNA ratios (7.5:1, 8.5:1, and 9.5:1). Polyethylenimine (PEI) at a polymer-to-DNA ratio of 25:1 was used as a positive control. After 4 h of incubation, luciferase activity was quantified as relative light units per milligram of protein following a 44 h post-transfection period. (B) Cytotoxicity of the gene delivery systems was evaluated under the same conditions as the transfection studies, using the MTT assay to compare the metabolic activity of treated cells with that of unstimulated control cells. (C) Transfection efficiency was evaluated by incubating 1 pg of pDNA with chitosan derivatives that were modified with both gluconic acid and mannose (CGA-MAN) for 4 h. Furthermore, polyplexes formed by the complexation of pDNA and chitosan modified solely with gluconic acid were also assessed, using two different degrees of gluconic acid modification. In total, twelve distinct polyplex conditions were tested, representing three different polymer-to-DNA ratios (6:1, 7.5:1 and 8.5:1) across various chitosan formulations. These conditions were subsequently evaluated for luciferase activity (RLU / mg protein) 44 h post-treatment. (D) A549 cell viability under the new conditions was assessed using the MTT assay and reported as a percentage relative to unstimulated cells. The findings are based on four separate experiments, with data presented as the mean ± SEM. Statistical analyses indicate significant differences (## at p<0.01) between the CL-MAN 0.8:0.2 formulation and the uncomplexed DNA control. Additionally, there are statistically significant differences (* p<0.05, ** p<0.01, and *** p<0.001) between PEI (positive control) and all the chitosan-functionalized formulations.

[0056] Figure 4: Illustration of the results of the effect of chitosan-based polyplexes on transfection efficacy and cell viability of A549 cells. (A) Transfection activity in A549 cells assessed by incubating 1 pg of pDNA complexed with non-modified or modified chitosan (C10LA10 and C5LA10) or PEI as a positive control for 4 h, followed by luciferase activity evaluation (RLU / mg protein) after 44 h. Results represent nine different polyplex preparation conditions using three PolymenDNA ratios with various chitosan formulations. (B) A parallel experiment was conducted to evaluate the corresponding cell viability of the various gene delivery systems using the MTT metabolic activity assay, expressed in comparison to unstimulated control cells. Additionally, HSA and laminarin at two concentrations (25 and 50 pg / mL) were added to the two most promising polyplexes identified in the previous assay to improve results. (C) Transfection activity in A549 cells evaluated by incubating 1 pg of pDNA complexed with the most modified chitosan, C5LA10, in the presence of soluble p-glucan (laminarin) or combined with HSA, or complexed with PEI as a positive control for 4 h, followed by luciferase activity assessment (RLU / mg protein) after 44 h. (D) Cell viability was determined by MTT metabolic activity assay and expressed as percentage relative to unstimulated control cells for the new conditions. Negative controls included cells with no stimulus or cells transfected with naked pDNA. The results represent six independent experiments and data expressed as mean ± SEM. **** at p<0.0001 denote statistical differences between 7.5:1 LAM 25 and the 7.5:1 alone, both obtained from the complexation with C5LA10 polymer; # p<0.05, ### p<0.001,and #### at p<0.0001 denote statistical differences between the positive control PEI and all the chitosan- based formulations.

[0057] Figure 5: Illustration of the results of the analysis of mannose receptor (MR) and macrophage galactose-type lectin (MGL) receptor expression, and polyplex internalization in human monocyte-derived dendritic cells. Basal levels of MR and MGL receptor expression were assessed in dendritic cells derived from human peripheral blood monocytes using flow cytometry. (A) The induction of MGL receptor expression in dendritic cells was quantified by measuring the mean fluorescence intensity (MFI) of the MGL marker following exposure to lactobionic acid-functionalized chitosan polyplexes (7.5:1 and LAM25 formulations, at a concentration of 1 pg of DNA per well) compared to appropriate controls. (B) Similarly, MR receptor expression was analyzed by determining the MFI of the MR marker in dendritic cells treated with mannose-functionalized chitosan polyplexes (C6M9 and AG40 formulations, at a concentration of 1 pg of DNA per well). Non-specific staining was controlled for using the appropriate isotype controls in both cases. (C) Uptake of polyplexes by dendritic cells was examined using flow cytometry following a 4 h incubation period with FITC-labeled chitosan-based polyplexes. Results were presented as the percentage of cells labeled with FITC. (D) To evaluate whether receptor-mediated uptake was occurring, dendritic cells were pre-incubated for 1 h with purified receptor-specific antibodies to competitively block MR or MGL before polyplex exposure. Each data point represents a sample from an independent donor. Statistical analyses were performed using the Kruskal-Wallis test, with significant differences relative to unstimulated controls indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (E) Representative confocal microscopy images further illustrate polyplex uptake by moDCs. The DNA was labeled with Cy5 (pink), while cell nuclei were stained with Hoechst 33342 (blue), and the cell membrane was labeled with Alexa Fluor 488 WGA (green). Stacked images compare untreated dendritic cells (iDCs) and cells exposed to uncomplexed DNA with those stimulated with functionalized chitosan polyplexes, providing a visual representation of DNA uptake and localization. The formulation previously coded as CL- MAN 0.9-0.1 is referred to as C6M9, 7.5:1 LAM25 as LAM25 and CGA-MAN 0.3-0.7 as AG40 in this figure.

[0058] Figure 6: Illustration of the results of the functional and phenotypic analysis of human monocyte- derived dendritic cells exposed to carbohydrate-functionalized chitosan polyplexes. (A) The production of ROS in moDCs was assessed following a 2 h exposure to polyplexes at a final concentration of 1 ug of DNA per well. Lipopolysaccharide (LPS, 20 ng / mL) was included as a positive control, while unstimulated cells served as the negative control. The fluorescence fold increase was calculated as the ratio of the mean fluorescence intensity (MFI) of the treated samples to that of the negative control. The expression of surface activation markers on moDCs was evaluated after 24 h of stimulation with polyplex formulations, uncomplexed DNA, or LPS. The MFI increase was analyzed for MHCII (B), MHCI (C), CD40 (D), CD86 (E), CD80 (F), CCR7 (G), and CXCR3 (H) using flow cytometry. Each data point represents an independent donor. (I) Cell viability under the experimental conditions used for surface marker analysis was confirmedvia the resazurin assay. Cytokine and chemokine secretion by moDCs was measured in cell culture supernatants collected after 24 h of stimulation with either LPS or chitosan-based formulations. The levels of IL-12 (J), IP-10 (K), RANTES (L), IL-6 (M), GRO-a (N), and TNF-a (O) were quantified using ELISA. Statistical analyses were conducted using the Kruskal-Wallis test, with significant differences relative to unstimulated controls indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. The formulation previously coded as CL-MAN 0.9-0.1 is referred to as C6M9, 7.5:1 LAM25 as LAM25 and CGA- MAN 0.3-0.7 as AG40 in this figure.

[0059] Figure 7: Illustration of the results of the humoral immune response induced by intranasal immunization with chitosan-based DNA vaccines against SARS-CoV-2. (A) Serum anti-SARS-CoV-2 total IgG titers were measured in individual vaccinated mice 14 days after the fifth vaccine dose. Subclass analysis of IgGl, lgG2c, lgG3, and IgE was conducted at the end of the study (day 63). Titers were determined as the highest dilution of plasma that produced an absorbance reading at least twice that of the non-immune plasma. (B) Cross-reactivity of serum SARS-CoV-2-specific IgG titers was evaluated against both the Omicron and Wuhan variants. (C) Neutralizing antibody levels were assessed by measuring ACE2-binding inhibition in sera from vaccinated and non-vaccinated (naive) mice at the final endpoint. The dotted line indicates the inhibition threshold (28.3 %). Secretory anti-SARS-CoV-2 IgA was analyzed in vaginal (D) and nasal (E) washings from all groups on day 63, expressed as the ratio of anti-SARS-CoV-2 IgA to total IgA. Data are presented as mean ± SEM. Statistical significance was determined using the Kruskal-Wallis test, with *p < 0.05 and **p < 0.01 compared to the naive group. The formulation previously coded as CL-MAN 0.9-0.1 is referred to as C6M9, 7.5:1 LAM25 as LAM 25 and CGA-MAN 0.3-0.7 as AG40 in this figure.

[0060] Figure 8: Illustration of the results of the SARS-CoV-2 DNA vaccines elicit S protein-specific type 1 cellular immune responses. C57BL / 6 mice received five intranasal doses of carbohydrate-functionalized chitosan DNA vaccines. On day 63, spleen and lung tissues were collected and stimulated with SARS-CoV- 2 S protein peptide pools, followed by flow cytometric analysis. The frequencies of TNF-a (A, B) and IFN- y (C, D) expressing CD4+ and CD8+ T cells, as well as Granzyme B-positive (E) CD8+ T cells, were determined. Memory T cell phenotypes were assessed after 6 h of peptide restimulation, with CD44 and CD62L expression analyzed in CD4+ (F, H) and CD8+ (G, I) T cells. (J) Cell proliferation was evaluated using a BrdU assay to measure BrdU incorporation in restimulated spleen and lung cells. Data are presented as mean ± SEM. Statistical significance was determined using the Kruskal-Wallis test, with *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control group. The formulation previously coded as CL-MAN 0.9- 0.1 is referred to as C6M9, 7.5:1 LAM25 as LAM25 and CGA-MAN 0.3-0.7 as AG40 in this figure.

[0061] Figure 9: Illustration of the results of the cytokine profile of vaccinated mice following SARS-CoV- 2 peptide restimulation of spleen cells. (A) Heatmap illustrating cytokine levels in spleen cell supernatants after restimulation with 1 pg / mL of SARS-CoV-2 S protein peptides. The heatmap shows the Iog2 fold change relative to the negative control (spleen cells from naive mice also restimulated with peptides).Each row represents a distinct cytokine, while each column corresponds to a different vaccinated group. Data are displayed as median values for all animals within each group. Cytokine concentrations associated with Thl (B), Th2 (C), and Th9 / Thl7 / Th22 / Treg (D) immune responses were quantified in spleen cell supernatants after 96 h of peptide restimulation (pg / mL). Statistical significance was assessed using the Kruskal-Wallis test for each cytokine subset. Significant differences relative to the naive group are indicated as **p < 0.01 and ***p < 0.001. The formulation previously coded as CL-MAN 0.9-0.1 is referred to as C6M9, 7.5:1 LAM25 as LAM25 and CGA-MAN 0.3-0.7 as AG40 in this figure.

[0062] Figure 10: Illustration of the results of the cytokine profile of vaccinated mice following SARS-CoV- 2 peptide restimulation of lung cells. (A) Heatmap illustrating cytokine levels in lung cell supernatants after restimulation with 1 pg / mL of SARS-CoV-2 S protein peptides. The heatmap shows the Iog2 fold change relative to the negative control (lung cells from naive mice also restimulated with peptides). Each row represents a distinct cytokine, while each column corresponds to a different vaccinated group. Data are displayed as median values for all animals within each group. Cytokine concentrations associated with Thl (B), Th2 (C), and Th9 / Thl7 / Th22 / Treg (D) immune responses were quantified in lung cell supernatants after 96 h of peptide restimulation (pg / mL). Statistical significance was assessed using the Kruskal-Wallis test for each cytokine subset. Significant differences relative to the naive group are indicated as *p < 0.05, **p < 0.01 and ***p < 0.001. The formulation previously coded as CL-MAN 0.9-0.1 is referred to as C6M9, 7.5:1 LAM25 as LAM25 and CGA-MAN 0.3-0.7 as AG40 in this figure.

[0063] Figure 11: Illustration of the results of the physicochemical characterization of chitosan-based vaccine formulations. The particle size, polydispersity index (PDI), and zeta potential of mannosylated chitosan formulations— either with SARS-CoV-2 spike-encoding DNA complexed within the polymer matrix or adsorbed onto the surface of nanoparticles were analyzed in sodium acetate buffer (used for complex formation) and water (for NP formulation). Data are presented as mean ± standard error of the mean (SEM) from at least five independent experiments (n > 5). Representative TEM images depict the morphology of both the complex and NP formulations, with a scale bar of 1000 nm.

[0064] Figure 12: Illustration of the results of the physicochemical characterization and structural analysis of chitosan-based particles. The particle size, polydispersity index (PDI), and zeta potential of both carbohydrate-functionalized and unmodified chitosan formulations were assessed in pyrogen-free water— the original medium of the nanoparticles— as well as after resuspension in DMEM and RPMI cell culture media (A). Data are reported as mean ± standard error of the mean (SEM), based on a minimum of four independent experiments (n > 4). (B) Representative TEM images illustrate the morphology of the chitosan-based nanoparticles in sterile water, with a scale bar corresponding to 200 nm.

[0065] Figure 13: Illustration of the results of the evaluation of humoral immune responses following intranasal immunization with mannosylated chitosan-based DNA vaccines against SARS-CoV-2. (A) Serum titers of SARS-CoV-2-specific total IgG were quantified in individual vaccinated mice 14 days after the finaldose. IgG subclasses, including IgGl, lgG2c, lgG3, and IgE, were analyzed at the conclusion of the study (day 56). The highest dilution of plasma that yielded absorbance measurements at least double those of non-immune plasma samples was defined as the titer. (B) The cross-reactivity of serum IgG titers was assessed against SARS-CoV-2 Omicron and Wuhan variants. (C) Neutralizing antibody activity was determined by evaluating the inhibition of ACE2 receptor binding in sera from vaccinated and unvaccinated (naive) mice at the final endpoint, using a surrogate virus neutralization test (sVNT) kit from GenScript. The dashed line represents the threshold for inhibition (28.3 %). Results are expressed as mean ± SEM, and statistical differences were evaluated using the Kruskal-Wallis test, with ** p < 0.01 relative to the naive group.

[0066] Figure 14: Illustration of the results of the mucosal immune response induced by intranasal immunization with mannosylated chitosan-based DNA vaccines against SARS-CoV-2. The ratio of anti- SARS-CoV-2 secretory IgA (slgA) to total IgA was evaluated in vaginal (A) and nasal (B) washes from all experimental groups on day 56. Data are expressed as mean ± SEM (n = 5). Statistical analysis was conducted using the Kruskal-Wallis test, with *p < 0.05 and **p < 0.01 indicating significance compared to the naive group.

[0067] Figure 15: Illustration of the results of the SARS-CoV-2 DNA vaccines trigger Thl cellular immune responses in spleen and lung tissues. Female C57BL / 6 mice were intranasally immunized with mannose- functionalized chitosan DNA vaccines, receiving four doses. On day 56, spleen and lung tissues were collected, restimulated with SARS-CoV-2 S protein peptide pools, and analyzed by flow cytometry. The frequencies of TNF-a-producing (A, B), IFN-y-producing (C, D), and Granzyme B-expressing (E) CD4+ and CD8+ T cells were measured. (F) Cellular proliferation was evaluated by BrdU incorporation in spleen and lung cells after restimulation. Data are shown as mean ± SEM (n = 5). Statistical significance was determined using the Kruskal-Wallis test, with *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the naive group.

[0068] Figure 16: Illustration of the results of the memory T cell phenotypes following vaccination with mannosylated DNA vaccines and subsequent SARS-CoV-2 S protein peptide pool restimulation. CD4+ and CD8+ T cell populations from spleen and lung tissues were analyzed for effector (TEM, CD44+ / CD62L-) (A, B) and central (TCM, CD44+ / CD62L+) (C, D) memory phenotypes after 6 h of peptide restimulation. Data are expressed as mean ± SEM (n = 5). Statistical significance was determined using the Kruskal-Wallis test, with *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control group.

[0069] Figure 17: Illustration of the results of the cytokine production in spleen cells following vaccination with mannosylated DNA vaccines and subsequent peptide restimulation. (A) A heatmap depicting relative cytokine levels in spleen cell supernatants after 96 h of exposure to SARS-CoV-2 S protein peptides (1 pg / mL), with color intensities reflecting variations in cytokine concentrations. The heatmap illustrates the Iog2 fold change in comparison to the negative control group. Each rowcorresponds to a unique cytokine, while each column denotes a distinct vaccinated cohort. The reported values represent the median responses observed across all mice within each experimental condition. The concentration of cytokines associated with Thl (B), Th2 (C), and Th9 / Thl7 / Th22 / Treg (D) immune responses were quantified in spleen cell supernatants after restimulation (pg / mL), providing a more detailed quantitative analysis. Statistical significance was evaluated using the Kruskal-Wallis test for each cytokine panel, with *p < 0.05 and **p < 0.01 indicating significant differences compared to the naive group.

[0070] Figure 18: Illustration of the results of the cytokine production in lung cells following vaccination with mannosylated DNA vaccines and subsequent peptide restimulation. (A) A heatmap depicting relative cytokine levels in lung cell supernatants after 96 h of exposure to SARS-CoV-2 S protein peptides (1 pg / mL), with color intensities reflecting variations in cytokine concentrations. The heatmap illustrates the Iog2 fold change in comparison to the negative control group. Each row corresponds to a unique cytokine, while each column denotes a distinct vaccinated cohort. The reported values represent the median responses observed across all mice within each experimental condition. The concentration of cytokines associated with Thl (B), Th2 (C), and Th9 / Thl7 / Th22 / Treg (D) immune responses were quantified in lung cell supernatants after restimulation (pg / mL), providing a more detailed quantitative analysis. Statistical significance was evaluated using the Kruskal-Wallis test for each cytokine panel, with *p < 0.05, **p < 0.01 and ***p < 0.001 indicating significant differences compared to the naive group.

[0071] Figure 19: Illustration of the results of the evaluation of internalization of carbohydrate- functionalized and unmodified nanoparticles in human monocyte-derived macrophages. (A) The uptake of NPs by macrophages was quantified following a 4 h incubation with FITC-conjugated chitosan-based NPs, with the results expressed as the percentage of FITC-positive cells, as determined by flow cytometry. (B) To assess receptor-mediated internalization, macrophages were pre-treated with purified antibodies against the MR or MGL for 1 h to block receptor-specific uptake before exposure to the particles. Each data point represents an independent donor. Statistical analysis was performed using the Kruskal-Wallis test, with significance denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (C) Representative confocal microscopy images showing NP uptake by macrophages. FITC-labeled particles are shown in green, nuclei are stained with Hoechst 33342 (yellow), and cell membranes are labeled with Alexa Fluor 594 WGA (red). Stacked images compare untreated macrophages (iMs) with cells exposed to FITC alone and those treated with functionalized and unmodified chitosan NPs, providing a visual representation of particle internalization and localization.

[0072] Figure 20: Illustration of the results of the functional and phenotypic activation of human monocyte-derived macrophages upon exposure to carbohydrate-functionalized chitosan-based vaccine formulations. (A) Macrophage production of reactive oxygen species (ROS) was measured after a 2 h incubation with NPs (5 pg / mL). Lipopolysaccharide (LPS, 1 pg / mL) was used as a positive control, whileunstimulated cells were included as a negative control. (B) Nitric oxide (NO) levels were quantified after 24 h of exposure to the NP formulations using a fluorescence probe. LPS served as the positive control. The fluorescence fold increase for both ROS and NO was calculated by comparing the mean fluorescence intensity (MFI) of the stimulated samples with the negative control. (C) Cell viability was confirmed under the same conditions as in (B) using the resazurin assay (n = 4). The expression of key macrophage surface markers was evaluated 24 h after stimulation. Flow cytometric analysis was used to measure the MFI increase for CD68 (D), CD86 (E), CD80 (F), CD163 (G), and CD206 (H) in cells treated with chitosan-based formulations or LPS (1 pg / mL), with untreated cells as controls. (I) The viability of macrophages during surface marker expression analysis was confirmed using the resazurin assay. Secretion of chemokines by macrophages was measured in the cell culture supernatants after 24 h of stimulation with LPS, CHIT, CHIT- LA, or CHIT-MAN NPs. The levels of RANTES (J) and IP-10 (K) were quantified. Statistical differences were determined using the Kruskal-Wallis test, with significance indicated relative to the unstimulated control as follows: *p < 0.05, **p < 0.01, and ***p < 0.001.

[0073] Figure 21: Illustration of the results of the humoral (systemic and mucosal) immune responses induced by intranasal vaccination with chitosan-based protein-subunit formulations against SARS-CoV-2. (A) Systemic humoral responses were evaluated by measuring total SARS-CoV-2-specific IgG levels in serum from individual mice 14 days after the third vaccine dose. Subclass analyses of IgGl, lgG2c, lgG3, and IgE were performed on day 42, with titers defined as the highest plasma dilution producing absorbance values at least twice those of non-immune plasma controls. (B) Neutralizing antibody activity was determined in sera from vaccinated and unvaccinated (naive) mice at the study endpoint. ACE2- binding inhibition was quantified using a surrogate virus neutralization test, with the dotted line representing the inhibition threshold (28.3 %). (C) The cross-reactivity of serum IgG titers was assessed against both the Omicron and Wuhan SARS-CoV-2 variants to evaluate the extent of the systemic immune response. Mucosal immune responses were analyzed by quantifying secretory IgA (slgA) in vaginal (D) and nasal (E) wash samples collected on day 42. Results are expressed as the ratio of SARS-CoV-2-specific slgA to total IgA for all experimental groups. All data are presented as mean ± SEM (n = 5). Statistical significance was determined using the Kruskal-Wallis test, with differences compared to the naive group indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0074] Figure 22: Illustration of the results evidencing that the intranasal SARS-CoV-2 protein-subunit vaccines induce S protein-specific type 1 cellular immune responses in spleen and lung tissues. Female C57BL / 6 mice were immunized intranasally with carbohydrate-functionalized chitosan-based vaccines, receiving three distinct doses. On day 42, spleen and lung tissues were harvested and stimulated ex vivo with SARS-CoV-2 S protein peptide pools for 6 h. Immune responses were assessed by flow cytometry. The frequencies of TNF-a-producing (A, B) and IFN-y-producing (C, D) CD4+ and CD8+ T cells, as well as Granzyme B-expressing (E) CD8+ T cells, were quantified. (F) Cell proliferation was assessed using a BrdUincorporation assay in spleen and lung cells after peptide restimulation to evaluate the expansion of antigen-specific cells. Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using the Kruskal-Wallis test, with significant differences relative to the naive group indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0075] Figure 23: Illustration of the results of the memory T cell responses induced by chitosan-based protein-subunit vaccines following SARS-CoV-2 S protein peptide pool restimulation. Effector memory (TEM, CD44+CD62L-) (A, B) and central memory (TCM, CD44+CD62L+) (C, D) phenotypes were analyzed in CD4+ and CD8+ T cell populations from spleen and lung tissues after 6 h of ex vivo restimulation with SARS-CoV-2 S protein peptide pools. Data are presented as mean ± SEM (n = 5). Statistical comparisons were performed using the Kruskal-Wallis test, with significant differences relative to the control group denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0076] Figure 24: Illustration of the results of the cytokine production in spleen cells following vaccination with chitosan-based protein-subunit vaccines after SARS-CoV-2 peptide restimulation. (A) A heatmap showing relative cytokine levels in spleen cell supernatants collected after 96 h of stimulation with 1 pg / mL of SARS-CoV-2 S protein peptides. The data are presented as the Iog2 fold change relative to the negative control group (spleen cells from naive mice also restimulated with peptides), with color intensities reflecting variations in cytokine concentrations. Each row represents a specific cytokine, while columns correspond to different vaccinated groups, with median values displayed for each condition. Cytokines linked to Thl responses (B), Th2 responses (C) and Th9, Thl7, Th22, and Treg pathways (D) were quantified in the supernatants (pg / mL) to provide a detailed quantitative assessment of immune polarization. Statistical analysis was conducted using the Kruskal-Wallis test, and significant differences compared to the naive group are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0077] Figure 25: Illustration of the results of the cytokine production in lung cells following vaccination with chitosan-based protein-subunit vaccines after SARS-CoV-2 peptide restimulation. (A) A heatmap showing relative cytokine levels in lung cell supernatants collected after 96 h of stimulation with 1 pg / mL of SARS-CoV-2 S protein peptides. The data are presented as the Iog2 fold change relative to the negative control group (lung cells from naive mice also restimulated with peptides), with color intensities reflecting variations in cytokine concentrations. Each row represents a specific cytokine, while columns correspond to different vaccinated groups, with median values displayed for each condition. Cytokines linked to Thl responses (B), Th2 responses (C) and Th9, Thl7, Th22, and Treg pathways (D) were quantified in the supernatants (pg / mL) to provide a detailed quantitative assessment of immune polarization. Statistical analysis was conducted using the Kruskal-Wallis test, and significant differences compared to the naive group are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0078] Figure 26: Illustration of the results of the functional and phenotypic characterization of human moDCs exposed to TLR agonist-loaded and unloaded carbohydrate-functionalized chitosan NPs. (A) ROSproduction in moDCs was evaluated after a 2 h incubation with NPs at 5 pg / mL per well. Lipopolysaccharide (LPS, 20 ng / mL) served as positive control, while unstimulated cells were used as the negative control. Free TLR agonists were also tested at concentrations equivalent to those encapsulated in the particles. The fluorescence fold increase was calculated as the ratio of the mean fluorescence intensity (MFI) of treated samples to that of the negative control. Surface activation marker expression was measured by stimulating moDCs for 24 h with various formulations— either the NP formulations (with or without SARS-CoV-2 antigen), free TLR agonists, free antigen, or LPS— and then analyzing MFI changes for CD86 (B), CD80 (C), CD40 (D), MHCII (E), MHCI (F), and CCR7 (G) via flow cytometry, with each data point representing an independent donor. (H) Cell viability under the conditions used for surface marker analysis was confirmed using a resazurin assay. Statistical significance, determined by the Kruskal-Wallis test, is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0079] Figure 27: Illustration of the results of the cytokine and chemokine levels in the culture supernatants were measured 24 h after moDCs were stimulated with either LPS, TLR agonist-loaded functionalized NPs (with or without SARS-CoV-2 antigen), or unloaded particles. Free TLR agonists and free antigen were also tested at concentrations equivalent to those used in the NP formulations. Levels of IP-10 (A), RANTES (B), GRO-a (C), IL-12 (D), IL-6 (E), and TNF-a (F) were quantified through ELISA. Statistical significance was determined using the Kruskal-Wallis test, with differences relative to unstimulated controls denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0080] Figure 28: Illustration of the results of the functional evaluation of stimulated dendritic cells. DCs were exposed for 24 h to both TLR agonist-loaded and unloaded carbohydrate-functionalized NPs and then co-cultured with autologous T cells at a ratio of 1:10. (A) T cell activation was monitored by measuring CD69 expression after one day of co-culture and CD25 expression after six days. (B) T cell polarization toward regulatory (CD25+FoxP3+), Th2 (GATA-3+), and Thl (Tbet+) phenotypes were determined via flow cytometry, focusing on CD4+T cell populations. (C) The cytotoxic potential induced by the stimulated DCs was assessed by quantifying Perforin* and Granzyme B+cells within the CD8+T cell subset. (D) Cytokine and chemokine levels were measured in the supernatants after a six-day autologous mixed lymphocyte reaction (MLR), wherein T cells were co-cultured with DCs matured with either TLR agonist-loaded or unloaded functionalized NPs— all associated with SARS-CoV-2 antigen— as well as with free TLR agonists or free antigen serving as controls. A heatmap displays the log2fold change in cytokine and chemokine expression relative to the negative control (T cells co-cultured with immature DCs), with each row representing a distinct cytokine / chemokine and each column a different experimental condition. Data are expressed as median values from at least four independent experiments, and statistical significance was evaluated using the Kruskal-Wallis test, with *p < 0.05 and **p < 0.01 compared to unstimulated controls.

[0081] Figure 29: Illustration of the results of the systemic and mucosal humoral immune responses induced by intranasal vaccination with TLR agonist-loaded and unloaded functionalized chitosan protein-subunit vaccines against SARS-CoV-2. (A) Serum total anti-SARS-CoV-2 IgG titers were measured in individual vaccinated mice 14 days after the third dose, with further subclass analysis (IgGl, lgG2c, lgG3, and IgE) performed at day 42. Titers were defined as the highest plasma dilution yielding an absorbance value at least twice that of non-immune plasma. (B) Neutralizing antibody activity was assessed via a surrogate virus neutralization test by measuring ACE2-binding inhibition in sera from both vaccinated and naive mice at the final endpoint; the dotted line represents the inhibition threshold (28.3 %). Mucosal responses were evaluated by quantifying secretory anti-SARS-CoV-2 IgA in vaginal (C) and nasal (D) wash samples on day 42, expressed as the ratio of specific IgA to total IgA. The dose-dependent neutralizing activity of serum antibodies against SARS-CoV-2 pseudotyped lentiviruses corresponding to the Omicron (E) and Wuhan (F) variants was determined by measuring luciferase activity, which was normalized to cell viability using a resazurin assay. Data are presented as mean ± SEM (n = 5). Statistical significance was determined using the Kruskal-Wallis test, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to the naive group.

[0082] Figure 30: Illustration of the results of the evaluation of T cell responses in spleen and lung tissues of female C57BL / 6 mice following SARS-CoV-2 protein-subunit vaccination. Mice were immunized intranasally with carbohydrate-functionalized chitosan-based vaccines, both with and without incorporated TLR agonists, administered in three doses. On day 42, spleen and lung tissues were collected and then stimulated ex vivo with SARS-CoV-2 spike protein peptide pools for 6 h. T cell responses were analyzed by flow cytometry, with quantification of TNF-a-producing CD4+and CD8+T cells (A, B), IFN-y- producing CD4+and CD8+T cells (C, D), and Granzyme B-expressing CD8+T cells (E). Additionally, antigenspecific cell proliferation was measured using a BrdU incorporation assay in both spleen and lung cells following peptide restimulation (F). Data are expressed as mean ± SEM (n = 5), and statistical significance relative to the naive group was determined using the Kruskal-Wallis test (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

[0083] Figure 31: Illustration of the results of the memory T cell responses were evaluated following a 6 h restimulation with SARS-CoV-2 spike protein peptide pools in mice immunized with chitosan-based protein-subunit vaccines, with or without TLR agonists. Effector memory T cells (TEM, CD44+CD62L“) (A, B) and central memory T cells (TCM, CD44+CD62L+) (C, D) were quantified within CD4+and CD8+T cell subsets from both spleen and lung tissues. Data are expressed as mean ± SEM (n = 5), with statistical significance determined by the Kruskal-Wallis test (*p < 0.05, **p < 0.01) compared to the control group.

[0084] Figure 32: Illustration of the results of the cytokine production determined in the supernatants of spleen cells following vaccination with TLR agonist-loaded and unloaded chitosan-based protein-subunit vaccines after SARS-CoV-2 peptide restimulation. (A) A heatmap displays the relative cytokine levels in spleen cell supernatants collected 96 h after stimulation with 1 pg / mL of SARS-CoV-2 spike protein peptides. Data are expressed as log2fold changes relative to the negative control (restimulated spleencells from naive mice), with varying color intensities indicating differences in cytokine concentrations. Each row represents a specific cytokine, while each column corresponds to a different vaccinated group, with median values from the 5 animals in each group shown for each condition. Cytokines linked to Th2 responses (B), Th9 / Thl7 / Th22 / Treg pathways (C), and Thl responses (D) were quantified (in pg / mL) to provide a detailed assessment of immune polarization. Statistical significance was determined by the Kruskal-Wallis test, with comparisons to the naive group denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0085] Figure 33: Illustration of the results of the cytokine production determined in the supernatants of lung cells following vaccination with TLR agonist-loaded and unloaded chitosan-based protein-subunit vaccines after SARS-CoV-2 peptide restimulation. (A) A heatmap displays the relative cytokine levels in lung cell supernatants collected 96 h after stimulation with 1 pg / mL of SARS-CoV-2 spike protein peptides. Data are expressed as log2fold changes relative to the negative control (restimulated lung cells from naive mice), with varying color intensities indicating differences in cytokine concentrations. Each row represents a specific cytokine, while each column corresponds to a different vaccinated group, with median values from the 5 animals in each group shown for each condition. Cytokines linked to Th2 responses (B), Th9 / Thl7 / Th22 / Treg pathways (C), and Thl responses (D) were quantified (in pg / mL) to provide a detailed assessment of immune polarization. Statistical significance was determined by the Kruskal-Wallis test, with comparisons to the naive group denoted as *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0086] Figure 34: Illustration of the results of the evaluation of internalization of carbohydrate- functionalized and unmodified nanoparticles in human monocyte-derived dendritic cells. (A) The induction of Macrophage Galactose-Type Lectin (MGL) receptor expression in dendritic cells was quantified by measuring the mean fluorescence intensity (MFI) of the MGL marker following exposure to lactobionic acid-functionalized chitosan NPs compared to appropriate controls. (B) Similarly, Mannose Receptor (MR) receptor expression was analyzed by determining the MFI of the MR marker in dendritic cells treated with mannose-functionalized chitosan NPs. Non-specific staining was controlled for using the appropriate isotype controls in both cases. (C) To assess receptor-mediated internalization, DCs were pretreated with purified antibodies against the MR or MGL for 1 h to block receptor-specific uptake before exposure to the particles. Each data point represents an independent donor. Statistical analysis was performed using the Kruskal-Wallis test, with significance denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (D) Representative confocal microscopy images showing NP uptake by DCs. FITC-labeled particles are shown in green, nuclei are stained with Hoechst 33342 (blue), and cell membranes are labeled with Alexa Fluor 594 WGA (red). Stacked images compare untreated DCs (iDCs) with cells exposed to FITC alone and those treated with functionalized and unmodified chitosan NPs, providing a visual representation of particle internalization and localization.

[0087] Figure 35: Illustration of the results of the functional and phenotypic analysis of human monocyte- derived dendritic cells exposed to carbohydrate-functionalized chitosan NPs. (A) The production of ROS in moDCs was assessed following a 2 h exposure to NPs at a final concentration of 5 ug / mL per well. Lipopolysaccharide (LPS, 20 ng / mL) was included as a positive control, while unstimulated cells served as the negative control. The fluorescence fold increase was calculated as the ratio of the mean fluorescence intensity (MFI) of the treated samples to that of the negative control. The expression of surface activation markers on moDCs was evaluated after 24 h of stimulation with NP formulations, free antigen, or LPS. The MFI increase was analyzed for CD86 (B), CD40 (C), CD80 (D), MHCII (E), MHCI (F), CCR7 (G), and CXCR3 (H) using flow cytometry. Each data point represents an independent donor. (I) Cell viability under the experimental conditions used for surface marker analysis was confirmed via the resazurin assay. Statistical analyses were conducted using the Kruskal-Wallis test, with significant differences relative to unstimulated controls indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0088] Figure 36: Illustration of the results of the humoral (systemic and mucosal) immune responses induced by subcutaneous vaccination with chitosan-based protein-subunit formulations against SARS- CoV-2. (A) Systemic humoral responses were evaluated by measuring total SARS-CoV-2-specific IgG levels in serum from individual mice 14 days after the second vaccine dose. Subclass analyses of IgGl, lgG2c, lgG3, and IgE were performed on day 28, with titers defined as the highest plasma dilution producing absorbance values at least twice those of non-immune plasma controls. (B) Neutralizing antibody activity was determined in sera from vaccinated and unvaccinated (naive) mice at the study endpoint. ACE2- binding inhibition was quantified using a surrogate virus neutralization test, with the dotted line representing the inhibition threshold (28.3 %). (C) The cross-reactivity of serum IgG titers was assessed against both the Omicron and Wuhan SARS-CoV-2 variants to evaluate the extent of the systemic immune response. Mucosal immune responses were analyzed by quantifying secretory IgA (slgA) in vaginal (D) and nasal (E) wash samples collected on day 28. Results are expressed as the ratio of SARS-CoV-2-specific slgA to total IgA for all experimental groups. All data are presented as mean ± SEM (n = 5). Statistical significance was determined using the Kruskal-Wallis test, with differences compared to the naive group indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0089] Figure 37: Illustration of the results of the subcutaneous SARS-CoV-2 protein-subunit vaccines induce S protein-specific type 1 cellular immune responses in spleen and lung tissues. Female C57BL / 6 mice were immunized subcutaneously with carbohydrate-functionalized chitosan-based vaccines, receiving two distinct doses. On day 28, spleen and lung tissues were harvested and stimulated ex vivo with SARS-CoV-2 S protein peptide pools for 6 h. Immune responses were assessed by flow cytometry. The frequencies of TNF-a-producing (A, B) and IFN-y-producing (C, D) CD4+ and CD8+ T cells, as well as Granzyme B-expressing (E) CD8+ T cells, were quantified. (F) Cell proliferation was assessed using a BrdU incorporation assay in spleen and lung cells after peptide restimulation to evaluate the expansion ofantigen-specific cells. Data are presented as mean ± SEM (n = 5). Statistical analysis was performed using the Kruskal-Wallis test, with significant differences relative to the naive group indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0090] Figure 38: Illustration of the results of the memory T cell responses induced by chitosan-based protein-subunit vaccines following SARS-CoV-2 S protein peptide pool restimulation. Effector memory (TEM, CD44+CD62L-) (A, B) and central memory (TCM, CD44+CD62L+) (C, D) phenotypes were analyzed in CD4+ and CD8+ T cell populations from spleen and lung tissues after 6 h of ex vivo restimulation with SARS-CoV-2 S protein peptide pools. Data are presented as mean ± SEM (n = 5). Statistical comparisons were performed using the Kruskal-Wallis test, with significant differences relative to the control group denoted as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0091] Figure 39: Illustration of the results of the cytokine production in spleen cells following vaccination with chitosan-based protein-subunit vaccines after SARS-CoV-2 peptide restimulation. (A) A heatmap showing relative cytokine levels in spleen cell supernatants collected after 96 h of stimulation with 1 pg / mL of SARS-CoV-2 S protein peptides. The data are presented as the Iog2 fold change relative to the negative control group (spleen cells from naive mice also restimulated with peptides), with color intensities reflecting variations in cytokine concentrations. Each row represents a specific cytokine, while columns correspond to different vaccinated groups, with median values displayed for each condition. Cytokines linked to Thl responses (B), Th2 responses (C) and Th9, Thl7, Th22, and Treg pathways (D) were quantified in the supernatants (pg / mL) to provide a detailed quantitative assessment of immune polarization. Statistical analysis was conducted using the Kruskal-Wallis test, and significant differences compared to the naive group are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

[0092] Figure 40: Illustration of the results of the cytokine production in lung cells following vaccination with chitosan-based protein-subunit vaccines after SARS-CoV-2 peptide restimulation. (A) A heatmap showing relative cytokine levels in lung cell supernatants collected after 96 h of stimulation with 1 pg / mL of SARS-CoV-2 S protein peptides. The data are presented as the Iog2 fold change relative to the negative control group (lung cells from naive mice also restimulated with peptides), with color intensities reflecting variations in cytokine concentrations. Each row represents a specific cytokine, while columns correspond to different vaccinated groups, with median values displayed for each condition. Cytokines linked to Thl responses (B), Th2 responses (C) and Th9, Thl7, Th22, and Treg pathways (D) were quantified in the supernatants (pg / mL) to provide a detailed quantitative assessment of immune polarization. Statistical analysis was conducted using the Kruskal-Wallis test, and significant differences compared to the naive group are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.DETAI LED DESCRI PTION

[0093] The present disclosure relates to a polymeric-carbohydrate conjugate or nanoparticle comprising a chitosan derivative and the use of said conjugate or nanoparticle as a vaccine delivery system. Moreover, it relates to the use of said conjugate or nanoparticle in immunization or gene therapy, in particular, for the treatment of infectious diseases.

[0094] In an embodiment, it is described glyco-modified chitosan polymeric-carbohydrate conjugate (or polyplex) synthesis, namely four distinct polymer / DNA complexes were prepared. Two were synthesized from chitosan modified with lactobionic acid: one at a 7.5:1 ratio (7.5:1) and the other at a 7.5:1 ratio with the addition of laminarin (LAM25). The third polyplex was prepared by mixing chitosan modified with both lactobionic acid and mannose (C6M9), while the fourth polyplex was synthesized by mixing chitosan modified with gluconic acid and mannose (AG40). Briefly, purified chitosan, free of lipopolysaccharides, was dissolved in 1 % acetic acid overnight under continuous magnetic agitation. The mass and volume of the chitosan were adjusted to achieve the desired ratios for sugar-modified chitosan. The pH of the chitosan solution was then regulated to 4.6-4.8 for gluconic acid or lactobionic acid modification, and to 5.5 for mannose modification, using 10 M sodium hydroxide. To activate gluconic acid, 600 mg of gluconic acid, 242 mg of / V-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), and 145 mg of N- Hydroxysuccinimide (NHS) were dissolved in 15 mL of lipopolysaccharides (LPS)-free water and stirred for 1 h. For lactobionic acid activation, 200 mg of lactobionic acid, 128.5 mg of EDC, and 77 mg of NHS were dissolved in 20 mL of LPS-free water. Following activation, the gluconic acid solution or lactobionic acid solution was added dropwise to the chitosan solution under slow stirring, and the mixture was incubated at room temperature for 24 h. For the preparation of mannose-functionalized chitosan, 135 mg of D- mannose and 32 mg of NaBH(OAc)3 were dissolved in 15 mL of endotoxin-free water. This solution was magnetically stirred for 1 h to activate the D-mannose. Subsequently, this mannose solution was added dropwise to 15 mL of the chitosan solution under slow magnetic agitation and incubated for 48 h at room temperature. The resulting solutions were then dialyzed for 72 h, to remove any unreacted components. Finally, the various sugar-modified chitosan solutions were freeze-dried for 48 h. Following chitosan functionalization, polyplexes were prepared as follows. For the 7.5:1 and LAM 25 formulations, 375 pL of 200 pg / mL lactobionic acid-modified chitosan dissolved in 25 mM sodium acetate buffer was combined with 200 pL of 50 pg / mL plasmid DNA encoding the SARS-CoV-2 spike protein (DNA spike), which was dissolved in a 50 mM sodium sulfate solution. The resulting mixture was vortexed vigorously for 30 s at 55 °C. In the case of the LAM25 formulation, laminarin at 25 pg / mL was directly added to the 50 mM sodium sulfate solution used to dissolve the DNA spike prior to mixing. For the C6M9 formulation, a mixture of lactobionic acid-modified chitosan and mannose-modified chitosan was prepared in a 9:1 ratio and combined with DNA spike. Specifically, 475 pL of 200 pg / mL of the functionalized chitosan solution was mixed with 200 pL of 50 pg / mL DNA spike solution, followed by vortexing at 55 °C for 30 s. In the caseof the AG40 formulation, a combination of mannose-modified chitosan and gluconic acid-modified chitosan was prepared in a 7:3 ratio and then mixed with the DNA spike. Here, 375 pL of 200 pg / mL functionalized chitosan solution was combined with 200 pL of 50 pg / mL DNA spike in a 50 mM sodium sulfate solution and vortexed under the same conditions. The key modification in this study was the substitution of the plasmid DNA encoding luciferase with plasmid DNA encoding the Omicron variant of the SARS-CoV-2 spike protein. All other preparation steps remained unchanged.

[0095] Table 1 summarizes the chitosan polyplexes of the present disclosure.Table 1. Glyco-modified chitosan.

[0096] In an embodiment, several ratios of chitosan modified with lactobionic acid, gluconic acid, and mannose were tested.

[0097] In an embodiment, for lactobionic acid, chitosan concentrations of 5-10 mg / mL were tested with a fixed 10 mg / mL of lactobionic acid.

[0098] In an embodiment, for mannose, chitosan concentrations of 3-6 mg / mL were tested with a fixed 9 mg / mL of mannose.

[0099] In an embodiment, for gluconic acid, a fixed 10 mg / mL of chitosan was tested with 20-40 mg / mL of gluconic acid.

[0100] In an embodiment, all chitosan modifications were successfully achieved, yielding functional polymers capable of forming stable complexes with DNA for vaccine formulation.

[0101] The polyplexes complexed with DNA are listed in Table 2.Table 2. List of the polyplexes complexed with DNA.LA - Lactobionic Acid; GA - Gluconic Acid; MAN - Mannose; C10AG20 - chitosan modified with gluconic acid at a ratio of 10 mg / mL chitosan to 20 mg / mL gluconic acid; C10AG40 - chitosan modified with gluconic acid at a ratio of 10 mg / mL chitosan to 40 mg / mL gluconic acid; C6M9 - chitosan modified with mannose at a ratio of 6 mg / mL chitosan to 9 mg / mL mannose; C3M9 - chitosan modified with mannose at a ratio of 3 mg / mL chitosan to 9 mg / mL mannose

[0102] In an embodiment, varying the mass of the different functionalized chitosan conjugates from 40 to 180 pg, when using 10 pg of DNA, resulted in efficient complex formation and good transfection performance

[0103] Fig. 1 shows the results of the size and zeta potential measurements of the polyplexes listed on Table 2.

[0104] In an embodiment, the polyplexes are stable after 1 month of storage (Fig. 2).

[0105] Fig. 3 and Fig. 4 reveal the transfection results of the polyplexes listed on Table 2.

[0106] Fig. 5 evidences the targeting ability of CL-MAN 0.9-0.1, CGA-MAN 0.3-0.7, 7.5:1, 7.5:1 LAM 25 in human dendritic cells.

[0107] Fig. 6 reveals that CL-MAN 0.9-0.1, CGA-MAN 0.3-0.7, 7.5:1, 7.5:1 LAM25 induce maturation and pro-inflammatory signaling in human dendritic cells.

[0108] Fig. 7 shows that the intranasal delivery of CGA-MAN 0.3-0.7 and 7.5:1 LAM25, elicited robust mucosal and systemic antibody responses, with neutralizing activity detected in mice serum.

[0109] Fig. 8 shows that CGA-MAN 0.3-0.7 and 7.5:1 LAM2 promotes effector memory T-cell development and enhances cytotoxic activity and induces balanced Thl / Th2 / Thl7 immune activation in spleen and lung cells (Fig. 9 and Fig. 10);Synthesis of Functionalized Chitosan Nanoparticles Loaded with TLR Agonists

[0110] In an embodiment, to synthesize functionalized chitosan NPs, the chitosan polymer was subjected to a two-step modification process. First, purified chitosan was modified by conjugating it with gluconic acid through carbodiimide chemistry, utilizing EDC and NHS reagents. Separately, mannose was attached to chitosan via reductive amination using sodium triacetoxyborohydride. These two chitosan derivatives were then blended in a 7:3 ratio of mannose-functionalized to gluconic acid-functionalized chitosan to create a composite material for further formulation steps. Sugar-functionalized chitosan NPs, either loaded or unloaded with two toll-like receptor (TLR) agonists, were synthesized using a coacervation / precipitation technique with sodium tripolyphosphate (TPP) as the cross-linking agent. Unmodified chitosan NPs were prepared as controls to assess the effects of functionalization and TLR agonist incorporation. Polymer solutions were prepared at a concentration of 0.1 % (w / v) in 1 % (v / v) acetic acid, and the pH was adjusted to 4.6-4.8 using 10 N sodium hydroxide to facilitate NP formation. Cross-linking was initiated by adding a specific volume of a 0.16 % (w / v) TPP solution to the polymer solution while homogenizing at high speed for 1.5 min. The volumes of TPP used were 1750 pL for chitosan NPs (CHIT NPs) and 1600 pL for the mannose- and gluconic acid-modified chitosan blend NPs (CHIT-MAN NPs). For TLR agonist-loaded NPs, two distinct agonists were incorporated: for CHIT-MAN IC NPs, 160 pL of a 5 mg / mL poly l:C solution was mixed with 1440 pL of the 0.178 % (w / v) TPP solution, and for CHIT- MAN CL NPs, 150 pL of a 1 mg / mL CL097 solution was combined with the same volume of TPP. These mixtures were then added to the functionalized polymer solution while being homogenized at high speed, and all the particles were allowed to mature for 1 h. Following synthesis, the NPs were concentrated and washed with pyrogen-free water using a Vivaspin 20 centrifugal concentrator (MWCO 300 kDa) at 3000 x g to remove the preparation medium.

[0111] In an embodiment, for in vivo experiments, the spike protein of the SARS-CoV-2 Omicron variant was adsorbed onto the NPs by incubating 500 pg of the prepared NPs with 10 pg of antigen for 10 min under agitation. Protein adsorption was confirmed using the Micro BCA™ Protein Assay Kit, ensuring efficient antigen loading onto the NP surface for further experimental use.

[0112] Table 3 and Table 4 summarize the physicochemical characterization of the nanoparticles of the present disclosure, so-called chitosan-based vaccine formulations.

[0113] Table 3. Particle characterization of chitosan-based vaccine formulations with the immunogen. The size, polydispersity index (PDI), and zeta potential of the delivery systems were assessed in both pyrogen-free water and supplemented RPMI medium. The loading efficiency (LE) of TLR agonists in each NP formulation was determined using a spectrophotometric assay. Measurements were performed across at least three independent experiments, and the results are expressed as mean ± standard error of the mean (SEM).Table 4. Particle characterization of chitosan-based vaccine formulations. The size, polydispersity index (PDI), and zeta potential of the delivery systems were assessed in both pyrogen-free water and supplemented RPMI and DMEM media. Measurements were performed across at least three independent experiments, and the results are expressed as mean ± standard error of the mean (SEM).

[0114] The nanoparticles complexed with DNA or incorporating an antigenic protein administered in vivo using the intranasal route are listed on Table 5.Table 5. List of the nanoparticles complexed with DNA or incorporating an antigenic protein (intranasal route).CHIT - Chitosan; LA - Lactobionic Acid; GA - Gluconic Acid; MAN - Mannose

[0115] The nanoparticles incorporating an antigenic protein administered in vivo using the subcutaneous route are listed on Table 6.Table 6. List of the nanoparticles incorporating an antigenic protein (subcutaneous route).CHIT - Chitosan; LA - Lactobionic Acid; GA - Gluconic Acid; MAN - Mannose

[0116] In an embodiment, a direct comparison was performed between the AG40 complex (CGA-MAN 0.3-0.7) described previously and the NP formulation (chitosan modified with gluconic acid and chitosan modified with mannose), which was also prepared using the same functionalized chitosan.

[0117] Fig. 11 shows the physicochemical characterization of the AG40 complex (chitosan modified with gluconic acid and chitosan modified with mannose) and the chitosan modified with gluconic acid and mannose nanoparticle.

[0118] In an embodiment, the nanoparticles of the present disclosure reveal consistent size and zeta potential (Table 3, Table 4 and Fig. 12)

[0119] In an embodiment, the intranasal delivery of the nanoparticles of the present disclosure elicited robust mucosal and systemic antibody responses, with neutralizing activity detected in mice serum (Fig.13 and Fig. 14).

[0120] In an embodiment, the nanoparticles of the present disclosure promoted the effector memory T- cell development and enhanced cytotoxic activity (Fig. 15 and Fig. 16).

[0121] In an embodiment, the nanoparticles of the present disclosure boost the antiviral cytokine activity in key immune organs (Fig. 17 and Fig. 18).

[0122] In an embodiment, the nanoparticles of the present disclosure drive targeted uptake and Ml activation in human macrophages (Fig. 19 and Fig. 20).

[0123] In an embodiment, the control NP shows no targeted uptake in macrophages (Fig. 19).

[0124] In an embodiment, the intranasal delivery of the nanoparticles of the present disclosure elicited modest mucosal and systemic antibody responses, with neutralizing activity detected in mice serum (Fig. 21).

[0125] In an embodiment, the nanoparticles of the present disclosure evidence modest cellular immune activation (Fig. 22, Fig. 23, Fig. 24 and Fig. 25).

[0126] In an embodiment, CHIT-LA NPs and CHIT-MAN NPs promote effector memory T-cell development and enhances cytotoxic activity (Fig. 22 and Fig. 23)

[0127] In an embodiment, the nanoparticles of the present disclosure induce a balanced Thl / Th2 / Thl7 immune activation in spleen and lung cells (Fig. 24 and Fig. 25).

[0128] In an embodiment, the nanoparticles of the present disclosure induce maturation and pro- inflammatory signalling in human dendritic cells (Fig. 26 and Fig. 27).

[0129] In an embodiment, CHIT-MAN IC NPs and CHIT-MAN CL NPs activate DCs that promote Thl polarization in a mixed lymphocyte reaction (Fig. 28).

[0130] In an embodiment, CHIT-MAN IC NPs and CHIT-MAN CL NPs intranasal delivery elicited robust mucosal and systemic antibody responses, with neutralizing activity detected in mice serum (Fig. 29).

[0131] In an embodiment, CHIT-MAN IC NPs and CHIT-MAN CL NPs promote effector memory T-cell development and enhances cytotoxic activity (Fig. 30 and Fig. 31).

[0132] In an embodiment, CHIT-MAN IC NPs and CHIT-MAN CL NPs boost antiviral cytokine activity in key immune organs (Fig. 32 and Fig. 33).

[0133] In an embodiment, CHIT-LA NPs and CHIT-MAN NPs drive targeted uptake and induces maturation and pro-inflammatory signaling in human dendritic cells (Fig. 34 and Fig. 35). Control NP shows no targeted uptake in dendritic cells (Fig. 34).

[0134] In an embodiment, the subcutaneous delivery of the present disclosure elicited strong systemic antibody responses, with neutralizing activity detected in mouse serum, but no mucosal response (Fig. 36).

[0135] In an embodiment, CHIT NPs show modest cellular immune activation (Fig. 37, Fig. 38, Fig. 39 and Fig. 40).

[0136] In an embodiment, the subcutaneous delivery of CHIT-LA NPs and CHIT-MAN NPs promote effector memory T-cell development and enhances cytotoxic activity (Fig. 37 and Fig. 38).

[0137] In an embodiment, the subcutaneous delivery of CHIT-LA NPs and CHIT-MAN NPs induce balanced Thl / Th2 / Thl7 immune activation in spleen and lung cells (Fig. 39 and Fig. 40).

[0138] The term "degree of deacetylation" used herein represents the extent to which / V-acetyl groups (-COCH3) in chitin have been removed to form free amino groups (-NHZ) in chitosan. The degree of deacetylation is a dimensionless parameter, commonly expressed as a percentage (%). It may be determined by any suitable analytical technique, including but not limited to1H nuclear magnetic resonance (NMR) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy, conductometric titration, or elemental analysis. For example, a chitosan having a degree of deacetylation of 80% indicates that 80% of the monomer units possess free amino groups, while 20% remain / V-acetylated.

[0139] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0140] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise

[0141] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The abovedescribed embodiments are combinable.

[0142] The following dependent claims further set out particular embodiments of the disclosure.References1. Islam, M.A., A review of SARS-CoV-2 variants and vaccines: Viral properties, mutations, vaccine efficacy, and safety. Infectious Medicine, 2023. 2(4): p. 247-261 DOI: 10.1016 / j.imj.2023.08.005.2. Hadj Hassine, I., Covid-19 vaccines and variants of concern: A review. Reviews in Medical Virology, 2021. 32(4) DOI: 10.1002 / rmv.2313.3. Hu, B., et al., Characteristics of SARS-CoV-2 and COVID-19. Nature Reviews Microbiology, 2020. 19(3): p. 141-154 DOI: 10.1038 / s41579-020-00459-7.4. Ong, S.W.X., T. Chia, and B.E. Young, SARS-CoV-2 variants of concern and vaccine escape, from Alpha to Omicron and beyond. Expert Review of Respiratory Medicine, 2022. 16(5): p. 499-502 DOI: 10.1080 / 17476348.2022.2057299.5. Bian, L., et al., Effects of SARS-CoV-2 variants on vaccine efficacy and response strategies. Expert Review of Vaccines, 2021. 20(4): p. 365-373 DOI: 10.1080 / 14760584.2021.1903879.6. Lycke, N., Recent progress in mucosal vaccine development: potential and limitations. Nature Reviews Immunology, 2012. 12(8): p. 592-605 DOI: 10.1038 / nri3251.7. Jang, Y.H. and B.L. Seong, Cross-Protective Immune Responses Elicited by Live Attenuated Influenza Vaccines. Yonsei Medical Journal, 2013. 54(2): p. 271 DOI 10.3349 / ymj.2013.54.2.271.8. Kehagia, E., P. Papakyriakopoulou, and G. Valsami, Advances in intranasal vaccine delivery: A promising non-invasive route of immunization. Vaccine, 2023. 41(24): p. 3589-3603 DOI:10.1016 / j.vaccine.2023.05.011.9. Tang, J., et al., Nanotechnologies in Delivery of DNA and mRNA Vaccines to the Nasal and Pulmonary Mucosa. Nanomaterials, 2022. 12(2): p. 226 DOI: 10.3390 / nanol2020226.

Claims

C L A I M S1. A polymeric-carbohydrate conjugate or a nanoparticle, wherein said polymeric-carbohydrate conjugate or nanoparticle, comprises chitosan conjugated with a carbohydrate residue selected from a list consisting of: mannose, gluconic acid, lactobionic acid, or mixtures thereof in other to form a modified chitosan; and a genetic material or an immunogen; with the proviso that, if the carbohydrate residue is mannose, the chitosan is additionally conjugated with at least one further carbohydrate residue selected from a list consisting of: gluconic acid, lactobionic acid, or mixtures thereof.

2. The conjugate or nanoparticle according to the previous claim, comprising a mixture of two or more modified chitosan; preferably with a mass ratio ranging from 1:5 to 20:1; more preferably with a mass ratio ranging from 1:5 to 15:1.

3. The conjugate or nanoparticle according to any of the previous claims, wherein the mixture of the two or more modified chitosan is chitosan-mannose and chitosan-gluconic acid.

4. The conjugate or nanoparticle according to the previous claim, wherein the mass ratio between chitosan-mannose and chitosan-gluconic acid ranges from 1:3 to 10:3; preferably the mass ratio is 7:3.

5. The conjugate or nanoparticle according to the any of the previous claims, wherein the mixture of the two or more modified chitosan is chitosan-lactobionic acid and chitosan-mannose.

6. The conjugate or nanoparticle according to the previous claim, wherein the mass ratio between chitosan-lactobionic acid and chitosan-mannose ranges from 1:1 to 10:1; preferably the mass ratio is 9:1.

7. The conjugate or nanoparticle according to any of the previous claims, wherein the mixture of the two or more modified chitosan is chitosan-lactobionic acid and chitosan-gluconic acid.

8. The conjugate or nanoparticle according to any of the previous claims, wherein the chitosan has a molecular weight ranging from 100 to 200 kDa; preferably 150 to 180 kDa; more preferably 160 to 170 kDa.

9. The conjugate or nanoparticle according to any of the previous claims, wherein the chitosan degree of deacetylation of at least 50%; preferably ranging from 60 to 95%; more preferably 65 to 94%.

10. The conjugate or nanoparticle according to any of the previous claims, wherein the degree of substitution of mannose, gluconic acid, or lactobionic acid ranges from 10 to 50% measured by nuclear magnetic resonance; preferably ranges from 12 to 30%; more preferably ranges from 15 to 28%.

11. The conjugate or nanoparticle according to any of the previous claims, wherein the substitution occurs via / V-linkage.

12. The conjugate or nanoparticle according to any of the previous claims, wherein the nanoparticle further comprises a crosslinker.

13. The conjugate or nanoparticle according to any of the previous claims, wherein the crosslinker is selected from: sodium tripolyphosphate, sodium sulfate, sodium phosphate, aluminum sulfate, or mixtures thereof.

14. The conjugate or nanoparticle according to any of the previous claims further comprising laminarin or human serum albumin.

15. The conjugate or nanoparticle according to any of the previous claims, wherein the conjugate or nanoparticle exhibits a particle size ranging from 200 nm to 8 pm.

16. The conjugate or nanoparticle according to the previous claim, wherein the nanoparticle exhibits a particle size ranging from 100 nm to 500 nm; preferably from 200 to 450 nm.

17. The conjugate or nanoparticle according to any of the previous claims, wherein the conjugate or nanoparticle exhibits a zeta potential of -10 to 60 mV.

18. The conjugate or nanoparticle according to the previous claim, wherein the nanoparticle exhibits a zeta potential ranging from -6 to 57 mV.

19. The conjugate or nanoparticle according to any of the previous claims, wherein the conjugate or nanoparticle exhibits a polydispersity index ranging from 0.1 to 0.35.

20. The conjugate or nanoparticle according to any of the previous claims, wherein the loading efficiency of the genetic material or immunogen is at least 24%.

21. The conjugate or nanoparticle according to any of the previous claims, wherein the genetic material is a plasmid DNA; preferably a plasmid DNA encoding a SARS-CoV-2 protein; more preferably a plasmid DNA encoding the SARS-CoV-2 protein.

22. The conjugate or nanoparticle according to the previous claim, wherein the mass ratio of modified chitosan to genetic material ranges from 4:1 to 10:1.

23. The conjugate or nanoparticle according to any of the previous claims, wherein the immunogen is selected from a list consisting of: toll-like receptor agonists, STING agonists, RIG-l-like receptor agonists, NOD-like receptor agonists, or mixtures thereof.

24. The conjugate or nanoparticle according to any of the previous claims, wherein the conjugate or nanoparticle is administered intranasally, intramuscularly, orally, or subcutaneously; preferably intranasally or subcutaneously.

25. The conjugate or nanoparticle described in any of the previous claims for use as a vaccine delivery system; preferably as a DNA vaccine.

26. The conjugate or nanoparticle described in any of the previous claims for use in gene therapy or immunization.

27. The conjugate or nanoparticle described in any of the previous claims for use in the treatment of infectious diseases; preferably respiratory diseases; more preferably COVID-19.

28. Method for producing the nanoparticle described in any of the previous claims 1-24 comprising the steps of: providing a solution comprising chitosan, modified chitosan, or a mixture of two or more modified chitosan; adding a crosslinker solution to induce coacervation or precipitation; obtaining said nanoparticle; optionally isolating said nanoparticle by centrifugation or filtration.

29. The method according to the previous claim further comprising the step of adding the immunogen with the crosslinker solution to encapsulate the immunogen.

30. The use of the conjugate or nanoparticle described in any of the previous claims 1-23 for the manufacture of a medicament for the treatment of infectious diseases; preferably respiratory diseases; more preferably COVID-19.

31. A method for treating or preventing infectious diseases in a subject, the method comprising administering the conjugate or nanoparticle of claim 1 to the subject.

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