Nanoparticles, vaccine compositions, procedures, uses, and methods of administration thereof

Abstract

The present disclosure is directed to adjuvant nanoparticles for vaccines comprising a molar TPP / PAH ratio of 0.01 / 0.6, a diameter from 80nm to 526nm, a diameter polydispersity from 0.04 to 0.25, and a surface Z potential from +70mV to -20mV. The charge and size of the nanoparticles will depend on the molar ratio, concentration, or quantity of TPP and PAH compounds, the initial pH of the procedure, and the medium in which the procedure is carried out. Additionally, vaccine compositions formulated with different immunogens are disclosed, in which the nanoparticles encapsulate the antigens, target them to immune sites of interest, and activate the immune system; and preparation methods thereof.

Description

[0001] NANOPARTICLES, VACCINE COMPOSITIONS, PROCEDURES, USES, AND METHODS OF ADMINISTRATION THEREOF

[0002] The present invention relates to adjuvant nanoparticles for vaccines, vaccine compositions, procedures, uses, and methods of administration. The nanoparticles may comprise a molar TPP / PAH ratio of 0.01 / 0.6, a diameter from 80nm to 526nm, a diameter polydispersity from 0.04 to 0.25, and a surface Z- potential from +70mV to -20mV. The charge and size of the nanoparticles will depend on the molar ratio, concentration, or quantity of TPP and PAH compounds, the initial pH of the procedure, and the medium in which the procedure is carried out. In a preferred embodiment, the nanoparticles comprise 5mM PHA, 3mM TPP, R=0.6, and an initial pH (pH0) of 8.5.

[0003] BACKGROUND

[0004] Both traditional systemic vaccines and the modem ones contain two essential components in their formulation: an immunogenic component or active ingredient and an adjuvant (Gause et al. ACS Nano, 11 , 1 , 54-68, 2017; Genito et al. Adv. NanoBiomed Res., 1 , 2000041 , 2021 ). The adjuvant is responsible for activating the immune system (typically through its action on innate immunity) and enhancing immunogen specific recognition to induce cellular and / or humoral effector mechanisms that confer protection (Nguyen et al. Npj Vaccines 6, 70, 2021 ). Therefore, the immunogen determines the specificity of the vaccine or immunotherapy. Mucosal vaccines, in contrast to systemic ones, require a more pronounced presence of a carrier to protect both the immunogen and the adjuvant. Mucosal degradation is common, requiring the protection of vaccine components to enable the administration of low doses and promote immune system activation (Xu et al. International Journal of Pharmaceutics, 609, 121180, 2021 ; Alu ef a / . eBioMedicine, 76, 103841 , 2022).

[0005] Advancements in nanotechnology have allowed for the optimization of vaccine platforms through the development of a new generation of vaccines that incorporate a carrier capable of encapsulating, stabilizing, and safeguarding vaccine components. There are certain antigen transport systems, such as lipid nanoparticles (vesicles and liposomes), polymeric nanoparticles, and micelles (Guerrini et al. Nature Nanotechnology, 17, 570-576, 2022). Often, the formation processes of such systems require multi-step synthesis methods and the use of organic solvents, which complicate the manufacturing process (Akbarzadeh et al. Nanoscale Res. Lett., 8, 102, 2013; Guimaraes et al. International Journal of Pharmaceutics, 601 , 120571 , 2021 ; Sercombe et al. Frontiers in Pharmacology, 6, 286, 2015). On the other hand, lipid nanoparticles show limited stability over time and require cold chain storage at ultra-freezer temperatures (Giddam et al. Nanomedicine, 7, 1877-1893, 2012; Jin et al. Int. J. Pharm. 572, 118731 , 2019).

[0006] Nanoparticles composed of polymers that can carry a positive charge, such as chitosan, have been explored in vaccine formulations. Electrostatically cross-linked chitosan nanoparticles have been studied as carriers for antigens and other bioactive agents (Zhao et al. ACS Biomaterials Science & Engineering 5, 4937-4950, 2015). The main property of chitosan nanoparticles is their tolerogenic adjuvant capacity, i.e., their immunomodulatory effect that has made them potential adjuvants for inducing immune tolerance instead of activation. However, chitosan has very poor solubility in water, requiring its dissolution in the presence of acetic acid as a solvent. Furthermore, chitosan- based nanoparticles show very low stability under physiological conditions (Mazancova et al. Carbohydr. Polym. 192, 104-110, 2021 ; Huang et al. J. Mater. Chem. B, 3, 5957-5970, 2015). On the other hand, they often display a highly dispersed size distribution with more heterogeneous distributions.

[0007] Other systems formed by the combination of two polymers (one of them cationic) through layer-by-layer ionic assembly have been informed (Jewell et al. ACS Nano, 9, 6465-6477, 2015; Jewell et al. ACS Appl. Mater. Interfaces, 8, 18722-18731 , 2016). For instance, in the article published in Biomed Microdevices on June 28, 2021 ; 23(3):32, multilayer gelatin nanoparticles are disclosed, where the antigen is tetanus toxoid.

[0008] Another system is based on gold nanorods that have been surface- modified with diallyldimethylammonium polychloride and polyethyleneimine (Xu et al. Nano Lett., 12, 4, 2003-2012, 2012).

[0009] BRIEF DESCRIPTION OF THE INVENTION

[0010] This invention provides adjuvant nanoparticles for vaccines, comprising a molar TPP / PAH ratio of 0.01 / 0.6, a diameter from 80nm to 526nm, a diameter polydispersity from 0.04 and 0.25, and a surface Z-potential from +70mV to - 20mV. As further detailed below, charge and size of the nanoparticles will depend on the molar ratio, concentration, or quantity of TPP and PAH compounds, the initial pH of the procedure, and the medium in which the procedure is carried out. In a preferred embodiment, the nanoparticles comprise 5mM PHA, 3mM TPP, R=0.6, and an initial pH (pH0) of 8.5. All preferred embodiments can be found in Tables 1 and 2 below. Nanoparticles may comprise a diameter from 80nm to 300nm.

[0011] A vaccine composition is provided, comprising nanoparticles in which the nanoparticles comprise a molar TPP / PAH ratio of 0.01 / 0.6, a diameter from 80nm to 526nm, a diameter polydispersity from 0.04 to 0.25, and a surface Z- potential from +70mV to -20mV, and at least one antigen. The antigen can be of any suitable nature for formulating the vaccine composition, such as viral and bacterial antigens for the treatment of infectious diseases, like RBD protein typical of the SARS-CoV-2 virus or the Btaf protein from the zoonotic bacterium Brucella suis. They can also be tumor or allergenic antigens, e.g., milk proteins, for the treatment of non-infectious diseases. The antigen may be in free form in the liquid or gel medium of the vaccine composition with the nanoparticles, acting as an adjuvant, or it could be encapsulated within the nanoparticle, acting as both an adjuvant and a carrier for the antigen in a vaccine composition. The composition can be in a spray, liquid, or gel form. In a preferred embodiment, the vaccine composition is a suspension.

[0012] A method is provided for preparing the nanoparticles described in the above paragraphs, the procedure involving the following steps: a) Preparing a PAH solution in an aqueous medium; b) Adjusting the pH; c) Preparing a TPP solution in an aqueous medium and adjusting the pH to the same pH value as in step b); d) Adding to a volume of step a) (VPAH) solution, a volume of water (VH2O) and subsequently a volume of the step c) (VTPP) solution, where the addition of the volume of the step c) solution is carried out under stirring.

[0013] The pH in steps b) and c) can range from 7 to 8.5. The pH of the suspension obtained in step d) ranges from 7.75 to 10.3. In a preferred embodiment, the final pH of the nanoparticle suspension is 10.3 times the pH0. After step d), 0 to 2mM of NaCI can be added. The molar TPP / PAH ratio is 0.01 / 0.6. As an example, without limitation, the procedure for calculating the volumes is shown: for a final volume Vf and a final concentration of PAH=CPAH, the value R will determine the volumes VPAH, VH2O, and VTPP. For instance, if V 1 0 ml, CPAH=5 mM, and R=0.6, the volumes to use would be: VPAH=1 .25 ml; VH2O=7.5 ml, and VTPP=1 .25 ml. Hence, the volumes are calculated as follows:

[0014] VpAH=(Vf*CpAH) / 40 mM

[0015] VTpp=(Vf*CpAH*R) / 24 mM

[0016] VH2o=Vf- VPAH - VTPP

[0017] A method for preparing a vaccine composition is provided, comprising the steps of: a) Preparing a PAH solution in an aqueous medium; b) Adjusting the pH; c) Preparing a TPP solution in an aqueous medium and adjusting the pH to the same pH value as in step b); d) Preparing an antigen solution and adjusting the pH to the same pH value as in step b); e) Adding a volume of water (VH2O) to a volume of step a) (VPAH) solution, then adding a volume of step d) (Vprot) solution, and finally adding a volume of step c) (VTPP) solution, where the addition of the volume of step c) solution is carried out under stirring.

[0018] The pH in steps b), c), and d) ranges from 7 to 8.5. The suspension obtained in step e) has a pH from 7.75 to 10.3. After step e), 0 to 2mM of NaCI can be added. The molar TPP / PAH ratio is 0.01 / 0.6. In a preferred embodiment, the protein antigen is RBD. In a embodiment, the final concentration of PAH CPAH, R, the final volume Vf, and the final protein concentration Cprot are known, thus the volumes can be calculated using the following ratios:

[0019] VPAH— (Vf*CpAH) / 40 mM

[0020] VTpp=(Vf*CpAH*R) / 24 m M

[0021] VProt=(Vf*CProt) / CProt, i

[0022] VH2O= Vf - VPAH - VTPP - VProt Nanoparticles can be used as vaccine adjuvants or as vaccines when they comprise an encapsulated antigen.

[0023] A method of immunization is provided, which comprises administering an amount of the vaccine composition to a mammal, which can be a mouse, a rat, a rabbit or a human. The administration of the vaccine composition can be carried out by various routes, for example, via mucosal, systemic, transdermal, parenteral, intravenous, subcutaneous, intradermal, intratumoral, or other routes.

[0024] DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 shows: a) The chemical structure of the constituents of the NPADs nanoparticles (PAH and TPP) of the invention and a scheme of NPADs synthesis by direct mixing of the solutions, b) Formation of NPADs at the molecular level, ionic cross-linking of PAH chains by TPP ions, and a photograph of a plastic cuvette containing 2 ml of NPADs in colloidal dispersion (a black background was used to create contrast).

[0026] Figure 2 shows: a) TEM micrograph of NPADs prepared using, for example, CPAH=5 mM, R=0.6, and pH°=8.5. b) Particle hydrodynamic diameter distribution obtained by DLS for a NPAD sample prepared with the same parameters.

[0027] Figure 3 shows: the variation in size (left axis) and polydispersity index PDI (right axis) for NPAD samples prepared at different initial pH (pH0), using CPAH=5 mM and R=0.6. Solid circles represent hydrodynamic diameter values measured by DLS, and open triangles represent PDI values.

[0028] Figure 4 shows: a) The final pH of NPAD solutions prepared at different initial pH (pH0), b) Difference between the final pH and pH0of NPAD solutions prepared at different initial pH (pH0). All samples were prepared using CPAH=5 mM and R=0.6.

[0029] Figure 5 shows: The hydrodynamic diameter (left axis) and PDI (right axis) graphs as a function of R=[TPP] / [PAH] for NPAD samples prepared at pH°=8.5 and PAH concentrations of 5 mM (a), 10 mM (b), 15 mM (c), and 20 mM (d). The gray-shaded areas correspond to NPAD dispersions that tend to precipitate (unstable). The data points on the graphs represent the average of measurements taken over 3 to 4 days after the samples were prepared. The error bars represent the standard deviation of the successive measurements.

[0030] Figure 6 shows: The Z-potential graphs as a function of R=[TPP] / [PAH] for NPAD samples with a PAH concentration of CPAH=1 0 mM in a 50 mM HEPES buffer at pH=7.4 (a) and a 50 mM Tris buffer at pH=9 (b). The potentials become zero for R=0.27 and R=0.20, respectively. The data points on the graphs represent the average of 3 measurements. The error bars correspond to the standard deviation of the measurements.

[0031] Figure 7 shows: The percentage of PAH* in the form of NPAD (%PAH*NPAD) as a function of R=[TPP] / [PAH] for samples with C AH=10 mM at pH°=8.5 without buffer (a) and at pH=7.4 in a 50 mM HEPES buffer (b).

[0032] Figure 8 shows: The hydrodynamic diameter (left axis) and PDI (right axis) measured by DLS for NPAD samples prepared in the presence of NaCI at different concentrations. CPAH=5 mM, R=0.6, pH°=8.5. The data points on the graphs represent the average of measurements taken over 3 to 4 days after the samples were prepared. The error bars correspond to the standard deviation of the successive measurements.

[0033] Figure 9 shows: a) A tracking graph of the hydrodynamic diameter and polydispersity index (PDI) measured by DLS for a sample of NPADs with CPAH=5 mM, R=0.6, and pH°=8.5 during the first 40 days after preparation, b) A tracking graph of the hydrodynamic diameter of the same sample after 9 months of preparation, c) A tracking graph of the hydrodynamic diameter of the same sample after the first hour of preparation and the values of the initial and final Z-potential. In all cases, storage and measurements were conducted at room temperature.

[0034] Figure 10 shows: a) A graph of the average hydrodynamic size measured by DLS for a sample of NPADs with CPAH=5 mM, R=0.6, and pH°=8.5, prepared by adding the TPP solution in various ways (all at once, in 2 steps, in 5 steps, in 10 steps, and dropwise from a burette), b) A photograph of the solutions after preparation.

[0035] Figure 11 shows: a) Hydrodynamic diameter (a), Polydispersity index (b), and size histogram (c) of 10 samples of NPADs with CPAH=5 mM, R=0.6, and pH°=8.5 prepared using the previously described protocol. All samples were measured by DLS after 6 hours of stabilization. Figure 12 shows: a) Graph of the hydrodynamic diameter of NPAD samples dispersed in RPMI cell culture medium (0.3 ml of NPADs + 1 ml of RPMI) at 25 °C and 37 °C at different times, b) Size distribution of nanocomplexes of NPADs dispersed in RPMI at 25°C (blue line) and 37°C (red line) after 24 hours of synthesis, c) Hydrodynamic diameter (left axis) and PDI (right axis) of a sample of NPADs dispersed in RPMI cell culture medium (0.3 ml of NPADs + 1 ml of RPMI) at 37 °C as a function of time. The initial time on the graph (t=0) corresponds to 24 hours after dispersing the NPADs in RPMI.

[0036] Figure 13 shows the graphs of protein adsorption efficiencies on NPADs based on the type of protein, a) Adsorption efficiencies for BSA, OVA, RBD, Lysozyme, and Cytochrome c. b) Adsorption efficiencies for the same proteins as in (a), arranged according to their isoelectric point.

[0037] Figure 14 shows the results of in vitro and in vivo cellular toxicity and activation. A mouse macrophage cellular line (J774) exposed to various MPAD amounts was assessed, a) IL-ip levels in the cell culture supernatant determined by ELISA at different concentrations of NPAD (positive control: LPS+Alum, and negative control: BASAL culture medium), b) LDH enzymatic activity in the cell culture supernatant as a function of the amount of NPAD and the time of the enzymatic reaction (positive control of cell activation: LPS+Alum, and control for cell lysis with lysis buffer), and c) body weight monitoring of mice immunized with NPADs.

[0038] Figure 15 shows the results of the NPADs interaction with various cell lines analyzed by flow cytometry to assess and quantify cellular activation by means of the expression of class II MHO and CD11b molecules on the cell surface, a) Morphological change in the human monocytic cell line THP1 exposed to various amounts of NPADs in culture, b) Histogram showing the MHClTCDU b* cell populations of the murine macrophage cell line RAW when incubated with medium (black histogram), or 6 hours of stimulation with LPS and NPADs-FITC (dark gray), or with NPADs only (light gray).

[0039] Figure 16 shows the internalization of NPADs-FITC by phagocytic cells analyzed by confocal microscopy, a) Images of different phagocytic cells (macrophages J774, RAW, and JAWS II dendritic cells) stimulated for 4 hours with NPADs-FITC, and b) Colocalization (white arrows) of NPADs-FITC in late endosomes with LAMP1 after 4 hours of stimulation in the J774 cell line. Figure 17 shows the mechanisms of NPADs-FITC internalization a) by phagocytosis, using the inhibitor of this process, cytochalasin D (30 minutes before stimulation with NPADs) b) By endocytosis, 4 hours of stimulation at 4°C (inhibition of endocytosis) or 37°C. The black histogram corresponds to the basal control, light gray to NPADs-FITC at 37°C, and dark gray to NPADs-FITC at 4°C or with the inhibitor cytochalasin D.

[0040] Figure 18 shows the activation of murine bone marrow-derived dendritic cells, a) Expression of CD86 on the cell surface of the CD11c+CD11 b+cell population, a positive control was used with LPS followed by aluminum hydroxide (black histogram corresponds to the basal condition, light gray to LPS+ALUM, and dark gray to NPADs; b) IL-1 p quantification by ELISA in the culture supernatant after 24 hours of stimulation (ANOVA **P<0.01 ).

[0041] Figure 19 shows cellular activation by quantifying cytokines in the culture supernatant of antigen-presenting cells using the ELISA method after 24 hours of stimulation with NPADs. a) mlL-113 and mlL-6 quantification in the culture supernatant of mouse bone marrow-derived macrophages (BMDM) using LPS+DNA as a positive control, b) hlL-113 and hlL-8 quantification in the culture supernatant of the human monocytic cell line THP1 using LPS+ATP as a positive control, c) m IL-1 [3 and m IL-18 quantification in the culture supernatant of mouse bone marrow-derived dendritic cells (BMDC) using LPS+ALUM as a positive control (ANOVA *P<0.05; **P<0.01 ; ***P<0.001).

[0042] Figure 20 shows the internalization of NPAD-FITC into non-phagocytic cells. Human colonic epithelial cells HT29 were used for this. EpCAM was used as a specific lineage marker, and different conditions (basal with culture medium and activation with TNF) were applied, a) Analysis of NPAD-FITC fluorescence in epithelial cells by confocal microscopy (Z-scan). b) hlL-8 quantification in the culture supernatant by ELISA (ANOVA ***P<0.001 ).

[0043] Figure 21 displays the analysis of inflammasome activation in THP1 - ASC-GFP reporter cells using microscopy and flow cytometry. ASC formation is observed as concentrated fluorescence spots or cytosolic specks, a) Scheme of the work protocol, b) Fluorescence microscopy with white arrows indicating specks, representative images are shown, c) Quantification of specks relative to the total cell count per field, d) hlL-1 [3 quantification by ELISA in the culture supernatant, e) Gate strategy for selecting ASC+cells and quantification of the percentage of ASC+cells. (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 ; t-Student, #p<0.05; ##p<0.01 ).

[0044] Figure 22 shows the analysis of inflammasome pathway functionality by measuring the levels of IL-1 p and IL-18 secreted into the medium and the use of inhibitors of the cytosolic pathway, a) Mouse bone marrow-derived dendritic cells, b) Murine J774 cell line, c) Human THP1 monocyte cell line. Inhibitors of the inflammasome pathway used: the phagocytosis inhibitor Cytochalasin D, the caspase-1 inhibitor CA-74Me, the caspase-1 inhibitor Z-VAD-FmKQ, and the IL- 1 R antagonist Anakinra (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 ).

[0045] Figure 23 shows the results that allow confirmation of inflammasome activation when phagocytic cells are exposed to NPADs. The figure presents the analysis of inflammasome activation in cells derived from knockout mice for NLRP3, Caspase-1 , Caspase-1 / 11 , and Gasdermin D. mlL-1 [B A) and TNF-a B) was quantified by ELISA, and LDH enzymatic activity (enzymatic assay) was assessed in the culture supernatants of bone marrow-derived macrophage (BMDM) from KO and wild-type mice. D) Immunoblotting of cell lysates for active Caspase-1 (20 kDa) detection, with p-actin analyzed as a protein loading control. The various experimental conditions were carried out in triplicate, the mean is plotted with its standard deviation, and the graphs represent at least 2 independent experiments (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 ).

[0046] Figure 24 shows monitoring of the fluorescence of NPADs-FITC administered via intranasal (upper panel) and intragastric (lower panel) routes. Flow cytometry was used to assess fluorescence in different organs at different times (light grey histogram for control and black histogram for NPADs-FITC).

[0047] Figure 25 analyzes the presence of LPS in NPADs and their components. A reporter cell line that generates and secretes alkaline phosphatase upon LPS exposure was used. The measurement of alkaline phosphatase activity (SEAP) in the culture supernatant of HekTLR4 with different LPS concentrations and the main components of NPADs is shown. These results confirm that NPAD and PAH do not contain LPS.

[0048] Figure 26 shows the characterization of the mucosal adjuvant function of NPADs by assessing humoral and cellular immunity in mice immunized with NPADs-OVA via the intranasal route, a) Scheme of intranasal immunization in Balb / c mice. 10 pg of OVA in 20 pl of NPADs were administered once a week for three weeks, b) Measurement of delayed-type hypersensitivity or DTH response as the ratio between inflammation in the antigen-administered footpad and the PBS-administered footpad, c) Measurement of OVA-specific IgG levels in serum (1 / 100 dilution) and bronchoalveolar lavage (BAL) (1 / 2 dilution) by ELISA, d) Measurement of OVA-specific IgA levels in BAL and saliva samples by ELISA, e) IFN-y quantification in the culture supernatant of splenocytes after 48 hours of stimulation with OVA and NPADs (ANOVA *P<0.05).

[0049] Figure 27 shows the results of intragastric immunization with NPADs- OVA and the analysis of the specific immune response, a) Immunization scheme, b) Weekly monitoring of body weight, c) Detection of OVA-specific isotypes in the blood by ELISA. Analysis of dendritic cell activation in d) mesenteric lymph node (MLN) and e) Peyer's patches (PP) (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 , t-Student, #P<0.05).

[0050] Figure 28 shows characterization of the systemic adjuvant function of NAPDs. Aluminum hydroxide was used as a control adjuvant, a) Immunization scheme via intraperitoneal route with 2 doses of 100 pg of OVA in 200 pL of NPADs suspension (500 pg / mL OVA), b) Detection of specific IgG and isotypes in the serum of animals immunized with NPADs-OVA by ELISA, c) IFN-y quantification in the supernatant of splenocyte cultures after 48 hours of OVA stimulation, d) Flow cytometry and gating strategy to select populations of CD4+IFN-y+or CD8+IFN-y+T lymphocytes, e) Mean fluorescence intensity (MFI) (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 , t-Student, #P<0.05).

[0051] Figure 29 shows the results in wild-type animals (C57BL / 6) and animals lacking components of the inflammasome pathway (NLRPS'7', Caspase- and IL-1 R’ / _) systemically immunized with NPAD and OVA, and the analysis of humoral and cellular immunity, a) Immunization scheme used, b) Detection of OVA-specific antibodies in the blood and c) IFN-y level quantification in the culture supernatant of splenocytes after 72 hours of culture and stimulation with OVA or NPADs-OVA (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 , t-Student, #P<0.05; ##P<0.01 ).

[0052] Figure 30 shows the analysis of specific humoral immune response of nanoparticles, PAH polymer, and OVA in animals systemically immunized with NPAD and OVA. Measurements of the levels of specific IgG for OVA, NPADs, and PAH using ELISA plates sensitized with PAH, NPADs, and OVA (ANOVA ***P<0.001 ).

[0053] Figure 31 shows the results related to humoral immune response in mice immunized through systemic and / or mucosal combined routes with NPADs- OVA. a) Scheme of immunization protocol, b) Quantification of specific IgG and isotypes levels in the serum, and c) Quantification of IgG, isotypes, and IgA in BAL (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 , t-Student, #P<0.05).

[0054] Figure 32 shows the adjuvant function of NPADs in mice systemically immunized with NPAD and RBD. a) Immunization scheme involving two doses of 40 pg of RBD antigen in 500 pL of NPADs (NPADs-RBD); b) and c) Titers of RBD-specific IgG and lgG2a in serum and BAL assessed by ELISA, d) Quantification of IFN-y levels in the culture supernatant of splenocytes after 48 hours of stimulation with RBD (10 pg / mL). (ANOVA *P<0.05; **P<0.01 , t- Student, #P<0.05).

[0055] Figure 33 shows parameters related to immune memory to assess both humoral and cellular immunity, a) Immunization scheme involving two doses and sacrifice on day 91 after the first immunization with NPAD via systemic route, b) Titer of RBD-specific IgG in serum, c) and d) Levels of IgA in serum and BAL, respectively, e) Quantification of IFN-y levels in the supernatant of splenocyte cultures after 48 hours of stimulation with RBD (10 pg / mL). Mice immunized with Alum+RBD were used as a control group. (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 ).

[0056] Figure 34 shows evaluation of NPAD-RBD and Alum+RBD formulations via intramuscular administration in heterologous regimens following a primary regimen of two doses of the Pfizer / BioNTech bivalent vaccine administered via intramuscular route. Therefore, the present disclosure assessed the formulation containing NPAD as a booster dose, delivered via intramuscular route, to induce specific immunity against RBD. a) Immunization scheme involving two doses separated by 21 days, with a booster dose on day 49. b) and c) Titers of RBD-specific IgG in serum and BAL, respectively, d) Selection and quantification of CD4+IFN+and CD8+IFN+T lymphocyte populations from splenocyte cultures by flow cytometry, e) Quantification of IFN-y levels in the supernatant of splenocyte cultures after 48 hours of stimulation with RBD (10 pg / mL). (ANOVA *P<0.05; **P<0.01 ; ***P<0.001 , t-Student, #P<0.05;

[0057] ##P<0.01 ).

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] As used herein, the term nanoparticle or NPADs refers to the nanoparticles of the invention. NPADs-antigen means nanoparticles comprising encapsulated antigens, for example, NPADs-RBD refers to NPADs that encapsulate the RBD antigen, or NPADs-OVA refers to nanoparticles that encapsulate ovalbumin (OVA).

[0060] As used herein, the term RBD refers to the receptor-binding domain of a virus, particularly a coronavirus, more particularly SARS-CoV-2.

[0061] For the purposes of the present application, the terms "encapsulation" and "adsorption" have the same meaning and are interchangeable. It is understood that nanoparticles can encapsulate or adsorb antigens and function as a vaccine per se.

[0062] The present invention relates to multifunctional nanoparticles (NPADs) that can be used in vaccine formulations as carriers or adjuvants. NPADs exhibit excellent adjuvant activity and high versatility.

[0063] As shown hereinbelow, NPADs are capable of encapsulating antigens of various sizes, ranging without limitation, for example, from 66 kDa to 12 kDa. Encapsulation efficiency will depend on the charge (zeta potential) of the nanoparticles and the isoelectric point of the protein antigens. Therefore, to obtain highly efficient NPADs-immunogen, the charge of NPADs can be adjusted according to the isoelectric point of the desired protein to be encapsulated as an immunogen.

[0064] NPADs can be used as a vaccine composition per se because they are capable of encapsulating different antigens and thus serve both as antigen or immunogen carriers and adjuvants. A vaccine formulated with vehicle and immunogen NPADs does not require the addition of an adjuvant due to the dual role of NPADs.

[0065] Preparing NPADs is quite simple: two or more components are directly mixed under mild conditions (aqueous solution, room temperature, and atmospheric pressure). Their possibility of being obtained in an aqueous medium, using two structural components, in the absence of organic solvents and chemical synthesis, renders the manufacturing process cost-effective and environmentally friendly.

[0066] The polymers used in NPADs are also employed in other biomedical uses, and the cross-linking agent is a widely used food additive. NPAD nanoparticles exhibit long-term stability and can be easily stored in vials and at room temperature. They also display a suitable colloidal stability at physiological pH.

[0067] Nanoparticles exhibit great versatility and adaptability to modify their properties and characteristics to achieve a specific therapeutic goal. The size, charge, and type of encapsulated molecules can be easily and rapidly adjusted to induce the desired immune responses against a specific antigen.

[0068] The adjuvant capacity of nanoparticles distinguishes them from other commonly used carrier systems. This is particularly so because of their ability to activate the inflammasome pathway through caspase-1 activation and the production and secretion of IL-1 p and IL-18. This imparts the aforementioned adjuvant properties, as they activate phagocytic cells of the innate immune system to generate an inflammatory process and Th1 -dependent mechanisms of adaptive immunity. Moreover, it is worth mentioning that NPADs are exclusively internalized by phagocytic cells, which are antigen-presenting cells. The fact that dendritic cells incorporate them and subsequently become activated, determines that they have the capacity to generate the cells necessary to induce the activation of naive T lymphocytes. This determines that NPADs not only activate cells of the innate immune response but also promote the activation of T lymphocytes (CD4+and CD8+T lymphocytes). These cells play a central role in the induction of specific humoral and cellular immunity.

[0069] Nanoparticles can be administered through various routes, such as intranasal, sublingual, oral, and others. Therefore, nanoparticles can not only function as adjuvants in systemic vaccines, but they can also be used as mucosal adjuvants due to their role as carriers that protect antigens in mucosal administration routes.

[0070] The nanoparticles can be found as a colloidal solution based on ionically cross-linked cationic polymers and biopolymers. The nanoparticles exhibit a strong adjuvant capability because they stimulate the immune system enhancing the action and durability of administered antigens. Besides having adjuvant qualities perse, they can encapsulate a wide range of antigenic and / or therapeutic compounds. Therefore, they can be used with a dual function: 1 ) antigen carriers and 2) immune system stimulants. Their preparation is carried out through a straightforward, reproducible, scalable, and environmentally friendly process (the process only uses water as a solvent and is carried out at room temperature). The nanoparticles, as thus prepared, exhibit a controllable size, typically in the range of hundreds of nanometers, and a remarkable colloidal stability.

[0071] It is important to highlight that the nanoparticles did not demonstrate cellular toxicity in both in vitro and in vivo assays. Interestingly, when evaluated as nano-adjuvants in mice, these nanoparticles significantly enhanced the immunogenicity of systemic and mucosal vaccine formulations, leading to an enhanced specific immune response. The observed adjuvant capacity was higher than that of aluminum hydroxide, an approved adjuvant agent for human use and widely used in numerous vaccines. These results suggest that the nanoparticles hold substantial potential for application as adjuvants. Furthermore, their stability in mucosal environments is particularly important as they serve as carriers to protect the antigen and target it to the desired immune sites, where they have been detected within dendritic cells. In other words, the NPADs can function as mucosal adjuvants.

[0072] Moreover, the NPAD nanoparticles can be utilized as nano-transporters, or nanocarriers, as adjuvants for the development of vaccines and immunotherapies, in both human and veterinary medicine.

[0073] Preparation, chemical composition, and size of NPAD nanoparticles:

[0074] The constituents of the NPADs are a polyelectrolyte with positive charge (PAH) and the multivalent ion tripolyphosphate (TPP), as shown in Figure 1a. During the preparation of the NPADs, TPP ions act as ionic crosslinkers for PAH chains (Figure 1 b). In other words, several PAH chains bind in a non- covalently fashion, bridged through TPP ions. For this phenomenon to occur, both PAH and TPP must be adequately charged. As both of these are species involved in acid / base equilibria, the pH is a critical factor that determines whether NPADs will form or not. Since PAH is a linear chain of primary amines with a pKai / 2 (the pH value at which half of the amino groups are protonated) from 8 to 9, when the pH is sufficiently alkaline, the NPADs do not form (the PAH does not carry enough electric charge to be ionically crosslinked by TPP ions). In addition, TPP ions lose charge as the pH becomes more acidic (pKa1 =1 ; pKa2=2.2; pKa3=2.3; pKa4=5.7: pKa5=8.5), determining a lower pH limit at which NPADs can be prepared. Using turbidimetry measurements as a function of pH it has been established that the pH range in which NPADs can be formed ranges from pH=1 to pH=11 .

[0075] NPADs have particle sizes in the order of a few hundred nanometers. In particular, the most stable nanoparticles have sizes that range from 150 to 200 nm. While it is possible to modulate particle size (see below), the range in which the size can be modulated is restricted to 80 nm to 300 nm. Although it is feasible to prepare NPADs with sizes larger than 300 nm, the polydispersity of these solutions is usually high, and particles of these sizes tend to coagulate over time. Figure 2a shows a TEM micrograph where it can be observed that the particle morphology is spherical and the particle sizes within the same sample do not vary significantly. It is worth noting that TEM measurements were conducted using phosphotungstic acid as a staining agent to generate contrast, which is why some particles may appear darker than others due to the higher or lower staining agent adsorption. Figure 2b shows the particle size distribution obtained by DLS for a sample of NPADs with a final PAH concentration of 5 mM in monomer. A single peak centered at 186 nm is observed, indicating that there is no more than one size distribution in the sample. Furthermore, for this type of sample, polydispersity index (PDI) values from 0.04 to 0.25 were obtained, with typical values of 0.1 , ensuring the absence of NPAD aggregates.

[0076] Variation of particle size as a function of pH0at constant R and CPAH:

[0077] As mentioned in the previous paragraphs, the components that constitute the NPADs participate in acid / base equilibria, making the preparation pH (pH0) a parameter that affects the final result. The NPADs are stabilized in solution because they have a non-zero surface charge, generating electrostatic repulsive forces that prevent NPAD aggregation. Since the charge within NPADs must be neutral (electroneutrality principle), the surface charge and the amount of PAH and TPP units that make up each NPAD depend on the concentrations (CPAH and R) and the charge state of the constituents (degree of protonation). For example, if there is 5 mM of completely charged PAH (one positive charge per monomer), 5 mM of negative charges will be required to compensate that positive charge. If the TPP is slightly charged, for example, with 2 negative charges per TPP, it will be required a total of 2.5 mM of TPP to counteract the charge. As the charge state of the constituents depends strongly on pH, the relative amounts of each component within the NPADs will vary with pH, causing effects such as a variation in size.

[0078] Samples of NPADs were prepared at different pH0, with constant CPAH = 5 mM and R = 0.6. The samples were measured by DLS immediately after preparation. Figure 3 shows the results, where it can be observed that the particle diameters do not vary within a pH0range from 7.5 to 8.5 and become larger as pH0becomes more acidic. When pH0is below 7, the NPADs obtained are unstable over time.

[0079] Variation of solution pH as a function of pH0at constant R and CPAH:

[0080] During the process of ionic crosslinking, TPP ions bind to PAH chains, displacing the natural counterions of the polyelectrolyte (chloride ions). This phenomenon results in the cooperative ionization of uncharged species, leading to a shift in the pKa values of these species. In other words, the presence of TPP ions forces a portion of the uncharged amines to become protonated to form ion pairs with TPP ions. This synergistic protonation of the amino groups in PAH results in a change in the pH of the solution. In other words, the final pH of the NPADs solution is different from pH0(the pH at which the starting solutions of PAH and TPP were prepared). Figure 4 illustrates the pH variation of NPADs solutions for different pH0values with CPAH=5 mM and ?=0.6. It can be observed that as pH0increases, the final pH of the solution also increases. Additionally, it can be observed thar the pH increment occurs at a rate of 0.5 pH units for every unit increase in pH0, resulting in a variation of more than 1.6 pH units when starting with a pH of 8.5 (pHfinai=10.15). It is worth noting that pH changes also depend on the concentration of PAH and the molar ratio used.

[0081] Variation of particle size as a function of R at constant pH0:

[0082] As mentioned earlier, a change in the molar ratio, R=[TPP] / [PAH], leads to alterations in the composition of NPADs. This, in turn, results in changes in particle size and surface charges. To illustrate the impact of the molar ratio of the constituents on particle size, the variation of NPAD sizes was measured by varying R for different CPAH values ranging from 5 to 20 mM. In all cases, the pH0was set to 8.5. Figure 5 shows the hydrodynamic diameters and PDI obtained by DLS as a function of R for each case. At first glance, it can be observed that particle sizes fall within the range of 200 to 300 nm. Furthermore, it was observed that when CPAH is equal to or greater than 10 mM, the NPAD solutions tend to aggregate for R values greater than 0.1. This means that to maintain the NPADs stable across the entire range of R, the PAH concentration should be equal to or less than 10 mM. That is why the examples used PAH concentrations lower than 10 mM, such as 5 mM in PAH (see below and Figure 5).

[0083] Surface charge:

[0084] Although the NPADs are composed of cationic polymeric chains crosslinked with multivalent anions, depending on the molar ratio between PAH and TPP, the NPADs can exhibit a positive or negative surface charge. When PAH is in excess (small R), there is a deficiency of negative charges, and as a result, NPADs have a positive surface charge. By contrast, when PAH is in deficiency (high R), there is an excess of negative charges, and NPADs have a negative surface charge. Following this concept, there is a value of R at which positive charges are completely counterbalanced by negative charges, making the surface charge zero. In this case, since electrostatic repulsion between NPADs is minimized, systems tend to aggregate or precipitate (see Figures 5a and 5b in the proximity of R=0.2). The value of R at which the surface charge is zero depends on the charge state of the constituents. For example, if we assume that all PAH monomers are charged and that there are 5 negative charges for each TPP molecule (see structures in Figure 1 ), then the charges are cancelled when R=(CPAH / 5) / CPAH=1 / 5=0.2. Since PAH is not 100% charged over the entire pH range, and TPP is also not 100% charged over the entire pH range, the value of R at which the surface charge of NPADs is cancelled varies slightly around R=0.2, depending on the pH0at which they were prepared. Figure 6 shows the Z potential graphs (which scales with surface charge) as a function of R at CPAH=10 mM for different pH values. As mentioned earlier, during NPAD formation, the pH of the solution changes dramatically due to cooperative ionization of the species. To avoid pH changes and force the species to maintain a given charge state, NPADs were prepared in the presence of buffered solutions at a concentration of 50 mM (HEPES pH=7.4 and Tris pH=9). The first phenomenon observed in the graphs is that there are two regions: one where the potentials are positive and another where the potentials are negative, as described earlier. The value of R at which the Z potential becomes zero varies depending on the preparation pH. While at pH=7.4, this value is 0.27, at pH=9, the R value is 0.2. This indicates that a lower pH requires a greater amount of TPP to cancel the surface charges. In addition, when the pH is 9, the R at which the Z potential becomes zero is 0.2, indicating that all species are completely charged. Although PAH pKa1 / 2 is 8.5, it can be argued that PAH protonation is forced by the presence of charged TPP ions. Another interesting phenomenon that can be observed from the graphs is that when NPADs are positive, Z potentials are higher when the pH is more acidic, reaching values close to +70 mV. This characteristic makes NPADs suitable for use as building blocks in layer-by-layer self-assembled films. In addittion, the ability to modulate their surface charge allows for the modulation of their interaction with cells and biological organisms.

[0085] Variation of particle size as a function of component concentrations:

[0086] Table 1 shows the variation of the hydrodynamic diameter of particles based on the molar [TPP] / [PAH] ratio. All results correspond to samples of nanoparticles synthesized using a final PAH concentration of 5 mM in monomer and an initial pH (pH0) of 8.5. Measurements of the hydrodynamic diameter (d) were carried out both after one hour of synthesis (initial diameter) and after 72 hours.

[0087] Table 1

[0088]

[0089] Table 2 displays the variation in particle hydrodynamic diameter as a function of the initial pH (pH0). All results correspond to samples of nanoparticles synthesized using a final PAH concentration of 5 mM in monomer and a 3 mM TPP concentration. Hydrodynamic diameter (d) measurements were conducted 24 hours after the synthesis.

[0090] Table 2

[0091] PAH amount in NPADs:

[0092] Although a PAH concentration is set during the preparation of NPADs, not all the PAH added to the solution will be necessarily part of the NPADs. Under certain conditions, part of the PAH might be dissolved in the solution and not incorporated into the NPADs. To assess this possibility, NPADs were prepared using PAH labeled with fluorescein isothiocyanate (PAH*) at different R while keeping CPAH* constant at 10 mM. Each of the samples was centrifuged at 10,000 rpm for 20 minutes, and the concentration of PAH* in the supernatant was calculated using a PAH* calibration curve. Subsequently, the percentage of PAH* in NPADs was calculated as %PAH*NPAD = [(10 mM - CPAH*, supernatant ) / 10 mM]*100. Figure 7a shows the %PAH*NPAD values as a function of R for samples prepared at pH°=8.5 without buffer, and Figure 7b shows the values for samples prepared at pH=7.4 using 50 mM HEPES buffer. As observed in the graphs, there is an increasing linear relationship between %PAH*NPAD at low R until it reaches a maximum and constant value of about 95%, which remains constant. This means that if R is sufficiently high, virtually all the PAH will be contained within the NPADs. The critical value of R at which %PAH*NPAD is maximum (marked with a dashed line) depends on the pH and becomes smaller as the pH becomes more alkaline. When observing the graphs of Figures 6 and 7, it can be concluded that it is possible to obtain NPADs with a positive surface charge and a high %PAH*NPAD only in limited regions of R. In fact, for pH=7.4, only one point meets these conditions, and it is the point at R=0.2 (%PAH*NPAD=95% and a Z potential of +45 mV). For an alkaline pH, on the other hand, there is a region between R=0.1 and R=15 for which the NPADs have a positive Z potential and a high %PAH*NPAD. Furthermore, for R values at which the Z potentials are negative, %PAH*NPAD remains at maximum values. This result is important because the toxicity of PAH packaged in the form of NPADs is not significantly affected. In addition, it is estimated that the encapsulation efficiency of proteins is maximized when %PAH*NPAD is maximum. For these reasons, in some cases, it might be convenient to prepare NPAD solutions with R>0.2.

[0093] Range of CPAH concentrations in which the NPADs are stable:

[0094] Although the stability of NPADs depends on multiple factors, the concentration of PAH at which the NPADs are prepared is one of the most critical factors in maximizing their stability. When the concentration (CPAH) is too high, NPADs tend to fuse through a coalescence process, leading to coacervates. Conversely, when CPAH is maintained at low levels, NPADs remain dispersed in solution for long periods. While it is difficult to establish a critical aggregation concentration, in general, when CPAH is greater than 5 mM, the NPADs tend to exhibit aggregation phenomena. On the other hand, if the PAH concentration is less than 0.5 mM, the NPADs also tend to aggregate. Therefore, it can be argued that the optimal stability range for NPADs is from CPAH=0.5 mM to CPAH=5 mM (using pH°=8.5 and R=0.6). In particular, we have observed that for CPAH=5 mM, R=0.6, and pH°=8.5, the stability of the NPADs is maximum.

[0095] Range of ionic strength in which NPADs are stable:

[0096] Since the stability (no aggregation) of NPADs is linked to electrostatic repulsions between particles, it is necessary to minimize the ionic strength to maximize stability. The addition of monovalent ions to the medium (e.g., Cl' and Na+) shields the surface charges of NPADs, causing an increase in the coalescence process between pairs of NPADs. For this reason, the maximum stability of NPADs is achieved in the absence of added salt. To evaluate the range of NaCI concentrations in which NPADs remain stable without altering their size, a series of samples containing increasing amounts of NaCI was prepared. The addition of salt was included as a preliminary step before adding TPP, namely, the order of additions was: 1 ) PAH, 2) H2O, 3) NaCI, and 4) TPP. A fixed volume of NaCI of 133 pL was used, with variable NaCI concentrations ranging from 5 to 100 mM. For a final volume of 2 mL, the volumes used in each step were 250 pL of 40 mM PAH pH=8.5, 1367 pL of H2O, 133 pL of x mM NaCI (x=0, 5, 10, 15, 25, 50, 100), and 250 pL of 24 mM TPP pH=8.5. Figure 8 shows the hydrodynamic diameter and PDI for the samples with salt. The NaCI concentration is expressed as the final NaCI concentration in the solution. From the graph, it can be observed that for NaCI concentrations below 2 mM, particle sizes remain in the range of 100 to 300 nm. Then, when the salt concentration is increased further, particle sizes and PDI increase significantly, reaching values of about 600 nm and PDI=0.35. It is necessary to clarify that the points in Figure 8 correspond to an average of measurements made from days 1 to 4 after preparing the samples, therefore, these are not long-term measurements. As a general rule, the size and stability of NPADs are affected by the presence of NaCI, and it is recommended not to exceed levels higher than 2 mM of NaCI in order to maximize NPAD stability.

[0097] Stability of NPADs at different temperatures and media:

[0098] To assess the long-term stability of NPADs, the hydrodynamic particle diameter was measured at different time points in a sample of NPADs using CPAH=5 mM, R=0.6, and pH°=8 5 (Figure 9). The sample was stored in a glass vial at room temperature. Figure 9a shows that the particle size and polydispersity index remain relatively stable for 40 days. 24 hours after being prepared, the size increases to 180 nm and then remains at a value close to 200 nm for more than 9 months (Figure 9b). Figure 9c shows that the size increases from 100 to 120 nm during the first hour, and its Z potential is about - 30 mV. This result indicates that, compared to other similar systems, the NPADs are suitable for long-term storage and that they maintain their size virtually unalterable.

[0099] To study the effect of the TPP addition rate, we prepared five solutions, varying the rate of TPP addition. When TPP is added dropwise from a burette, the hydrodynamic diameter increases significantly, and the colloidal dispersion is highly unstable (Figure 10a), resulting in a very turbid mixture (Figure 10b). Similar results were obtained when adding the TPP solution in 5 or 10 steps. When TPP is added in 5 or 10 steps, a molar [TPP] / [NH2] ratio=0.24 is achieved in the second or fourth step, respectively. When TPP is added in 2 steps, the system behaves almost identically to when the TPP solution is added all at once. After the first step, a molar [TPP] / [NH2] ratio=0.3 is achieved, which is far enough from the critical point. Therefore, the most critical step of the process is the rate at which the TPP solution is added to the PAH solution. When [TPP] / [NH2]=0.2, the system tends to aggregate very quickly, as all the charges of PAH are compensated by TPP anions. Thus, in order to obtain a stable colloidal dispersion, the final molar ratio ([TPP] / [NH2]=0.6) must be reached quickly, avoiding the system being close to [TPP] / [NH2]=0.2 during the mixing of the components.

[0100] In order to assess reproducibility, the hydrodynamic diameter and polydispersity index of 10 independently prepared NPAD samples were measured (Figure 11 ). As can be observed, the formation of NPADs is highly reproducible and exhibits minimal fluctuations in sizes from batch to batch.

[0101] To investigate the stability of the NPADs in complex media and at different temperatures, the particle size of the NPADs was measured after dispersing them in RPMI culture medium. For this purpose, 0.3 mL of NPADs (CPAH=5 mM, R=0.6, and pH°=8.5) were added to 1 mL of RPMI medium to obtain 1.3 mL of NPADs dispersed in RPMI. Figure 12a shows the evolution of particle sizes at different temperatures and different times, with average values of around 270 nm. This value is somewhat higher than the equivalent in water (see Figure 9), indicating the possible surface adsorption of proteins and biomolecules present in the culture medium. Figure 12b shows the size distribution of NPADs dispersed in RPMI at 25°C and 37°C after 24 hours of synthesis. Both samples showed a single distribution of hydrodynamic diameters of nanocomplexes after 24 hours of synthesis. Figure 12c shows the evolution of particle size over time at 37°C after 24 hours of preparation. As can be observed, the size remains nearly constant over time and shows a polydispersity of less than 0.3.

[0102] Incorporation of Proteins into NPADs: Efficiency of protein adsorption or encapsulation in NPADs according to the type of protein:

[0103] Since NPADs are essentially drops of polymeric coacervates in colloidal suspension, they can act as carriers for various molecules, such as peptides, proteins, and drugs in various configurations. The ability of NPADs to encapsulate (or adsorb) different protein antigens was assayed. Proteins share structural similarities with polyelectrolytes (they are charged macromolecules), and the incorporation of proteins into NPADs is due to non-covalent polymer- protein interactions, similar to interactions in polymer-polymer or polymer- crosslinker systems. Therefore, it is necessary to know protein charge and maximize electrostatic attraction interactions, both with the polymer and the crosslinking agents of NPADs. Five different proteins were used: BSA (66 kDa, pl=4.75), OVA (44.3 kDa, pl=4.5), RBD (26 kDa, pl=8.8), Lysozyme (14.3 kDa, pl=11.0), and Cytochrome C oxidase (12.4 kDa, pl=10.25). The preparation parameters for NPADs were: CPAH=5 mM, R=0.6, pH°=8.5. The protocols for preparing NPADs containing proteins and quantifying the protein encapsulation efficiency in NPADs were described in the examples. The graph in Figure 13a shows the encapsulation percentages for the different proteins under study, which range from about 10% to 30%. When the proteins are sorted by their isoelectric point, as shown in Figure 13b, it is observed that the encapsulation percentages increase as the isoelectric point of the proteins decreases. It is also important to mention that sizes and stability of NPADs are not altered or modified by the presence of proteins.

[0104] Toxicity of NPADs:

[0105] Toxicity was measured by the activity of the enzyme lactate dehydrogenase (LDH) in the J774 cell line. For this purpose, different amounts of NPADs (CPAH=5 mM, R=0.6, pH°=8.5) were tested to create a dose-response curve. LDH is an enzyme found in the cytoplasm and is present in all cells. When the plasma membrane is damaged, this enzyme is rapidly released into the culture supernatant. Additionally, LDH is closely related to inflammasome activity. When the stimulus is too strong or long-lasting, the inflammasome is activated, leading to the release of IL-1 p, IL-18, and LDH into the extracellular medium due to the rupture of the cytoplasmic membrane, resulting in cell death. The detection of extracellular LDH activity can be measured by a simple and precise colorimetric method. In Figure 14a, it can be observed that the cells respond to different concentrations by increasing the release of IL-1 p and IL-18. On the other hand, the release of LDH (Figure 14b) does not appear to have a clear relationship with the increase in NPADs concentrations when compared to the controls.

[0106] The weight of the animals injected with different NPADs formulations was monitored, as shown in Figure 14c, and none of the animals experienced significant changes in their body weight. Therefore, the NPADs are non-toxic.

[0107] Cellular Internalization:

[0108] The behavior of cultured cells exposed to NPADs (CPAH=5 mM, R=0.6, pH°=8.5) was analyzed, including morphological changes and intracellular nanoparticle distribution. A morphological change was observed in the THP-1 cell line, which was independent of the number of nanoparticles in suspension in the culture, as compared to the basal control (Figure 15a). It was also observed that the macrophage cells RAW MHCII+CD11 b+ were fluorescent, implying an interaction with the NPADs (Figure 15b).

[0109] To distinguish whether the particle is bound to the cell surface or located inside it, cell cultures of various antigen-presenting cells such as macrophages (J774 and RAW) and dendritic cells (JAWS II) were performed, and the location of fluorescent particles was analyzed using confocal microscopy. Figure 16a shows the distribution of NPADs-FITC in the cytosol, with regions of more concentrated fluorescence that could be cellular organelles. In Figure 16b, colocalization with LAMP1 marker (a protein specific to late endosomes and lysosomes) indicates that NPADs-FITC are located within late endosomes.

[0110] Figure 17a shows, through both microscopy and flow cytometry, that the J774 macrophage cell line treated with phagocytosis inhibitor exhibits a decrease in fluorescence. This is because the cells cannot capture NPADs- FITC. In Figure 17b, when endocytosis of NPADs-FITC is inhibited at 4°C in bone marrow-derived macrophages, flow cytometry reveals no variation in fluorescence compared to stimulation at 37°C. However, when these same cells were examined under fluorescence microscopy, it was detected that the particles are attached to the cell surface and not inside cells. By contrast, in cells stimulated at 37°C, NPADs were observed inside the cells. These results indicate that NPADs can enter cells through both phagocytosis and endocytosis, and when these processes are inhibited, NPADs remain on the cell surface.

[0111] Cellular Activation:

[0112] Cellular activation parameters were studied by flow cytometry in bone marrow-derived dendritic cells (BMDCs) exposed to NPADs (CPAH=5 mM, R=0.6, pH°=8.5). Dendritic cells were characterized using different surface markers such as CD11c and CD11b, and with CD86 as a marker for cellular activation. To do this, BMDCs were stimulated with 0.5 mM of NPADs, and the expression of CD86 on the cell surface of the CD11c+CD11 b+cell population was evaluated. It was observed that the entry of NPADs into the cells not only led to morphological changes and localization in late endosomes but also had the capability to induce cellular activation, as evidenced by an increase in CD86 expression on the cell membrane (Figure 16a). Additionally, the production and secretion of the proinflammatory cytokine IL-1 p were measured in BMDCs in the culture supernatant using ELISA, and an increase was observed when the cells were exposed to NPADs. A positive control using LPS with aluminum hydroxide (Alum) was also included (Figure 18b).

[0113] IL-1 p, as well as IL-18, are proinflammatory cytokines closely associated with the activation of the multiprotein complex inflammasome. NPADs can create an inflammatory environment with the secretion of both cytokines. This cellular activation was observed in bone marrow-derived dendritic cells (BMDCs), bone marrow-derived macrophages (BMDMs), and the human monocytic cell line THP1. Exposure of these cells to NPADs induced increases in IL-i p and IL-18 in the culture supernatant compared to control cells with culture medium only (Figure 19). By contrast, NPADs-FITC were not internalized by non-phagocytic epithelial cells, as evidenced using the HT29 cell line, and there was no observed release of IL-8 in the culture supernatant (Figure 20). The secretion of the pro-inflammatory cytokine is a result of cell exposure to TNF, indicating that, even though they activate the epithelial cells, NPADs are not internalized.

[0114] To study inflammasome activation, a reporter cell line for the adaptor protein ASC (apoptosis-associated speck-like protein) was used. It was observed that resting cells were not fluorescent, but when the inflammasome was activated (LPS priming), fluorescence was observed throughout the cytoplasm (Figure 21 ). Using this cell line, well-defined ASC specks formation within the cells was observed by microscopy (Figure 21 b) when stimulated with NPADs, indicating inflammasome activation. Furthermore, the specks were counted under the microscope in 3 fields per condition, and an increase in IL-1 p in the culture supernatant was observed under the same conditions (Figure 21 d). To confirm these findings, fluorescence was quantified by flow cytometry (Figure 21 e).

[0115] To determine the inflammasome activation pathway, different inhibitors were used, including Cytochalasin D (inhibitor of phagocytosis), CA-74Me (an inhibitor of the enzyme Cathepsin B), Z-VAD-FMK (caspase inhibitor), and Anakinra (a monoclonal antibody blocking the IL-1 receptor, IL-1 R). The activity of the pathway was evaluated by measuring the release of IL-1 p and IL-18 in the culture supernatant using the capture ELISA technique in various cell lines. As shown in Figure 22, the release of IL-1 [3 and IL-18 considerably decreased when BMDCs (Figure 22a), J774 macrophage cell line (Figure 22b), and THP1 monocytes (Figure 22c) were incubated with NPADs and the different inhibitors, compared to activation with NPADs alone.

[0116] Figure 23 shows the results obtained in cultures of BMDMs from NLRP3' inflammasome KO mice, Caspase1 / 1 T / _and Caspase1 TAenzymes KO mice, and GSDMDApore forming pore KO mice. After priming with LPS and stimulation with 0.5mM of NPADs, the quantification of IL-1 f3 showed a significant decrease, directly dependent on NLRP3 / _and Caspase1 / 1 T / _(Figure 23a). IL-1 p secretion does not depend on Gasdermin D expression, as GSDMD' mice exhibited cytokine secretion. Figure 23b displays the levels of TNFa exposed to LPS, NPADs, and LPS+NPADs. As observed in KO and wild-type animals, TNFa levels are significantly higher under the culture conditions with LPS or LPS+NPADs. Cellular integrity was simultaneously evaluated by quantifying LDH release as a signal of cell death in the culture supernatant, where all experimental conditions resulted in reduced and comparable values between controls without stimulation and with LPS+NPADs (Figure 23c). In Figure 23d, we can observe that in the lysate of NLRP3-deficient BMDMs culture, there was no decrease in the band corresponding to active Caspase-1 (20 kDa). Therefore, these results confirm cellular activation through the inflammasome pathway after the phagocytosis of NPADs, involving cathepsins, caspase-1 , and the IL-1 receptor. Interestingly, Gasdermin D is not involved in the mechanism of mature cytokine secretion through pore formation in the plasma membrane. This indicates that the secretion of IL-i p and IL-18 is independent of this secretion mechanism, which is only induced through the inflammasome pathway.

[0117] Adjuvant Capacity:

[0118] 20 pL of NPADs-FITC (CPAH=5 mM, R=0.6, pH°=8.5) were administered intranasally to male Balb / c mice, and 200 pL were administered by intragastric route in order to detect fluorescence using flow cytometry at various time points (30, 60, 90, and 120 minutes and 4, 6 hours, depending on the administration route) and in different tissues. As shown in the upper panel of Figure 24, at short times of 30 and 60 minutes, NPADs-FITC were found in the nasal mucosa and cervical lymph nodes, and at longer times of 90 and 120 minutes, the particles were located in the mediastinal lymph nodes and lung epithelium. Furthermore, the transit of NPADs-FITC through the gastrointestinal tract from the intestinal epithelium to the mesenteric lymph nodes was monitored (Figure 24, lower panel). These results demonstrate that the particles remain stable during its passage through the nasal and intestinal mucosa, allowing access to lymphoid organs of interest to induce an immune response. It is important to highlight the significance of this result for using these particles as a vehicle in a mucosal vaccine. Protecting the immunogen in mucosal tissues is critical, as degradation and dilution (respiratory and digestive tracts) occur in these tissues.

[0119] LPS Content Analysis:

[0120] As a first test, an assay was conducted using HEKhTLR4 cells (human kidney cells) which act as reporters for the Toll-like receptor 4, specific for LPS. When these cells are stimulated with LPS, they synthesize and secrete alkaline phosphatase into the extracellular medium. Measuring its fluid-phase activity allows for the quantification of LPS presence. As shown in Figure 25, the NPADs components (pharmacological-grade water, PAH, and TPP), the proteins encapsulated within them (RBD and OVA), and the buffer in which the proteins are stable generated low enzymatic activity compared to the basal control. Solutions with different concentrations of LPS were used as positive controls, and culture medium served as a negative control. This result is important because it helps to understand the need to add LPS for NPADs to activate the inflammasome pathway (the canonical cellular activation pathway).

[0121] Nanoparticles as a Mucosal Adjuvant (Intranasal Administration) and Antigen Carrier in Vaccine Formulation (NPADs-OVA):

[0122] After confirming that NPADs are stable and properly distributed when administered intranasally, ovalbumin was encapsulated to administer it to mice according to the scheme shown in Figure 26a. Mice were inoculated once a week for three weeks with NPADs-OVA (each dose containing 10 pg of OVA in 20 pL of NPADs, CPAH=5 mM, R=0.6, pH°=8.5). One week after the last dose, animals were sacrificed, and both the cellular and humoral immune responses were evaluated. The delayed-type hypersensitivity (DTH) response was measured, and a stronger response was observed in mice immunized with NPADs-OVA compared to those receiving OVA alone (Figure 26b). In addition, specific IgG humoral responses, both in serum and bronchoalveolar lavage, were significantly higher in mice immunized with NPADs-OVA compared to mice immunized with OVA alone (Figure 26c). An increasing trend for specific IgA was observed in mice immunized with NPADs-OVA, especially in saliva samples (Figure 26d). Regarding the cellular response, the production of IFN-y in splenocyte culture supernatants 48 hours after stimulation with OVA or NPADs-OVA was measured. As shown in the graph, mice that generated the highest amount of IFN-y in cells stimulated with OVA in vitro are those that were inoculated with NPADs-OVA (Figure 26e). In conclusion, in this model, NPADs were able to protect the antigen during its transit through the mucosa and generated a specific antigen-specific cellular and humoral response.

[0123] Nanoparticles as a Mucosal Adjuvant (Intragastric Administration) and Antigen Carrier in Vaccine Formulation (NPADs-OVA):

[0124] To demonstrate the mucosal adjuvant properties of NPADs in intragastric administration, NPADs-OVA or OVA as a control (100 pg of OVA in 200 pL of solution) were administered once a week for three weeks in two groups of mice. The animals' body weights were monitored weekly (Figure 27a and b). Although there was an increase in OVA-specific IgG and lgG2a, this increase did not reach statistical significance when comparing mice immunized with NPADs- OVA to those immunized with OVA alone (Figure 27c). However, flow cytometry analysis showed that NPADs induced a significant increase in CD11c+CD11 b+MHCII+CD80+ and CD11 c+CD11 b+MHCII+CD86+ dendritic cells in mesenteric lymph nodes (MLN) and Peyer's patches (PP) (Figure 27d and e). Taken together, these results demonstrate the behavior of NPADs as a mucosal adjuvant and their ability to induce local and systemic humoral and cellular immunity.

[0125] Nanoparticles as a Systemic Adjuvant in Vaccine Formulation (NPADs-OVA):

[0126] The systemic adjuvant and antigen carrier capability of NPADs containing encapsulated protein antigen OVA, which would constitute a vaccine composition, was studied. Balb / c mice were intraperitoneally immunized with NPADs-OVA (OVA at a final concentration of 100 g in 200 pl of NPADs suspension, CPAH=5 mM, R=0.6, pH°=8.5). As a control, a combination of aluminum hydroxide (alum) with OVA and OVA alone was administered. As shown in the schematic in Figure 28a, two doses were administered, 21 days apart, and the animals were sacrificed two weeks after the final inoculation. Mice immunized with NPADs-OVA generated a specific IgG humoral response. Regarding the antibody isotypes, NPADs-OVA induced specific lgG1 antibodies, and only the administration of NPADs-OVA was able to generate specific lgG2a antibodies, characteristic of a Th1 response (Figure 28b). For the cellular response, it was determined that only mice immunized with NPADs- OVA produced high levels of IFN-y in the splenocyte culture supernatants (Figure 28c). Furthermore, through flow cytometry, it was determined that immunization with OVA-NPADs induced IFN-y-producing CD4+and CD8+lymphocytes (Figure 28d and e).

[0127] Hence, systemic administration of NPAD-OVA induces humoral and Th1 - dependent cellular immunity, leading to the production of specific IgG antibodies and the secretion of IFN-y by splenic T lymphocytes.

[0128] Systemic Immunization of Mice Deficient in Components of the Inflammasome Pathway with the NPAD-OVA Vaccine:

[0129] The induction and secretion of specific OVA IgG antibodies were significantly reduced in animals deficient in NLRP3 and caspase-1 compared to wild-type mice. At the same time, when analyzing the cellular immunity in these mice, it was observed that the secretion of IFN-y by in vitro expanded splenocytes with the NPAD-OVA candidate is suppressed in NLRP3 / _and Caspase- T / _mice. These results confirm the in vitro findings shown in Figure 23, allowing us to confirm that the NLRP3- and caspase-1 -dependent inflammasome pathway is involved in the induction of specific antibodies and the cellular activation with IFN-y secretion, necessary to promote a Th1- dependent specific immune response. Here, we observe that the response is independent of the functionality of the IL-1 receptor.

[0130] To investigate whether NPADs generate a specific response against themselves, antibodies against the particles and its components were measured using an indirect ELISA. ELISA plates were sensitized with OVA, NPADs, or PAH, and sera from NPADs-OVA immunized mice were used to reveal the presence of specific antibodies. We found that mice did not produce antibodies against any of these nanoparticle components but did produce antibodies against OVA (Figure 30).

[0131] Immunization Combining the Systemic (Intraperitoneal) and Mucosal (Intranasal) Routes with the NPAD-OVA Vaccine Candidate:

[0132] A slightly modified intranasal immunization protocol (2 doses separated by 15 days) combining the systemic or intraperitoneal (IP) route with the intranasal (IN) route. As a control, animals were injected with 2 doses of NPADs-OVA IP. Two days before sacrifice, an intranasal immunization with OVA alone (boost) was administered (Figure 31a). The analysis of humoral immunity revealed significantly higher serum levels of OVA-specific IgG in mice receiving the combined heterologous (IP+IN) scheme compared to those receiving the homologous (IP+IP) scheme or compared to unimmunized animals. This increase was also observed in bronchoalveolar lavage, along with an increase in IgA in those who received the heterologous scheme (Figure 31 b).

[0133] Systemic Immunization Using the NPAD-RBD Vaccine Candidate:

[0134] Following the systemic immunization scheme of two doses separated by 21 days (Figure 32a), the immunogen RBD was encapsulated, and the specific response to this component of SARS-CoV-2 was evaluated. Alum-RBD was used for comparison to assess the ability of the NPAD-RBD vaccine candidate to activate the immune system compared to the Alum-RBD vaccine as a positive control. A humoral response was observed, with a significantly higher induction of RBD-specific IgG and lgG2a in animals that received NPAD-RBD compared to those who received Alum+RBD (Figure 32b). Additionally, an increase in the lgG2a isotype was found, both in serum and in bronchoalveolar lavage (BAL), in mice immunized with NPAD-RBD (Figure 32b and c). Regarding the cellular response, it was determined that only those mice receiving NPAD-RBD generated splenocytes capable of secreting IFN-y (Figure 32d). Therefore, immunization of mice with NPAD-RBD generated a specific Th1 -dependent immunity.

[0135] Immune Memory in Animals Systemically Immunized with the NPADs-RBD Vaccine Candidate:

[0136] In order to study immune memory, an immunization scheme of 2 doses with NPADs-RBD separated by 21 days was used, and the animals were sacrificed on day 91 , using Alum+RBD as a positive control (Figure 33a). A significant increase in specific IgG titers for RBD in serum was found in mice immunized with NPADs-RBD (Figure 33b) compared to animals immunized with Alum+RBD. Additionally, an increase in IgA was detected in serum and in BAL in those animals immunized with NPADs-RBD (Figure 33c and d). Regarding the cellular response, an increase in the production and release of IFN-y in the culture of splenocytes from animals immunized with NPADs-RBD was observed (Figure 33e).

[0137] Boost Immunization and Under a Heterologous Scheme Using the NPAD-RBD Vaccine Candidate:

[0138] NPAD-RBD, Alum+RBD, and Alum / CpG+RBD formulations were evaluated as boosters in a heterologous scheme by intramuscular route after a primary immunization of two doses with the bivalent Pfizer / BioNtech commercial vaccine (Figure 34a). It was observed that mice receiving NPADs, Alum, or Alum / CpG as boosters exhibited higher titers of specific IgG in serum compared to the PBS control (without booster doses) and values comparable to those receiving Pfizer / BioNtech as a booster (Figure 34b). Likewise, an increase in antigen-specific IgG was observed in BAL (Figure 34c). Regarding the cellular response, an increase in the percentage of CD4+IFN-y+and CD8+IFN-y+T lymphocytes and high levels of IFN-y in the culture supernatant of splenocytes from animals immunized with the vaccine variants NPAD-RBD, Alum-RBD, and Alum / CpG-RBD was observed by flow cytometry (Figure 34d and e). These results indicate that the use of NPAD-RBD as a booster generates specific immunity similar to that induced by the systemic and mucosal bivalent Pfizer / BioNtech vaccine, although lower than that promoted by Alum / CpG+RBD. In summary, these results allow to conclude that NPADs act as a vehicle encapsulating the antigen, protecting the antigen when administered intranasally or intragastrically (mucosal vaccine), and generate a Th1- dependent humoral and cellular immunity with the induction of IgG antibodies and IFN-y-producing CD4+and CD8+T lymphocytes. Furthermore, the NPAD- antigen generated a specific humoral response with the induction of IgG and lgG2a in serum and BAL, both via intranasal and intraperitoneal routes. This Th1 -dependent response is responsible for generating IFN-y-secreting lymphocytes, which was observed systemically in splenocytes upon in vitro restimulation with the respective antigens. In addition, intramuscular administration (systemic route) of NPAD-RBD also generated specific immunity, and its use as a booster dose (third dose) improved the immunity previously established with the initial systemic homologous scheme using the Pfizer / BioNtech bivalent vaccine. It is also worth noting that NPADs exclusively activate phagocytic cells, particularly dendritic cells, which are essential for activating naive T lymphocytes. This implies that they target the antigen (OVA or RBD) to the cell they activate, creating an antigen-presenting cell with the necessary tools to induce T cell activation. At the same time, our findings demonstrate that inflammasome activation promotes a Th1- and IFN-y- dependent immunity with the expansion of CD8+T lymphocytes, which are crucial in antiviral immunity. This suggests that NPADs induce antigen crosspresentation, which was observed in macrophages and dendritic cells.

[0139] In conclusion, the NPADs have demonstrated adjuvant properties with 2 immunogens (OVA and RBD) and in two types of administration: systemic (intraperitoneal and intramuscular) and mucosal (intranasal and intragastric), and in addition they have a carrier function in order to protect and deliver the immunogen to the desired immune site.

[0140] This invention is better illustrated in the following examples, which should not be interpreted as a limitation of the scope thereof. On the contrary, it should be clearly understood that other embodiments, modifications, and equivalents may be resorted after reading this description, which may suggest to those skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims. MATERIALS AND METHODS

[0141] The following chemical reagents were used in the preparation of the adjuvant nanoparticles (NPADs): polyallylamine hydrochloride (PAH) (Sigma- Aldrich - Mw= 17500 - CAS No: 71550-12-4), polyallylamine hydrochloride : fluorescein isothiocyanate 50:1 (PAH*) (Sigma-Aldrich - Mw=56000), sodium tripolyphosphate (TPP) (Sigma-Aldrich - CAS No: 7758-29-4). For the preparation of solutions of each component, distilled and pharmacological- grade water were used for characterization experiments and for biological assays, respectively. The pH of the solutions was adjusted using 1 M HCI and 1 M NaOH. The following proteins were used for protein adsorption experiments on NPADs: Recombinant Receptor Binding Domain (RBD) protein from the S spike of SARS-CoV-2 (COVID-19 virus) (26000 Da, pl=8.8, provided by the Argentine anti-COVID consortium), Lysozyme (Liso) (14300 Da, pl=11.0, Sigma-Aldrich), Cytochrome c oxidase (Cit-C) (12384 Da, pl=10.0-10.5, Sigma- Aldrich), Ovalbumin (OVA) (44287 Da, pl=4.5, Sigma-Aldrich), Bovine Serum Albumin (BSA) (66000 Da, pl=4.5-5.0, Sigma-Aldrich).

[0142] Method for Preparing Adjuvant Nanoparticles (NPADs):

[0143] A solution of PAH in aqueous medium was prepared such that monomer concentration was 40 mM (MrpAH=93.5 g.mol-1). The pH was adjusted to the desired value (pH0) by adding NaOH. This solution was named "Solution A". On the other hand, a solution of TPP in aqueous medium was prepared such that the concentration was 24 mM (MrTPP=367.86), and the pH was adjusted with HCI to the pH0value (the same pH as Solution A). This solution was named "Solution B". To obtain a colloidal dispersion solution of NPADs, the following reagents were placed in a glass flask in the following order: 1 ) VPAH ml of Solution A; 2) VH2O ml of water; 3) VTPP ml of Solution B. To achieve a homogeneous solution of NPADs, the final addition (step 3) should be done quickly and with constant stirring. R is defined as the TPP to PAH monomers concentration ratio, i.e. , R=[TPP] / [PAH], At a final volume Vf and a final PAH concentration of CPAH, the value of R will define the volumes VPAH, VH2O, and VTPP. For example, if Vf=10 ml, CPAH=5 mM, and R=0.6, the volumes to be used will be: VPAH=1 .25 ml; VH2O=7.5 ml, and VTPP=1 .25 ml. In other words, the volumes are calculated as follows: VPAH= (Vf*CPAH) / 40 mM

[0144] VTpp=(Vf*CpAH*R) / 24 m M

[0145] VH2O=Vf - VPAH - VTPP

[0146] Example 2: Protein Encapsulation in NPADs, Vaccine Preparation Method:

[0147] To incorporate proteins into NPADs and prepare a vaccine composition, the following quantities were added to a flask in the following order: 1 ) PAH ml of Solution A; 2) VH2O ml of water; 3) Vprot ml of the protein solution; 4) VTPP ml of Solution B. In this protocol, the protein solution used in step 3 is an aqueous stock solution of the protein to be incorporated into the NPADs, with a concentration of Cprot, i. This solution (protein solution) can be prepared in pure water or in Tris or HEPES buffer. The use of phosphate buffer should be avoided as phosphate ions interfere with the formation of NPADs. Similar to the previous case, the final step (step 4) should be performed quickly and with constant stirring. Knowing the final PAH concentration CPAH, R, the final volume Vf, and the final protein concentration Cprot, the volumes are calculated using the following relationships:

[0148] VPAH=(Vf*CpAH) / 40 mM

[0149] VTpp=(Vf*CpAH*R) / 24 mM

[0150] Vprot— (Vf*Cprot) / Cprot, i

[0151] VH2o=Vt - VPAH - VTPP - Vprot

[0152] All preparations were carried out using non-pyrogenic materials and pharmacological-grade water to prevent contamination with lipopolysaccharides.

[0153] Example 3: Determination of the Protein Association Efficiency with NPADs:

[0154] To determine the efficiency of protein association with NPADs (EAprot), the samples containing protein at a concentration Cprot (see example 3) were allowed to rest for 2 hours, and then centrifuged at 10,000 rpm for 20 minutes. The supernatant was collected, and the concentration of protein not bound to NPADs (Cprot, supernatant) was calculated using UV-Vis spectrophotometry by means of a calibration curve at A=278 nm (absorption band of tryptophan and tyrosine). A calibration curve was prepared for each assayed protein, obtaining the following absorptivity values: £RBD= 2.46 Lcnr1mg'1; £cit-c= 1.52 Lcnr1mg'1; £OVA= 0.71 Lcm’1mg’1; £BSA= 0.46 Lcm'1mg'1; £uso= 3.57 Lcm’1mg’1. EAprot was calculated using the following expression:

[0155] EAprot— [ ( Cprot-C prot, su pern atan ) / Cpro t]*100

[0156] Each of the reported EAprot values was the result of the average of three experiments conducted under the same conditions.

[0157] Example 4: Characterization of NPADs:

[0158] Dynamic light scattering and zeta potential: Dynamic light scattering (DLS) and Zeta potential measurements were carried out using a ZetaSizer Nano instrument (ZEN3600, Malvern, UK) at 20 °C, using disposable cuvettes (DTS0012 for DLS and DTS1060 for Z potential). For hydrodynamic particle size measurements, a backscattering angle configuration of 173° was used with 10 cycles per measurement for 20 seconds per cycle for each sample. The Zeta potential of the particles was determined based on the electrophoretic mobility obtained by laser Doppler velocimetry using a general-purpose analysis method, and averaging 100 runs for each sample.

[0159] Transmission Electron Microscopy: The transmission electron microscopy (TEM) images were obtained using a JEOL JEM-1400PLUS LaB6-TEM (120 kV) equipped with a GATAN US1000 CCD camera (2 k x 2 k). Samples were stained with phosphotungstic acid on carbon grids to create contrast.

[0160] Spectrophotometry: The UV-Vis experiments were carried out using a Perkin Elmer Lambda 35 spectrophotometer, using 1 cm pathlength quartz cuvettes.

[0161] Example 5: In Vitro Biological Characterization of NPADs:

[0162] Cell Cultures and Culture Conditions:

[0163] For the biological characterization, a ratio of NPADs:cell culture medium of 1 : 10 was used in the following cell lines:

[0164] • RAW 264.7 (murine macrophages) were maintained in DMEM-10% FBS, 100 U / mL penicillin, 100 pg / mL streptomycin at 37°C in a 5% CO2 atmosphere, and seeded at a rate of 2x105cells / well in 48-well plates. They were stimulated with NPADs overnight, and the culture supernatant was collected and stored at -80°C.

[0165] • THP-1 (human peripheral blood monocytes) were maintained in RPMI- 10% FBS, 100 U / mL penicillin, 100 pg / mL streptomycin at 37°C in a 5% CO2 atmosphere and seeded at a rate of 2.5x105cells / well in 48-well plates. They were stimulated with NPADs overnight, and the culture supernatant was collected and stored at -80°C until use.

[0166] • J774 (murine macrophages) were maintained in DMEM-10% FBS, 100 U / mL penicillin, 100 pg / mL streptomycin at 37°C in a 5% CO2 atmosphere and seeded at a rate of 2.5*105cells / well in 48-well plates. They were stimulated with NPADs overnight, and the culture supernatant was collected and stored at -80°C until use for cytokine measurement.

[0167] • HEK reporter hTLR4 (human renal epithelial cells) were maintained in DMEM-10% FBS, 100 U / mL penicillin, 100 pg / mL streptomycin at 37°C in a 5% CO2 atmosphere and seeded in 96-well plates at a rate of 3*104cells / well. They were incubated overnight or until reaching 70% confluence, the medium was changed, and they were stimulated with NPADs overnight. The culture supernatant was collected and stored at - 20°C until use for alkaline phosphatase (SEAP) activity measurement.

[0168] • JAWS-II (mouse bone marrow dendritic / monocyte cell line) were maintained in IMDM-10% FBS, GM-CSF, 100 U / mL penicillin, 100 pg / mL streptomycin, and p-mercaptoethanol at 37°C in a 5% CO2 atmosphere. They were seeded in 100 mm Petri dishes at 6x106cells with 10 mL of complete medium. Both the suspended and adherent cells were harvested and seeded at a rate of 2.5*105cells / well in 48-well plates. The culture supernatant was collected and stored at -80°C until use for cytokine measurement.

[0169] • Bone Marrow (BM) extraction and differentiation and proliferation of dendritic Cells (BMDC): The femur and tibia were extracted from the mouse, the surrounding muscle was removed, and the ends of the bones were cut. Using a tuberculin syringe with a 23G needle, 0.5 mL of PSE (0.5% bovine fetal serum, 2 mM EDTA in PBS, pH 7.2) was injected to flush the bone marrow and collect it in a sterile eppendorf tube. The bone marrow suspension was centrifuged at 250xg for 10 minutes, resuspended in PSE, and then seeded in 100 mm Petri dishes at 6x106cells with 10 mL of RPMI / GM-CSF. On day 3, 10 mL of RPMI / GM-CSF medium was added. On day 7, 10 mL of the suspension was removed, centrifuged for 5 minutes at 500xg, the supernatant was discarded, and it was resuspended in 100 mL of new RPMI / GM-CSF and placed in the original plate. On day 8, the no adhered cells were harvested by gentle pipetting and centrifuged at 300xg for 5 minutes.

[0170] • DC Maturation: 1x106cells were seeded per well in a 24-well plate in RPMI. They were stimulated with NPADs and incubated for 24 hours at 37°C with 5% CO2. The culture supernatant was harvested and stored at -80°C until use for cytokine measurement.

[0171] Stimulants and inhibitors used:

[0172] LPS: 1 pg / mL

[0173] ATP: 2.5 nM / mL

[0174] Z-VAD-FmKQ: caspase inhibitor

[0175] Anakinra: monoclonal antibody blocking interleukin-1 p (IL-1 R) receptor Cytochalasin D: phagocytosis inhibitor

[0176] CA-74Me: catepsin B inhibitor

[0177] Aluminum hydroxide (Alum) or Alhydrogel

[0178] Flow Cytometry and Microscopy Cellular Internalization and Activation Assay:

[0179] The cell lines RAW, J774, THP1 , JAWS, and BMDC were stimulated with NPADs and NPADs-FITC at a 1 :10 ratio. They were incubated for 6 hours and prepared for analysis by flow cytometry, and for fluorescence microscopy they were incubated for 1 , 2, or 4 hours. For flow cytometry, the cells were washed with PBS, collected, and labeled with specific antibodies CD11 b, CD11c, MHCII, and CD86, or left unlabeled depending on the assay. After centrifugation at 500xg for 10 minutes, they were resuspended in 300 pL of FACS buffer. They were analyzed using a FACScalibur or FACSAria flow cytometer (BD®). For confocal or epifluorescence microscopy, the cells were stimulated in the same fashion with NPADs-FITC, washed with PBS, fixed with 2% paraformaldehyde (PFA), and the nucleus was stained with 10 pg / mL of propidium iodide or DAPI in PBS for 15 minutes at room temperature, washed with PBS, mounted on a slide and observed under a confocal or epifluorescence microscope.

[0180] CD11 c mouse Pe eBioscience (1 / 100)

[0181] CD11 b mouse PerCp5.5 eBioscience (1 / 100)

[0182] MHCII mouse APC eBioscience (1 / 650)

[0183] CD86 mouse FITC eBioscience (1 / 400)

[0184] Cytokine detection in culture supernatants by ELISA: The aforementioned cell lines were stimulated with 0.5 mM NPADs, and the following positive controls were used: 1 pg / mL of LPS for 3 hours followed by 2.5 nM ATP for 3 hours, 1 pg / mL LPS for 3 hours followed by 50 to 100 pg / mL of aluminum hydroxide for 24 hours, or 1 pg / mL LPS for 3 hours followed by DNA transfection with lipofectamine for 3 hours. The cells were incubated for 24 hours at 37°C in a 5% CO2 incubator. The culture supernatants collected from the various stimulated cells were thawed on the day of the assay (from -80°C) and used for the determination of hlL-1 p, hlL-8, mlL-6, mlL-18, and mlL-1[3 concentrations according to the manufacturer's specifications. For DNA transfection, a random unrelated gene DNA fragment was used, and Lipofectamine™ LTX Reagent with PLUSTM Reagent (Invitrogen®) was used as the transfection agent following the manufacturer's instructions.

[0185] Measurement of lactate dehydrogenase (LDH) activity: Lactate dehydrogenase enzymatic activity (assessment of cellular integrity) was measured in the culture supernatant and cell lysates (lysed with Cell Culture Lysis 5x Reagent from Promega®) using the LDH-L kit (WienerLab) according to the manufacturer's specifications. OD was measured at 340 nm using a plate spectrophotometer.

[0186] LPS Detection: The HEK-hTLR4 cell line expressing the Toll-like receptor 4 (TLR4) that recognize pathogen-associated molecular patterns and, in this case, lipopolysaccharides (LPS), was stimulated. Therefore, when these cells are stimulated with LPS they secrete the alkaline phosphatase enzyme (SEAP) into the extracellular medium, which was subsequently measured in the supernatant. The HEK-hTLR4 cell line was stimulated with NPADs, PAH, TPP, pharmacological-grade water, RBD, OVA, and LPS as a positive control for 24 hours at 37°C and 5% CO2. The NPADs and their components were used in a 1 :10 ratio, and the antigens were used at 1 pg / mL. The supernatant was stored at -20°C.

[0187] Measurement of alkaline phosphatase (SEAP) activity: The enzymatic activity of alkaline phosphatase was measured in 30 pL of cell culture supernatant from HEK-hTLR4 cells using 1 mg of PNPP as substrate dissolved in 1 mL of FAL buffer (60 pL of the substrate dissolved per condition), incubated for 10 minutes at 37°C, and the reaction was quenched with 30 pL of 0.1 M EDTA, and the OD was measured at 405 nm using a plate spectrophotometer.

[0188] Example 6: In Vivo Biological Characterization

[0189] Laboratory Animals: Pathogen-free male Balb / c mice 5 to 7 weeks old were provided by the Faculty of Veterinary Medicine at the National University of La Plata. The animals were housed in the own animal facility of the IIFP Institute and handled following international guidelines for animal experimentation. The Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of the School of Exact Sciences (University of La Plata) approved the protocol (006-37-21 ).

[0190] In Vivo biodistribution monitoring of NPADs-FITC: NPADs were administered to BALB / c mice via intranasal (IN) or intragastric (IG) routes, and at different time points the animals were sacrificed, and the presence of fluorescence was examined in various tissues using flow cytometry.

[0191] Isolation of enterocytes and lamina propria mononuclear cells: The first section of the small intestine (approximate length of 10 cm) was extracted, placed in a Petri dish with PBS 1 X, and the external fat surrounding the intestinal tube was removed. Peyer's patches were removed, and the intestine was cut longitudinally to clean the interior, and it was collected in Solution I. Subsequently, it was transferred to Solution II and kept in cold for 10 minutes. It was then transferred to Solution III and incubated at 37°C for 15 minutes with stirring, and after the stablished time, it was vigorously agitated to detach epithelial cells, which were separated by centrifugation (5 minutes at 500xg) and stored on ice. The tissue was cut with a scalpel on a Petri dish and placed in Solution IV, incubated with stirring at 37°C for 30 minutes. To complete the tissue disintegration and obtain lamina propria cells, a syringe, needle, and a cell strainer with 40 pm pore size were used, and the it was collected in RPMI to halt the action of collagenase. The cells were centrifuged at 500xg for 5 minutes, the supernatant was discarded, they were resuspended in FACS buffer, and analyzed by flow cytometry.

[0192] Isolation of sublingual mucosal cells: The mucosa located in the oral cavity beneath the tongue was extracted and placed in Solution I. It was then transferred to Solution III and incubated for 20 minutes at 37°C with stirring. After incubation, the tissue was vigorously agitated several times to detach the epithelial cells, which were separated by centrifugation (5 minutes at 500xg) and stored on ice. The tissue was cut with scissors to complete the disaggregation and then transferred to Solution IV and incubated at 37°C for 30 minutes with stirring. If necessary, the tissue was further disaggregated using a needle, syringe, and cell strainer. The cells were then centrifuged for 5 minutes at 500xg, resuspended in FACS buffer, and analyzed by flow cytometry.

[0193] SOLUTION I: HBSS, 2-5% SFB, 10mM Hepes, penicillin and Streptomicin, pharmacological-grade sterile water

[0194] SOLUTION II: SOLUTION I, 1 mM DTT

[0195] SOLUTION III: SOLUTION DE, 1mM EDTA

[0196] SOLUTION IV: complete RPMI, 10% SFB, 1 mg / ml Collagenase

[0197] Isolation of mesenteric, mediastinal, and lung lymph node cells: Lymph nodes and the lower left lung lobe were extracted and collected in 1 mL of complete RPMI (base RPMI, 10% Fetal Bovine Serum, 100 U / mL penicillin, and 100 U / mL streptomycin). They were kept on ice until further processing. Collagenase (1 mg / mL) was added, and the tissue was dissected with scissors. It was then placed on stirring at 37°C for 30 minutes, and fresh culture medium was added to deactivate the collagenase. The mixture was passed through a syringe and needle to ensure complete disintegration, and the cell suspension was filtered to isolate mononuclear cells from the lamina propria (LP). The cells were centrifuged at 500xg for 5 minutes and analyzed by flow cytometry.

[0198] Analysis of immunogenicity and adjuvanticity of NPADs: Animals were intranasally administered once a week for three weeks (days 0, 7, and 14) with NPADs-OVA (each dose contained 10 pg of OVA in 20 pL of NPADs (CPAH=5 mM, R=0.6, pH°=8.5). For intragastric immunization, the same three-dose schedule of 100 pg of OVA in 200 pL of NPADs was followed. Serum samples were collected before each immunization. One week after the final dose, the animals were sacrificed, and both cellular and humoral immune responses were evaluated. The delayed-type hypersensitivity (DTH) response was assessed.

[0199] Balb / c mice were intraperitoneally inoculated with NPADs-OVA, (OVA at a final concentration of 100 pg in 200 pL of NPADs suspension, CPAH=5 mM, R=0.6, pH°=8.5). As a control, a combination of aluminum hydroxide (alum) with OVA and OVA alone was administered. Two doses were given, separated by 21 days, and serum samples were collected before each immunization. The animals were euthanized two weeks after the final inoculation, using a carbon dioxide chamber, and serum samples and bronchoalveolar lavage fluid were collected to measure the humoral response. Additionally, the spleen was extracted for splenocyte culture to assess the cellular response.

[0200] Following the same immunization scheme of two doses with 40 pg of the RBD antigen in 500 pL of NPADs, RBD was encapsulated in NPADs (NPADs- RBD) to evaluate the development of a vaccine composition against SARS- CoV-2 components. The same RDB protein with alum served as a positive control. Serum samples were collected before each immunization. The animals were euthanized two weeks after the final immunization, using a carbon dioxide chamber, and serum samples and bronchoalveolar lavage fluid were collected to measure the humoral response. Additionally, the spleen was extracted for splenocyte culture to assess the cellular response.

[0201] Serum and bronchoalveolar lavage (BAL) fluid collection: Whole blood was obtained by intracardiac puncture, then centrifuged at 1500xg for 10 minutes, and serum was separated using a pipette. For bronchoalveolar lavage, 1 mL of 1X PBS was passed through the trachea using a syringe and a tube to collect the lung lavage. Both samples were stored at -20°C.

[0202] Detection of specific IgG, lgG1, and lgG2a by ELISA in serum and BAL: Maxi Sorp plates (NUNC, Maxisorp, Denmark) were sensitized with 1 pg / 1 OO pL of antigen in carbonate buffer, pH 9.6, overnight at 4°C. Sensitized plates were blocked with 5% normal equine serum in PBS for 1 hour at 37°C, followed by incubation with the sera for 1 hour at 37°C. Antigen-immunoglobulin binding was detected with mouse gammaglobulin-specific and isotype-specific antibodies. TMB was used as the substrate for color development, and the color reaction was quantified by optical density with a spectrophotometer (OD 450 nm). The colorimetric reaction was quenched with 2M H2SO4.

[0203] Spleen cell primary culture for cellular response assay: Spleens of immunized mice were disintegrated, and cells were plated at 4x106cells / mL in complete RPMI medium (base RPMI, 10% FBS, 100 U / mL penicillin, 100 U / mL streptomycin). Cells were stimulated with 10-100 pg / mL of antigen for 48 or 72 hours, depending on the antigen. IFN-y was measured in the supernatants using a commercial ELISA kit according to the manufacturer's specifications.

[0204] Delayed-type hypersensitivity (DTH) test: Twenty-one days after the final immunization, the footpad inflammatory response was measured after a subcutaneous injection of 10 pg of antigen in 20 pL of PBS in one hind footpad. As a negative control, saline solution was injected in a similar manner in the contralateral footpad. The swelling of the footpad was measured 48 hours after injection using a digital micrometer with a minimum increment of 0.01 mm.

Claims

CLAIMS:Having thus described and determined the nature of the present invention and how it is to be practiced, it is hereby claimed as our exclusive property and right:

1. Nanoparticles, characterized by comprising a molar TPP / PAH ratio of 0.01 / 0.6, a diameter from 80nm to 526nm, a diameter polydispersity from 0.04 to 0.25, and a surface Z potential from +70mV to -20mV.

2. The nanoparticles according to claim 1 , characterized by comprising a diameter equal to or less than 300nm.

3. The nanoparticles according to claim 2, characterized by comprising a diameter from 80nm to 300nm.

4. The nanoparticles according to claim 1 , characterized by comprising a diameter polydispersity of 0.1 .

5. A vaccine composition, characterized by comprising nanoparticles, wherein the nanoparticles comprise a molar TPP / PAH ratio of0.01 / 0.6, a diameter from 80nm to 526nm, a diameter polydispersity from 0.04 to 0.25, and a surface Z potential from +70mV to -20mV, and at least one antigen.

6. The composition according to claim 5, characterized by comprising the RBD antigen.

7. The composition according to claim 5, characterized in that the antigen is encapsulated in the nanoparticle.

8. The composition according to claim 5, characterized in that the antigen is free in the medium.

9. The composition according to claim 5, characterized in that the composition is in a form selected from the group consisting of a spray, a liquid, and a gel form.

10. A procedure for preparing the nanoparticles of claim 1 , characterized by comprising the steps of: a) Preparing a PAH solution in an aqueous medium; b) Adjusting the pH; c) Preparing a TPP solution in an aqueous medium and adjusting the pH to the same pH value as in step b);d) Adding to a volume of the solution from step a) (VPAH) a volume of water (VH2O) and subsequently a volume of the solution from step c) (VTPP), wherein the addition of the volume of the solution from step c) is carried out under stirring.

11. The procedure according to claim 10, characterized in that the pH in steps b) and c) is from 7 to 8.5.

12. The procedure according to claim 10, characterized in that the suspension obtained in step d) has a pH from 7.75 to 10.3.

13. The procedure according to claim 10, characterized in that after step d), 0 to 2mM of NaCI are added.

14. The procedure according to claim 10, characterized in that the molar TPP / PAH ratio is 0.01 / 0.6.

15. A procedure for preparing the vaccine composition of claim 5, characterized by comprising the steps of: a) Preparing a PAH solution in an aqueous medium; b) Adjusting the pH; c) Preparing a TPP solution in an aqueous medium and adjusting the pH to the same pH value as in step b); d) Preparing a solution of the protein antigen and adjusting the pH to the same pH value as in step b); e) Adding a volume of the solution from step a) (VPAH) to a volume of water (VH2O), then adding a volume of the solution from step d) (Vprot), and then adding a volume of the solution from step c) ( TPP), wherein the addition of the volume of the solution from step c) is carried out under stirring.

16. The procedure according to claim 15, characterized in that the pH in steps b), c), and d) is from 7 to 8.5.

17. The procedure according to claim 15, characterized in that the suspension obtained in step e) has a pH from 7.75 to 10.3.

18. The procedure according to claim 15, characterized in that after step e), 0 to 2mM of NaCI are added.

19. The procedure according to claim 15, characterized in that the molar TPP / PAH ratio is 0.01 / 0.6.

20. The procedure according to claim 15, characterized in that the protein antigen is RBD.21 . Use of the nanoparticles of claim 1 as a vaccine adjuvant.

22. Use of the nanoparticles of claim 1 for preparing a vaccine composition.

23. An immunization method, characterized by comprising administering to a mammal an amount of the composition of claim 5.

24. The method according to claim 23, characterized in that the mammal is a human being.

25. The method according to claim 23, characterized in that the administration is selected from the group consisting of mucosal, systemic, transdermal, intratumoral, parenteral, intravenous, subcutaneous, intradermal, and intratumoral administration.

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