Compositions, methods and uses of extracellular vesicles of giardia spp

Abstract

The present disclosure relates to extracellular vesicles of Giardia spp, preferably Giardia lamblia, for use in medicine or veterinary. The present invention also relates to a composition and vaccine comprising a therapeutically effective amount of extracellular vesicles of Giardia spp, as is or encapsulated in a capsule comprising polysaccharide particles, preferably glucan.

Description

D E S C R I P T I O NCOMPOSITIONS, METHODS AND USES OF EXTRACELLULAR VESICLES OF GIARDIA SPPTECH N I CAL F I E LD

[0001] The present disclosure relates to extracellular vesicles of Giardia spp, preferably Giardia lamblia, for use in medicine or veterinary. It also refers to compositions or vaccines comprising a therapeutically effective amount of extracellular vesicles of Giardia spp, as is or encapsulated, and the method to obtain such capsules. The present invention is enclosed in the area of infection diseases, specifically diarrheal disease evoked by Giardia lamblia parasite.BACKG RO U N D

[0002] The present invention is enclosed in the area of infection diseases, specifically diarrheal disease evoked by Giardia lamblia parasite.

[0003] Diarrhoea is a common presentation of intestinal enteritis, and nearly 1.7 billion cases of diarrheal disease are reported globally each year. Importantly, acute enteritis or diarrheal disease is the second leading cause of death worldwide, accounting for over 1 million deaths annually with higher mortality rates in developing countries. Acute enteritis also imparts significant direct and indirect costs to society, including lost worker productivity and direct impacts on health care systems.

[0004] Enteropathogens such as Giardia lamblia (syn. G. intestinalis, G. duodenalis). cause diarrhoea and are commonly spread through the faecal-oral-route by contaminated water and food, or person-to- person contact. G. lamblia is the most common intestinal pathogenic parasite in humans, with an estimated 280 million cases of symptomatic giardiasis annually worldwide. Giardiasis has been included in the Neglected Diseases Initiative of the World Health Organization (WHO) since 2004 due to its impact on health.

[0005] For reasons that remain undisclosed, Giardia infections cause a spectrum of symptoms ranging from asymptomatic carriage to chronic diarrheal disease. Giardia trophozoites attach strongly to the intestinal epithelial cells via a ventral adhesive disc and cause significant damage and disruption to gastro epithelial cells in the absence of cell invasion and secreted toxins (Jimenez et al 2014). During the course of giardiasis in humans and experimental models, G. lamblia trophozoites express and secrete several proteins affecting structural, cellular and soluble components of the host intestinal milieu including proteinases of the cysteine type, variant surface proteins (VSPs), high-cysteine membrane proteins (HCMPs), arginine catabolism enzymes such as arginine deiminase (gADI) and ornithinecarbamoyl transferase (gOCT) and glycolytic ones as enolase (gENO) (Rodriguez-Fuentes et aL, 2006; Cabrera-Licona et al., 2017).

[0006] The eradication of and protection against Giardia are dependent on both B cell-mediated antibody production and T cell-mediated immune responses (Thl / Th2 / Thl7) (Singer et al. 2019; Saghaug et al., 2016; Serradell et al, 2018). The precise nature of parasite-immune cells interactions during giardiasis has important consequences for both immunopathology and immunity. Few studies have been focused in this area and, consequently, the molecular basis characterizing the modulation of the immune system by Giardia parasites remain to be explored.

[0007] A vaccine is not yet available and despite a commercial vaccine, GiardiaVax®, for dogs have been licensed, this was being discontinued in Europe by lack of scientific evidence of efficacy (Anderson et aL, 2004; Olson et aL, 2000). Thus, there has been a growing interest among the scientific community and pharmaceutics in the identification and characterization of Giardia antigens that may be used as vaccine targets, including variant-specific proteins (VSPs), the immunoglobulin binding protein (BIP), excretory- secretory products (ESPs), the annexin homolog al-giardin, and cell wall protein 2 (CWP-2) (Feng et aL, 2016; Jimenez et aL, 2014; Lee et aL, 2014; Lopez-Romero et aL, 2017; Serradell et aL, 2016; Serradell et aL, 2018).

[0008] In the last decades, Extracellular Vesicles (EVs) (exossomes, microvesicles and apoptotic bodies) have been well acknowledged as mediators of intercellular communications in prokaryotes and eukaryotes (Huda Nurul & Nurunnabi, 2022, Twu & Johnson 2014; Raposo & Stoorvogel, 2013; Wu et aL, 2019). Distinct EVs have been reported in most groups of parasitic protozoa, including flagellates (Twu et aL, 2013) and sporozoa (Martin et aL, 2011). Recently, the release and characterization of EVs from Giardia was described (Evan-Osses et aL, 2017). Giardia EVs are composed by proteins, DNA or RNA, lipids and metabolites (Khosravi et aL, 2020) and seem to play an important role in the communication between Giardia and the host cell, namely, through modulation of the host immune response (Evan- Osses et al. 2017; Gavinho et al, 2020; Khosravi et aL, 2020; Sabatke et aL, 2021; Zhao et al. 2021). Giardia EVs contain some important proteins to guarantee the survival of the species and to regulate the infection including VSPs, HSP70, f-tubulin, a tubulin, giardin, ADI, OCT, enolase, proteases (Evans-Osses et aL, 2017; Gavinho et aL, 2020).

[0009] Taking the above into consideration, namely the lack of effective strategies for the prevention of giardiasis, and focused on the market demand, herein we developed an innovative EVs-based vaccine capable of priming immune response in innate cells and T cells, and presenting immunogenicity in vivo. Of utmost importance, we developed a formulation for oral administration, encapsulating the EVs into glucan particles (GPs), and characterized the cargo of EVs responsible for the immune effects presented in this disclosure, which constitutes an innovative feature of this EVs-based vaccine.

[0010] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0011] The present disclosure relates to extracellular vesicles of Giardia spp, preferably Giardia lamblia, for use in medicine or veterinary. The present invention also relates to a composition and vaccine comprising a therapeutically effective amount of extracellular vesicles of Giardia spp, as is or encapsulated in a capsule comprising polysaccharide particles in the shell, preferably glucan, for use in medicine.

[0012] In an embodiment, the present disclosure relates to the development of Giardia EVs-based vaccine, using exossomes and microvesicles (MVs), with the ability to induce an inflammatory response in innate immune cells and on T cells, and with immunogenicity properties in vivo. Also the cargo of Giardia EVs responsible for the immunological response was characterized and a formulation for oral administration was developed, specifically the encapsulation of Giardia EVs into glucan particles (GPs).

[0013] Glucan particles are hollow, porous microspheres / capsules, comprising an outer shell of 1,3-0- glucan, and are used, for instance, for drug encapsulation to improve pharmacokinetic properties.

[0014] An aspect of the present disclosure relates to extracellular vesicles of Giardia spp for use in the prevention or treatment of an infection, in particular an infection with a parasite, bacterium, or virus; more in particular an infection with Giardia spp.

[0015] In an embodiment, the extracellular vesicles of Giardia spp are of Giardia lamblia.

[0016] In an embodiment, the extracellular vesicles of Giardia spp comprises exosomes and microvesicles.

[0017] In an embodiment, the extracellular vesicles of Giardia spp comprises a particle size ranging from 1-1000 nm; preferably 1-700 nm; more preferably ranging from 80-230 nm.

[0018] Another aspect of the present disclosure relates to the extracellular vesicles of Giardia spp for use as a vaccine adjuvant.

[0019] Another aspect of the present disclosure relates to a capsule with a shell and a core, preferably for use in medicine or veterinary, wherein the core comprises said extracellular vesicles of Giardia spp.

[0020] In an embodiment, the shell comprises a polysaccharide; wherein the polysaccharide is selected from a list consisting of: glucan, chitosan, chitin, laminarin, xylan, mannan, chrysolaminarin, arabinoxylan, fucoidan and galactomannan, amylopectin, amylose, or mixtures thereof.

[0021] In an embodiment, the capsule is glucan.

[0022] In an embodiment, the amount of extracellular vesicles of Giardia spp in the core ranges from 10-100 pg / mL; preferably 10-60 pg / mL; more preferably preferably 30- 60 pg / mL; even more preferably 60 pg / mL.

[0023] In an embodiment, the size of the capsule ranges from 0.5 - 20 pm.

[0024] Another aspect of the present disclosure relates to said capsule for use as an adjuvant for improving the immune response of other vaccines to treat infection diseases.

[0025] Another aspect of the present disclosure relates to a composition comprising a therapeutically effective amount of said extracellular vesicles of Giardia spp or of said capsule and a pharmaceutical acceptable carrier, for use in medicine or veterinary.

[0026] Another aspect of the present disclosure relates to a vaccine comprising a therapeutically effective amount of said extracellular vesicles of Giardia spp or of said capsule and a pharmaceutical acceptable carrier.

[0027] In an embodiment, the composition / vaccine may be used in the prevention of infections from Giardia spp.

[0028] In an embodiment, the composition / vaccine may be used in the prevention or treatment of a diseases that response positively to the increase of inateinnate cells and / or T cells.

[0029] In an embodiment, the composition / vaccine may be used as an immunogenic agent.

[0030] In an embodiment, the composition / vaccine is an oral, an intravascular, an intravenous or an intranasal composition / vaccine; preferably an oral composition / vaccine.

[0031] Another aspect of the present disclosure relates to a kit for use in medicine comprising said extracellular vesicles of Giardia spp, said capsule and / or said composition / vaccine.

[0032] Another aspect of the present disclosure relates to a method of producing a capsule comprising extracellular vesicles of Giardia spp comprising the following steps: diluting Giardia spp extracellular vesicles with aqueous solvent; more preferably water or saline buffer; preferably PBS; mixing the obtained diluted Giardia spp extracellular vesicles with a polysaccharide; preferably wherein the polysaccharide is glucan; incubating the previous mixture, in a period ranging from 1-3 h and in a temperature ranging from 2-6 °C; more preferably incubating for 2 h at 4 °C;freeze-drying the incubated mixture; wherein the temperature of freeze-drying ranging from -20 °C to +20 °C in a period ranging from 4-14 h; preferably for 12 h at an automatic program that gradually increases the temperature 80 °C; mixing said freeze-dried mixture with a first solution of Torula yeast RNA; preferably said Torula yeast RNA is solubilized in 50 mM Tris-HCI, pH 8, 2 mM EDTA, 0.15 M NaCI; incubating the previous mixture, preferably in a period ranging from 5 min - 2 h and a temperature ranging from 20 - 80 °C; more preferably incubating during 30 min at 50°C; adding a second solution of Torula yeast RNA to the mixture obtained in the previous step; incubating the previous mixture, preferably in a period ranging from 5 min - 2 h and in a temperature ranging from 20 °C to 80 °C; more preferably incubating during 1 h at 50°C; centrifugating the previous mixture; preferably 3 times during 10 min at 2000 x g; collecting the precipitated residue to obtain capsules comprising extracellular vesicles of Giardia spp.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0034] Figure 1: Characterization of Giardia EVs by TEM and nanoparticle tracking system (Nanosight). A: TEM image of Exosomes; B: TEM image of MVs; C: Estimated size distribution profiles of Exosomes using nanoparticle tracking system (Nanosight); D: Estimated size distribution profiles of MVs using nanoparticle tracking system (Nanosight).

[0035] Figure 2: Internalization of Giardia EVs in macrophage cells. The uptake of EVs were analysis by confocal microscopy (A-C) and through flow cytometry (D-F). A, D: Macrophage cells maintained in culture medium (control); B, E: Macrophage cells incubated with Exosomes labeled with PKH26; C, F: Macrophage cells incubated with MVs labeled with PKH26. Cells were stained for: red, PKH26-labeled EVs; green, host sialic acid and N-acetylglucosamine residues; blue, nuclei.

[0036] Figure 3A: Effect of Giardia EVs on the expression of cytokines triggered by LPS in murine macrophages and assessed by real time PCR (RT-PCR). The levels of mRNA were assessed by RT-PCR for IL16 (a), IL6 (b), INOS (c), TNF-a (d) IL10 (e), COX2 (f).

[0037] Figure 3B. Effect of Giardia EVs on the expression of cytokines triggered by LPS in murine macrophages and assessed by real time PCR (RT-PCR). The levels of mRNA were assessed by RT-PCR for PPARy (g) TLR4 (h), IL12 (i), ARG1 (j), IL4 (k), CD36 (I) and IDO (m).

[0038] Figure 4: Effect of Giardia EVs on MAPKs signaling pathways and assessed by western blot using antibodies against (A) phospho-p44 / p42, (B) phospho-JNK % and (C) phospho-p-38. An anti-tubulin antibody was used to confirm equal protein loading.

[0039] Figure 5: Effect of Giardia EVs on NF-kB signaling pathway and assessed by western blot. The activation of N F-KB was evaluated by determination of the levels of IKB-OI (A) and by assessment of nuclear translocation of the NF-KB p65RelA subunit (B-C).A: levels of protein, I K B-ot ; B:Cytoplasmic levels of RelA / p65. C: nuclear levels of RelA / p65.

[0040] Figure 6: Effect of Giardia EVs on INOS protein expression (A) and NO production (B) in macrophage cells and assessed by Western blot and by the Griess reagent, respectively

[0041] Figure 7: Effect of Giardia EVs on COX-2 expression and assessed by western blot. COX-2 expression was performed using a specific anti- COX-2 antibody and anti-tubulin antibody was used to confirm equal protein loading.

[0042] Figure 8: Effect of the Giardia EVs on the maturation status of human monocyte-derived dendritic cells (Mo-DCs) and assessed by the up-regulation of the major histocompatibility complex (MHC) type I (MHC-I) (A) and MHC type II (MHC-II) (B) and maturation markers CD86 (C), CD80 (D), CD54, through flow cytometry.

[0043] Figure 9A: Effect of the Giardia EVs on functional capacities of the stimulated human monocyte- derived dendritic cells (Mo-DCs). The polarization of T cells toward Thl (CD4+Tbet+), Th2 (CD4+GATA3+) and (G) "natural" Treg (CD4+CD25+FoxP3+) induced by DCs was evaluated by flow cytometry. A: General proliferation; B: CD4+ proliferation; C: CD8+ proliferation; D: CD4+Tbet+.

[0044] Figure 9B: Effect of the Giardia EVs on functional capacities of the stimulated human monocyte- derived dendritic cells (Mo-DCs). The polarization of T cells toward Thl (CD4+Tbet+), Th2 (CD4+GATA3+) and (G) "natural" Treg (CD4+CD25+FoxP3+) induced by DCs was evaluated by flow cytometry. E: CD4+GATA3+; F: CD4+CD25+FoxP3+; G: CD25+; H: CD4+; I: CD8+.

[0045] Figure 10: Effect of Giardia EVs on matured DCs cytokine production by ELISA. A: IL1 beta; B: IL12 (p70); C: I L10; D: IFN gama; E: IL4.

[0046] Figure 11: In vivo mice immunization with Giardia EVs. The specific serum IgG levels of mice subcutaneously immunized on days 0, 14 and 28 with 30 pg / dose of Giardia EVs and 60 pg / dose of G. lamblia trophozoites lysate (LYS) were determined by ELISA either using trophozoites lysates as capture antigens.

[0047] Figure 12: Giardia EVS trigger a specific immune response in mice. The specific serum IgG, IgG 1 and lgG2a levels of mice immunized on days 0, 14 and 28 with 30 pg / dose of Giardia EVs and 60 pg / doseof G. lamblia trophozoites lysate (LYS) were determined by ELISA, antigens. A: EVs as capture antigens; B: trophozoites lysates as capture antigens

[0048] Figure 13: Comparison of the capture antigen specificity when analyzing the immune response of SC (subcutaneous) immunized mice, assessed by ELISA.

[0049] Figure 14: Characterization of antigenic proteins in Giardia EVs through SDS-PAGE and Western blot. A: detection of proteins in MVs, EXO and LYS using a 10% SDS-PAGE, stained with Coomassie Brilliant blue G-250; B: Western blot identification of antigenic proteins reactive to the serum of mice previously immunized with EVs.

[0050] Figure 15: Internalization of encapsulate Giardia EVs in macrophage cells. The uptake of EVs were analysis by confocal microscopy. Cells were stained for: A: red, PKH26-labeled EV; B: green, host sialic acid and N-acetylglucosamine residues; C: combined the two image A and B.DETAI LED DESCRI PTION

[0051] The present disclosure relates to extracellular vesicles of Giardia spp, preferably Giardia lamblia, for use in medicine or veterinary. The present invention also relates to a composition and vaccine comprising a therapeutically effective amount of extracellular vesicles of Giardia spp, as is or encapsulated in a capsule comprising polysaccharide particles in the shell, preferably glucan, for use in medicine.

[0052] In an embodiment, the present disclosure relates to the development of EVs-based vaccine with the capacity of priming an effective immune response, protecting the development of giardiasis, comprise the following steps:- To isolate and characterize the Giardia EVs;- To evaluate in vitro the effect of Giardia EVs on innate immune cells and on T cells;- To evaluated in vivo the immunogenicity of Giardia EVs;- To characterize the proteome of EVs responsible for the immune response in animals;- To formulate the Giardia EVs into glucan particles (GPs).

[0053] Another aspect of the present disclosure relates to the use of the Giardia EVs as a new adjuvant for improving the immune response of other antigens (vaccines) to treat infection diseases. Knowing that the vaccine adjuvants are immunostimulatory components (adjuvant comes from latin word adjuvare = to help) that help the initiation, amplification and guidance of an appropriate adaptive immune response of sufficient magnitude and duration to a certain antigen.Materials

[0054] In an embodiment, Dulbecco's modified Eagles's medium (DMEM), lipopolysaccharide (LPS) from Escherichia coli (serotype 026:B6), Micro BCA Protein Assay Kit, PKH26 Red Fluorescent Cell Linker Mini Kit, Resiquimod (R848), penicillin and streptomycin, were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Glutamax, sodium pyruvate, MEM non-essential amino acids and RPMI 1640 were acquired from Gibco (MA, USA). Fetal bovine serum (FBS), Griess reagent, and both probes (WGA Alexa 633 and Hoechst 33342) were purchased from Invitrogen (Paisley, UK). Ficoll-Paque was acquired from GE Healthcare (Chalfont St. Giles, UK). UranyLess was obtained from Delta Microscopies. The protease and phosphatase inhibitor cocktails were obtained from Roche (Mannheim, Germany). Bicinchoninic acid protein assay (BCA) was from ThermoFisher Scientific (Rockford, IL, USA). Nuclear Extract Kit was purchased from Active Motif Inc. (Carlsbad, CA). CD14 and CD3 antibody-coated magnetic beads were acquired from Miltenyi Biotec. Antibodies against phospho-ERKl / ERK2, phospho-p38 MAPK, phospho- JNK, and NF-KB p65RelA were from Cell Signaling Technologies (Danvers, MA, USA). The iNOS antibody was from R&D Systems (Mineapolis, MN, USA) and COX-2 was from Abeam (Cambridge, UK). The antitubulin antibody was purchased from Sigma Chemical Co. (St. Louis, MO, USA). The alkaline phosphatase-linked secondary antibodies were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and the polyvinylidene difluoride (PVDF) membranes were from Millipore Corporation (Bedford, MA). Enhanced chemiluminescence (ECL) reagent, iScript Select cDNA Synthesis kit and Sso Fast Eva Green Supermix were purchased from BioRad (Hercules, CA, USA). NZYol reagent was purchased from NZYTech (Lisbon, Portugal). Primers were obtained from MWG Biotech (Ebersberg, Germany). GM-CSF and IL-4 were acquired from Peprotech (London, UK). Polyinosinic:polycytidylic acid (Poly l:C) was obtained from Novus Biologicals (Abingdon, UK). The p-slides 4 wells and fluorescent mounting medium were purchased from IBIDI GmbH, Germany. Legend MAX Human ELISA kit with precoated plates, Cyto- Fast™ fix / perm buffer set, CD80-PerCP / Cy5.5, CD86-Alexa Fluor 488, human leukocyte antigen (HLA)- DR-PE, HLA-ABC-APC, fluorescence-conjugated antibodies were obtained from Biolegend (San Diego, CA, USA). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA) or from Merck (Darmstadt, Germany).Cell culturesGiardia lamblia trophozoites culture

[0055] In an embodiment, the culture of G. lamblia trophozoites (strain WB, clone 6 (ATCC 30957)) was maintained as previously described (Sousa and Poiares-da Silva, 1999). Trophozoite forms were growth in axenic culture at 37°C in 10 ml of Keister's modified TYI-S-33 medium. Penicillin (lOOU / ml) and streptomycin (lOOpg / ml) were added during routine culture. Cells were incubated at 37°C in 5% CO2until a confluent cell monolayer was reached.Raw 264.7 Cell culture

[0056] In an embodiment, the mouse macrophage cell line Raw 264.7 (ATCC number: TIB-71) was cultured in DMEM supplemented with 10% FBS, lOOU / mL penicillin, and lOOpg / mL streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO2.Human monocyte-derived macrophages culture

[0057] In an embodiment, to obtain human monocytes, peripheral blood mononuclear cells (PBMCs) were firstly isolated by Ficoll-Paque density gradient centrifugation from buffy coats of healthy volunteers provided by the Portuguese Blood and Transplantation Institute (IPST) following an established protocol allowing access to buffy coats for scientific research. Monocytes were isolated by positive selection using CD14 antibody-coated magnetic microbeads, as described by the manufacturer. Monocytes (1 x 106cells / mL) were then cultured in 6-well microplates in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum and lOOng / mL GM-CSF, which induce their differentiation into macrophages. The medium was changed every two days and macrophages were collected at 7thday culture.Dendritic cells isolation and culture

[0058] In an embodiment, to obtain human monocytes and T cells, PBMCs were isolated by Ficoll- Paque gradient centrifugation from buffy coats of healthy volunteers. Buffy coats were provided by the Portuguese Blood and Transplantation Institute (IPST) following an established protocol allowing access to buffy coats for scientific research with academic purposes. Monocytes and T cells were isolated by positive selection using CD14 and CD3 antibody-coated magnetic beads, respectively, as described by the manufacturer. T cells were frozen at -80°C using a solution of FBS, 5% of glucose at 40%, and 10% of dimethyl sulfoxide (DMSO) until co-culture with DCs. Monocytes were cultured in RPMI 1640, supplemented with 10% FBS, 100 U / ml penicillin, 100 pg / mL streptomycin, 2 mM glutamax, 1 mM sodium pyruvate and MEM non-essential amino acids. Then, 1 x 106 monocytes / ml were differentiated into immature DCs (iDCs) in culture media supplemented with 250 U / ml of IL-4 and 400 U / ml of Granulocyte-macrophage colony-stimulating factor (GM-CSF). The medium was refreshed every 2 days and DCs maturation was induced at day 6 of culture, by adding 25 pg / mL of EVs (exosomes and MVs), or 20 pg / mL of polyinosinic: polycytidylic acid (Poly l:C) and 2.5 pg / mL of Resiquimod (R848).Isolation, purification and characterization of Giardia EVs

[0059] In an embodiment, Giardia EVs were obtained using differential ultracentrifugation as previously described (Gavinho et al., 2020). Briefly, G. lamblia cells in log-phase of growth were washed twice with warm PBS IX (37 C) to eliminate detached and dead parasites. Adherent parasites were collected by cooling of the culture vials on ice for 20 min and centrifuged at 400 x g for 5 min at 4°C. After that, G.lamblia was counted in a Neubauer cell-counter chamber and diluted to 1 x 106parasites / mL in TYI-S-33 medium without bovine serum in order to avoid serum-derived exosome contamination. ImM of CaCI2was added for EV induction. The parasites were incubated at 37°C for 1 h for EVs releasing (Evans-Osses et al., 2017). Then, the medium was centrifuged at 600 x g for 5 min at 4°C and the supernatant was further centrifugated at 4.000 x g for 30 min at 4°C to remove parasites and eliminate cellular debris. Afterward, the supernatant was recovered and filtered through a 0.45-pm sterilized filter (TPP). Following, the supernatant was ultracentrifugated at 15.000 x g for lh at 4°C, the pellet was washed once and then diluted in sterile filtered PBS. The remaining supernatant was then ultracentrifuged for 100.000 x g for lh30min, the collected pellet was washed once and then diluted in sterile filtered PBS. Both samples were kept at 4°C until further use. Beckman L80 (rotor 50.2 Ti) was used in the ultracentrifugation steps.Quantification and Characterization by Nanoparticle Tracking Analysis (NTA)

[0060] In an embodiment, the protein concentrations isolated EVs were estimated by the Micro BCA Protein Assay Kit, according to manufacturer's instructions, employing bovine serum albumin (BSA) as standard. Each sample was assayed in triplicate and blanks were included in all assays. The concentration of particles and size distribution of EVs were evaluated by Nanoparticle Tracking Analysis (NTA) using a Nanosight NS300 instrument (Malvern Instruments Ltd., Malvern, United Kingdom). Samples were diluted 100-fold in filtered PBS and captured in quintuplicate for 60s (20 frames per second) at room temperature. The camera level was set to 14, and the threshold used was always the same. Data were processed using the NTA 3.3 analytical software.Transmission electron microscopy (TEM)

[0061] In an embodiment, both populations of EVs (microvesicles and exosomes) produced by G. lamblia were characterized through transmission electron microscopy (TEM). First, the fresh EVs were fixed with 2% PFA. Subsequently, five microliters were deposited on Formvar-carbon coated grids (TAAB Laboratories) during 5 min at room temperature and then dried by touch with filter paper. For contrasting, the grids were put on a drop of UranyLess, for 5 min at RT. After removal of the redundant liquid, observations were carried out on a FEI Tecnai G2 Spirit Bio Twin at 100 kV.Effect of Giardia EVs on innate immune cells and on T cellsRaw 264.7 cells-and Giardia EVs interaction

[0062] In an embodiment, raw 264.7 cells (6 x 105 cells / well / ml) were culture in 24-well microplates in growth medium at 37°C for 14h. Following this period, macrophage cells were either maintained in culture medium (control), or pre-incubated with G. lamblia EVs (12.5pg / mL or 25pg / mL) for one hour.Later, LPS (lpg / ml) was used to activate macrophage cells during 30min or 14h depending on the experiments.

[0063] In an embodiment, to obtain the lysates, cells were washed in cold PBS and harvested in RIPA buffer (50mM Tris-HCI, pH 8.0, 1% Nonidet P-40, 150mM NaCI, 0.5% sodium deoxycholate, 0.1% SDS and 2mM EDTA) freshly supplemented with ImM DTT, protease and phosphatase inhibitor cocktails and sonicated (three times for 4s at 40pm peak to peak) to decrease viscosity. The nuclei and the insoluble cells debris were removed by centrifugation at 12.000 x g for 10 min at 4°C. The postnuclear extracts were collected and used as total cell lysates. Nuclear and cytosolic fractions were prepared using the Nuclear Extract Kit according to the manufacturer's instructions. Protein concentration was determined using the BCA and cells lysates were denatured at 95°C for 10 min in sample buffer (0.125mM Tris, pH 6.8, 2% (w / v) SDS, lOOmM DTT, 10% glycerol and bromophenol blue). Thereafter, western blot analysis was performed.Human macrophages-Giardia EVs interaction

[0064] In an embodiment, human macrophages for seven day cultures were maintained in medium (control cells), or exposure to Giardia EVs (12.5pg / mL or 25pg / mL) during one hour at 37°C. Later, human macrophage cells were activated with LPS (1 pg / ml) during 30 min. The macrophage cells lysates were obtained as described for Raw 264.7 cells.EVs Staining

[0065] In an embodiment, for uptake assays, both EVs populations (microvesicles or exosomes) were stained with PKH26 Red Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling (Xexc 551 nm Xem 567 nm), according to the manufacturer's instructions. Briefly, 15pg / ml of EVs populations were mixed with 500pL diluent C and 2pL of PKH26 dye. The EVs / dye solution was incubated for 5 min at room temperature in dark. After, 1 mL of 1% BSA was added to the mixture and incubated more 1 min at room temperature in dark, and samples were then washed in PBS. To obtain the EVs the mixtures were ultracentrifugated as previously described (see Section Isolation, purification and characterization of Giardia EVs).Cellular internalization of PKH26-positive EVsFlow Cytometry

[0066] In an embodiment, raw 264.7 cells were culture in 24-well microplates at 37°C for 14h (as described above). Following this period, macrophage cells were either maintained in culture medium (control), or incubated with 15pg of PKH26-labeled giardial EVs for 5h. Then, macrophages were washed three times in PBS, detached using a cell scraper, collected and quantified with a BD Accuri C6 cytometer (BD Biosciences, Franklin Lakes, NJ, USA).Confocal microscopy

[0067] In an embodiment, raw 264.7 cells were seeded on Ibidi p-slide 4 well at 37°C for 14h (as described above). Again, macrophage cells were either maintained in culture medium (control), or incubated with 15 pg of PKH26-labelled EVs for 5h. After, cells were washed three times with PBS, fixed with 4% paraformaldehyde at 37°C for 15 min, stained with WGA Alexa 633 (Xexc 632 nm Xem 647 nm) and Hoechst 33342 (Xexc 350 nm Xem 461 nm). Cells were preserved using IMM Ibidi Mounting medium and were viewed on a confocal microscope Zeiss LSM 710.Nitrite production

[0068] In an embodiment, the supernatants from co-culture of Raw 264.7 cells and G. lamblia EVs were collected and the levels of nitrite were determined using Griess reagent as previously described (Faria et aL, 2020). In brief, supernatants were centrifuged at 800 x g for 5 min and then diluted with equal volumes of Griess reagent [0.1% (w / v) N-(l-naphthyl)-ethylenediamine dihydrochloride and 1% (w / v) sulphanilamide containing 5% (w / v) H3PO ] and incubated at room temperature during 30 min, in the dark. The absorbance at 530 nm was measured in an automated microplate reader (Synergy™ HT, BioTek) and nitrite concentration was determined from a regression analysis using serial dilutions of sodium nitrite as standard.Western blot analysis

[0069] In an embodiment, Western blot analysis was performed to evaluate the effects of G. lamblia EVs on the activation of NF-KBp65RelA, phospho-ERKl / ERK2, phospho-p38 MAPK and phospho- SAPK / JNK MAPKs and on the expression of iNOS and COX-2 proteins. Briefly, 30 pg of protein sample was electrophoretically separated on a 10% (v / v) sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) at 130 V for 60-75 min, transferred to polyvinylidene difluoride (PVDF) membrane, and blocked with 5% (w / v) fat-free dry milk in Tris-buffered saline containing 0.1% (v / v) Tween 20 (TBS-T) for 1 hour at room temperature. Then blots were incubated overnight at 4°C with the primary antibodies against the different proteins to be studied as follow: COX-2 (1:5000), iNOS (1:1000), phospho-ERKl / ERK2 (1:1000), phospho-p38 MAPK (1:1000), phospho-JNK (1:1000), and NF-KB p65RelA (1:1000). After washing three times with TBS-T, membranes were incubated for 2 h at room temperature with alkaline phosphatase- conjugated anti-rabbit or anti-mouse antibodies (1:5000). The blots were visualized by chemiluminescence using ImageQuant LAS 500 (GE Healthcare, Chicago, IL, USA). The generated signals were analyzed using software TotalLab TL120 (Nonlinear Dynamics). Equivalent protein loading was verified by stripping the membranes and reprobing with antibodies to anti-tubulin antibody.Analysis of gene transcription by quantitative reverse transcription PCR (RT-qPCR)

[0070] In an embodiment, for assessment of gene transcription during macrophage-Giardia EVs interaction, Raw 264.7 cells (1.5 x 106 / well) were plated in 24-well microplates in 1.2 ml culture medium and incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2for 14 h. Subsequently, 12.5pg / mL or 25pg / mL EVs parasites were added to each well and the samples for RNA extraction were taken after 6 h of co-infection. Therefore, the microplate was chilled on ice for 20 min, supernatant was removed, and macrophage cells were washed several times in cold PBS. Total RNA was extracted from macrophage cells with NZYol reagent, according to the manufacturer's instructions, and the concentration was spectrophotometric determined by measurement of OD260 (NanoDrop, Thermo Scientific). RNA samples were stored in Storage Solution at -80°C until they were used. Total RNA (lpg) was reverse-transcribed using iScript Select cDNA Synthesis kit, and real-time reverse transcriptase- polymerase chain reaction (RT-PCR) reactions were performed, in duplicate for each sample, on CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA), using Sso Fast Eva Green Supermix. The results were normalized using Gapdh as a reference gene. Primer sequences were designed using Beacon Designer software version 7.7 (Premier Biosoft International, Palo Alto, CA, USA) (Table 1) and thoroughly tested. Table 1 depict the list of used primers.

[0071] Table 1. List of used primers.Calculation of qPCR results

[0072] In an embodiment, gene expression changes were calculated by the Pfaffl method, a variation of detla / delta CT method corrected for gene-specific efficiencies, and to report gene expression changes as relative fold changes compared to control samples (Pfaffl, 2001). Obtained fold changes were then processed as previously described (Neves et al., 2009). Briefly, as the qPCR results are presented as ratios of treated samples to untreated cells (control), the distribution of data does not follow a normal distribution. Therefore, a two-base logarithmic transformation was used to make observations symmetric and closer to a normal distribution. If x represents the fold change of the gene in one sample, then the two-base logarithmic transformation [Iog2(x)] is ln(x) / ln(2). Therefore, fold changes of 2 and 0.5 correspond to mean Iog2 values of 1 and -1, respectively.Dendritic cells maturation

[0073] In an embodiment, DC staining was performed using fluorescence-conjugated antibodies, specifically CD80-PerCP / Cy5.5, CD86-Alexa Fluor 488, human leukocyte antigen (HLA)-DR-PE and HLA- ABC-APC. Isotype-matched antibodies were used as controls. Briefly, DCs were washed and stained with 3pl of fluorescence-conjugated antibodies in phosphate-buffered saline (PBS) + 1% FBS for 30 min at 4°C, in the dark. Cells were subsequently washed, ressuspended in PBS + 1% FBS, and analyzed in an Accuri C6 flow cytometer (BD Bioscience, San Jose, CA, USA). Data were analyzed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) and the results are presented as mean fluorescence intensity (MFI), after subtraction of isotype control values.Mixed Lymphocyte Reaction (MLR)

[0074] In an embodiment, to assess T cell proliferation, autologous T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) before being co-cultured with matured DCs for 5 days at a 10:1 ratio. All co-cultures were carried out in U-bottomed 96- well plates in a final volume of 200pl of RPMI medium. The percentage of positive T cell subtypes and their activation and proliferation were analyzed by flow cytometry. At the end of the co-culture period, cells were stained with fluorescence- conjugated antibodies, namely CD4-PerCP / Cy5.5 and CD8-APC antibodies. Type 1 T cells (Thl), type 2 T cells (Th2), and regulatory T cells (Treg) subsets were also evaluated by flow cytometry after the coculture period with DCs for 5 days. The autologous T cells were stained using anti-CD4-PerCP / Cy5.5, anti-CD8-APC, anti-CD25-APC, anti-forkhead-box-P3 (FoxP3)-FITC, anti-GATA-binding protein 3 (GATA3)- FITC, and anti-T-box protein expressed in T-cells (T-bet)-PE. As some markers are intracellular, Cyto- Fast™ Fix / Perm Buffer Set, a fixation and cell permeabilization kit, was used for the intracellular staining, according to the manufacturer's instructions. Data were analyzed with GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA) and the results are presented as percentage of positive cells (%) after subtraction of isotype control values. Polyinosinic:polycytidylic acid (Poly l:C) and Resiquimod (R848) were used as positive controls.Cytokine detection by Enzyme-Linked Immunosorbent Assay (ELISA)

[0075] In an embodiment, IL-1 , IL-10, IL-12 (p70), IL-4 and IFN-y secretions in DCs and in primary human monocytes were measured with ELISA kit according to the manufacturer's instructions. Absorbance values were measured in a standard Synergy HT Multi Detection Microplate Reader (BioTek Instruments, Winooski, VT, USA) set to 450 nm and 570 nm wavelengths. IL-1 , IL- 10, IL- 12 (p70), IL-4 and IFN-y secreted levels were expressed as pg / mL. The secretion of I L12p70 by mature DCs and I FNg by T cells after co-culture with matured DCs was analyzed by ELISA Max Deluxe Kits (Biolegend, London, UK), according to the manufacturer instructions.In vivo studiesAnimals

[0076] In an embodiment, adult sixteen-week-old female CD1 mice were provided with food and water ad libitum and housed in feeding cages, kept under a 12h light / dark cycle. All experiments were in accordance with FELASA guidelines, approved by the Animal Care Committee (ORBEA) from the Faculty of Pharmacy from the University of Coimbra and approved by DGAV (Diregao-Geral da Alimentagao e Veterinaria) with reference 0421 / 000 / 000 / 2020.Subcutaneous vaccination studies

[0077] In an embodiment, groups of 3 mice were used to test the immunogenicity of Giardia lamblia secreted extracellular vesicles (EVs). For comparison, a second group of 3 mice were simultaneously immunized with trophozoites' lysates. A group of naive mice was used as the negative control. The detailed vaccination study schedule and formulations is described in Table 2 and Immunizations were performed with the formulations diluted to 120 pL in sterile phosphate buffer saline (PBS) pH 7.4, under isoflurane anesthesia. At day 42, mice were euthanized by cervical dislocation.Table 2: Details of the subcutaneous immunization: formulations and schedule.Determination of serum IgG, IgGl and lgG2a

[0078] In an embodiment, blood was collected from mice under slight isoflurane anesthesia, at day 14, 28 and 42, by the submandibular lancet method. After blood coagulation at room temperature for approximately 5 h, it was centrifuged at 4500 x g for 10 min, for serum collection. For antibody evaluation, high-binding 96-well plates (Nunc MaxiSorp®, Thermo Fisher Scientific Inc., Waltham, MA, USA) were coated with 1 pg / well of EVs diluted in PBS pH 7.2 and incubated overnight at 4 °C. Plates were washed 5 times with PBS-polysorbate 20 (0.05 %) and blocked with 200 pL of 5 % milk in PBS- polysorbate, for 1 h at 37 °C. After washing, serial dilutions of serum with a starting dilution of 1:16 were applied and incubated for 2 h at 37 °C. Specific antibodies were detected after extensive washing, using horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA), lgG2c (GeneTex, Irvine, CA, USA) or IgGl (Rockland Immunochemicals Inc., Limerick, PA, USA), according to manufacturer's instructions for 30 min at 37 °C. Next, the plates were washed and HRP was detected using o-phenylenediamine (OPD, Sigma-Aldrich Corporation, St. Louis, MO, USA). One OPD tablet (5 mg) was diluted in 10 mL citrate buffer and 10 pL H2O2 and 100 pL were added to each well and incubated for 10 min at room temperature. The reaction was stopped with 1 M H2SO4 and the samples optical density (OD) was determined at 492 nm with a microplate reader (Multiskan EX Microplate, Thermo Fisher Scientific Inc., Waltham, MA, USA).

[0079] In an embodiment, the serum IgG, IgGl and lgG2a titers were presented as the end-point titer, which is the antilog of the last log 2 dilution for which the OD was at least two-fold higher than the value of the naive sample equally diluted.Giardia EVs proteome characterization

[0080] In an embodiment, to characterize EVs' protein content, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB) analysis were performed. Primarily, EVs were processed by ultracentrifugation (Beckman, L-80 Ultracentrifuge) to separate exosomes (EXO) from microvesicles (MVs), and these were examined separately. For comparison, trophozoites lysates (LYS) were also assessed. EXO, MVs and LYS (30, 10 and 20 pg / well, respectively) were diluted in lysis buffer (3.5 % Sodium dodecyl sulfate (SDS), 0.1 M Tris buffer, pH 8.5) and then mixed (1:1 v / v) with the denaturing loading buffer (4 % SDS, 20 % glycerol, 200 mM dithiothreitol (DTT) in 0.25 M Tris buffer, pH 6.8 with Bromophenol blue as color maker) and incubated 10 min at 95 - 99 °C. The resulting samples were stacked on a 4 % polyacrylamide gel and further separated on 10 % polyacrylamide gel, both prepared from a 30 % Acrylamide / Bis-acrylamide solution (Bio-Rad, Hercules, CA, USA). A pre-stained protein marker (ProtoMarkers, National Diagnostics, Atlanta, GA, USA) was used to estimate proteins molecular weight. Gels run approximately for 2 h at 100 V in Tris-glycine-SDS running buffer.

[0081] In an embodiment, to observe the different protein pattern content of the samples after the SDS-PAGE, the polyacrylamide gels were stained with 0.08 % Coomassie colloidal staining (Comassie brilliante blue G-250, Sigma-Aldrich Corporation, St. Louis, MO, USA) overnight. The excess stain was removed with distilled water (several changes).

[0082] In an embodiment, to further identify immunogenic proteins, a western blot was performed using serum from immunized mice. Polyacrylamide gels (without stain) were transferred (100 V, 2 h, Tris-glycine-methanol transfer buffer) to nitrocellulose membranes (0.45 pm, Thermo Scientific, Rockford, IL, USA). The membranes were blocked for 1 h with blocking buffer (5 % milk in TBS with 0.1 % polysorbate 20). After blocking, membranes were washed 3 times (10 min each) and then incubated overnight with the serum from mice immunized with EVs (1: 50 dilution in 1 % milk in TBS-polysorbate 20). After incubation with the serum antibodies, the membranes were washed 3 more times and then incubated for 1 h with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (Bethyl Laboratories, Montgomery, TX, USA). Chemioluminiscence detection of bands was performed after washing with Clarity Max Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and detected with ImageQuant™ LAS 500 imaging system (GE Healthcare, Little Chalfont, UK). Subsequently, a quantitative label-free gel based proteomic approach adopted from previous studies of our group is used for GEVs proteomic mapping. Briefly, Giardia EVs proteins were separated by SDS-PAGE, the gels stained with colloidal Coomassie G-250 and the protein spots previously selected by immunoblotting, were manually excised from the gel and tryptic digestion was performed using an adapted method according to (Shevchenko et al, 2007). Spots were washed with 25 mM ammonium bicarbonate and acetonitrile followed by disulfide bonds reduction and acetylation with 10 mM DTT and 55 mM IAA respectively.Protein digestion was performed with Pierce™ Trypsin Protease (MS Grade, 90058) in 50 mM ammonium bicarbonate, overnight (37 °C), at an enzyme-to-substrate ratio of 1:30 (w / w). The tryptic peptide samples that resulted from in-gel digestion were reconstituted with 0.1% FA (formic acid) and analysed with a QExactive Orbitrap (Thermo Fisher Scientific, Bremen) through the EASY-spray nano ESI source (Thermo Fisher Scientific, Bremen) coupled to an Ultimate 3000 (Dionex, Sunnyvale, CA) HPLC system. The trap column (100 pm I.D. x 2 cm packed with Acclaim PepMap RSLC C18, 5 pm 100 A) and the EASY-spray analytical (75 pm I.D. x 75 cm packed with Acclaim PepMap RSLC C18, 2 pm 100 A) columns were from Thermo Fisher Scientific. Peptides were trapped at 30 pL / min in 96% solvent A (water with 0.1% formic acid). Elution was achieved with the solvent B (formic acid / acetonitrile, 0.1:80 (V / V)) at 300 nL / min. The 92 min gradient used was as follows: 0-3 min, 96% solvent A; 3-70 min, 4- 25% solvent B; 70-90 min, 25-40% solvent B; 90-92 min, 90% solvent B; 90-100 min, 90% solvent B; 101-120 min, 96% solvent A (Mauricio et al., 2022). The mass spectrometer was operated at 2.5 kV in the data-dependent acquisition mode. An MS2 method was used with an FT survey scan from 400 to 1600 m / z (resolution 70,000; AGC target 1E6). The ten most intense peaks were subjected to HCD fragmentation (resolution 17,500; AGC target 5E4, NCE 28%, max. injection time 100 ms, dynamic exclusion 35 s). Spectra were processed and analyzed using Proteome Discoverer (version 2.2, Thermo), with MS Amanda and Sequest HT search engines, with percolator validation (FDR<0.01). The search was performed against the Giardia lamblia ATCC 50803 Uniprot (Swiss-Prot) protein database, accessed on May 2022, Taxon identifier = 184922.Encapsulation of Giardia EVs into glucan particles (GPs)

[0083] For Giardia EVs encapsulation in GPs, first EVs were stained with PKH26 Red Fluorescent Cell Linker Mini Kit for General Cell Membrane Labeling (Xexc 551 nm Xem 567 nm), according to the manufacturer's instructions and as described by us in EVs Staining. Then PKH26-labeled giardial EVs were diluted in saline buffer (PBS) in two different concentrations (30 and 60 pg / mL) and 100 pL of both giardial EVs solutions were added to Eppendorf tubes containing 10 mg of powder GPs and incubated for 2 h at 4 °C. After incubation, GPs were kept at -80 °C and freeze-dried overnight. Next, 100 pL of 25 mg / mL torula yeast RNA (tRNA) solubilized in TEN buffer (50 mM Tris-HCI, pH 8, 2 mM EDTA, 0.15 M NaCI) were added to the freeze-dried GPs and incubated at 50 °C for 30 min. Then, 450 pL of 10 mg / mL tRNA were added and incubated at 50 °C for 1 h. GPs were centrifuged 3 times (10 min, at 2000 x g), adding 500 pL of 0.9 % saline in each wash. All supernatants were kept at -20 °C until further analysis. Finally, GPs were resuspended in 1 mL of 0.9 % saline and placed at -20 °C. Following encapsulation, the amount of EVs encapsulated was evaluated by the indirect quantification of free PKH26-labeled giardial EVs present in the supernatants (not encapsulated). Fluorescence was read at ex:530 / 25 nm, em.:590 / 35 nm (SHIMADZU UV-1700 PharmaSpec) and compared with the fluorescence of a standard curve of EVs. The encapsulation efficacy (EE %) was determined using the following Eq. (1):EE%=total EVs pg / mL - free EVs in supernatant pg / mL / total EVs pg / mLxlOO

[0084] The GPs loading capacity (LC %) with Giardia EVs was calculated following Eq. (2):(2) LC%=total EVspg / mL-free EVs in supernatant pg / mLtotal GPs pg / mLxlOOStatistical Analysis

[0085] In an embodiment, the results of Western Blot are expressed as mean ± SEM from at least three independent experiments. The results were analyzed by one-way analysis of variance (ANOVA), followed by Tukey's test, using GraphPad Prism, version 8.4.3 (GraphPad Software, San Diego, CA, USA). The data of qPCR are presented as mean ± SEM, and the means were statistically compared using the one-way ANOVA test, followed by Bonferroni's multiple comparison post-test. The significance level was *p<0.05, **p<0.01 and ***p<0.001.

[0086] In an embodiment, for dendritic cells maturation and MLR experiments, statistical analysis was performed using GraphPad Prism, version 8 (GraphPad Software San Diego, CA, USA). Data are shown as mean ± standard error of the mean (SEM) of the indicated number of experiments. Comparisons were made by the multiple group comparisons by one-way ANOVA analysis, with a Turkey multiple comparison post-test. Significance levels are as follows: *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.Results

[0087] Figure 1 shows that the size of the produced Giardia EVs corresponds to exosomes (Figure 1A) and microvesicles (MVs) (Figure IB) displaying typical rounded or cup-shaped structures by Transmission Electron Microscopy (TEM). The EVs isolated by sequential centrifugation were quantified by laser scattering using NanoSight. This analysis showed that the 100.000 x g fraction contains particle sizes smaller than lOOnm (exosomes) with a mean diameter of 82.6nm, 86% are <100 nm in size and concentration was 1,11 x 1011particles / mL (Figure 1C and Table 3). Whereas the 15.000 x g fraction contains particles larger than lOOnm size (MVs) with an average diameter of 230nm, 71.3% are between 151-700 nm in size and concentration was 1,9 x 1010 particles / mL (Figure ID and Table 4).Table 3. Particle size of the particles from the 100.000 g fractionTable 4. Particle size of the particles from the 15.000 g fraction

[0088] Table 5 depict the encapsulation efficacy (EE) of Giardia EVs in Glucan Particles (GPs). From the table 5 it is possible to conclude that by adding a greater concentration of exosomes of Giardia spp to the glucan-based microparticles, the encapsulation efficiency is maintained. Consequently, a greater amount of exosomes is associated or encapsulated within the same amount of microparticles. In conclusion, the loading capacity of these microparticles is not at its limit and it is possible to test experimental conditions that lead to an even greater loading of the microparticles.Table 5. Encapsulation efficacy (EE) of Giardia EVs in Glucan Particles (GPs)

[0089] Figure 2 shows that Giardia EVs are efficiently taken up by macrophage cells. Approximately 15pg of EVs (exosomes and MVs) labeled with PKH26 were incubated with 6 x 105 cells / well / ml macrophage cells for 5 h at 37°C and 5% CO2. (A, B, C): The cells were then collected for detecting the uptake percentage of EVs through flow cytometry. (D, E, F): Confocal microscopy photomicrographs confirmed the internalization of PKH26-labeled EVs by macrophage cells. Cells were stained for: red, PKH26-labeled EVs; green, host sialic acid and N-acetylglucosamine residues; blue, nuclei. (A, D): Macrophage cells maintained in culture medium (control); (B, E) Macrophage cells incubated with exosomes; (C, F) Macrophage cells incubated with MVs.

[0090] Figure 3A e 3B shows that Giardia EVs trigger the activation of canonical pro-inflammatory signaling cascades in macrophages, namely the transcription of cytokines / chemokines such as 1116 (a), 116 (b), 1110 (c), Cox2 (d), Nos2 (e) and Tnf-a (fj. qPCR analyses showed that Giardia EVs induced a significant transcription of 1116, 116, 1110, Cox2, Nos2 and Tnf-a (p < 0.01; p < 0.001; p < 0.0001). In contrast, a decrease in the transcription of Ppary (g) and Tlr4 (h) in the mRNA levels were observed (p < 0.05; p < 0.001). The interaction of macrophages with G. lamblia EVs had no significant effects on mRNA levels of Arg 1, 114, 1112, Cd36 and Ido (Figure 3B). Together, the results presented suggest that Giardia EVs elicit a proinflammatory response in cultured macrophages. Raw 264.7 cells (1.5 x 106cells) weremaintained in culture medium (control), or pre-incubated with Giardia EVs (12.5pg / mL or 25pg / mL) for 1 h, and then activated with 1 pg / ml LPS for 6 h. The levels of mRNA were assessed by RT-PCR, for 1116, 116, 114, 1110, Cox2, Nos2 and Tnf-a. Gene expression is indicated as Iog2 values of fold changes relatively to control. Each value represents the mean ± SEM. from three independent biological experiments run in duplicate (*p < 0.05, **p < 0.01, ***p < 0.001, compared to control; ns, not significant.

[0091] Figure 4 shows the effect of Giardia EVs on MAPKs signaling pathways. Giardia EVs trigger the activation of the canonical pro-inflammatory signaling cascades ERK1 / ERK2 (Figure 4A), SAPK / JNK (Figure 4B) and the pattern of p38 MAPK (Figure 4C). The effects of GiardiaEVs on the activation of the MAPK subfamilies, namely, Extracellular-signal Regulated Kinase (ERK) 1 / 2, p38 and Jun N-terminal Kinase (JNK), were evaluated by measuring their phosphorylated levels in response to macrophage stimulation with LPS. Raw 264.7 cells (6 x 105cells) were maintained in culture medium (control), or preincubated with G. lamblia EVs (25pg / mL) for 1 h, and then activated with 1 pg / ml LPS for 30 minutes. Total cell extracts were analyzed by Western blot using antibodies against phospho-p44 / p42 (A), phospho-JNK % (B) and phosphor-p-38 (C). An anti-tubulin antibody was used to confirm equal protein loading. The blot shown is representative of 3 blots yielding similar results. Results were expressed as percentage of phospho-p44 / p42 or phospho-JNK 1 / 2 or phosphor-p-38 protein levels relatively to control. Each value represents the mean ± SEM from at least 3 independent experiments (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001, compared to control; ns, not significant).

[0092] Figure 5 show the effect of Giardia EVs on NF-kB signaling pathways. The involvement of the transcription factor NF-KB was evaluated by determination of the protein levels of its inhibitory protein, IKB-O, and by assessing the nuclear translocation of the p65RelA subunit. Figure 5 (A) shows that LPS and Giardia EVs induced IKB-CI degradation. In addition, western blot analysis shows that treatment with LPS and with Giardia EVs decreased the cytoplasmic levels of NF-KB p65RelA (Figure 5B), while its nuclear levels were concomitantly increased (Figure 5C). Overall, Giardia EVs leads to the activation of NF-KB by IKB-CI degradation and p65 translocation into the nucleus. The Raw 264.7 cells (6 x 10 cells) were maintained in culture medium (control), or pre-incubated with Giardia EVs (12.5pg / mL or 25pg / mL) for 1 h, and then activated with 1 pg / ml LPS for 30 minutes. Total cell extracts were analyzed by Western blot using antibody against IkB-a and N F-KB p65RelA. Results were expressed as percentage of IkB-a protein levels relatively to control (A). Each value representes the mean ± SEM from at least 3 independent experiments (*p <0.05, compared to control in A; **p <0.01, ***p <0.001, compared to control (cytoplasmic fraction; in B) or LPS (nuclear fraction; in C); ns, not significant.

[0093] Figure 6 shows the activation of NO production by the Giardia EVs via upregulation of iNOS. The effect of Giardia EVs on iNOS expression 8 h after cells infection was analyzed by Western blot using a specific anti-iNOS antibody (Figure 6A). Giardia EVs stimulation in Raw 264.7 cells resulted in an increaseof the protein iNOS (p < 0.05). The effect of Giardia EVs on LPS-induced NO production in Raw 264.7 cells was also evaluated by measuring nitrite accumulation in macrophage culture medium (Figure 6B). In resting conditions, macrophage cells produced low levels of nitrites, which increased after LPS stimulation (Figure 6 B). Corroborating the data obtained for iNOS protein expression, interaction of Raw cells with Giardia EVs strongly increased the nitrite production (p < 0.0001). Raw 264.7 cells (6 x 105cells) were maintained in culture medium (control), or pre-incubated with G. lamblia EVs (25pg / mL) for 1 h, and then activated with 1 pg / ml LPS for 8 h. iNOS expression was analyzed by Western blot using a specific anti-iNOS antibody and anti-tubulin antibody was used to confirm equal protein loading. Results were expressed as percentage of iNOS protein levels relatively to control. (B) Raw 264.7 cells (6 x 105 cells) were maintained in culture medium (control), or pre-incubated with G. lamblia EVs (25pg / mL) for 1 h, and then treated with 1 pg / ml LPS for 8 h. Each value represents the mean ± SEM from at least 3 independent experiments (**p <0.01, ****p < 0.0001, compared to control; ns, not significant).

[0094] Figure 7 shows that Giardia EVs enhance COX-2 expression. Similarly, to the observed for iNOS expression, LPS treatment caused a strong increase in COX-2 protein levels, and Giardia EV stimulated Raw 264.7 cells increased COX-2 protein levels (p<0.01). Raw 264.7 cells (6 x 105 cells) were maintained in culture medium (control), or pre-incubated with G. lamblia EVs (25pg / mL) for 1 h, and then activated with 1 pg / ml LPS for 8 h. COX-2 expression was analyzed by Western blot using a specific anti- COX-2 antibody and anti-tubulin antibody was used to confirm equal protein loading. Results were expressed as percentage of COX-2 protein levels relatively to control. Each value represents the mean ± SEM from at least 3 independent experiments (**p <0.01, ****p < 0.0001, compared to control; ns, not significant).

[0095] Figure 8 shows that Giardia EVs increase the maturation status of human monocyte-derived dendritic cells (Mo-DCs). The expression of major histocompatibility complex (MHC), type I (MHC-I) (A) and MHC type II (MHC-II) (B), CD86 (C) and CD80 (D), was measured by assessing the Mean Fluorescence Intensity (MFI) by flow cytometry. DCs were treated with EVs (12.5 pg / mL protein) for 24 h and control cells (CTR) were not stimulated. The results demonstrated that EVs significantly increased the surface expression of all marker molecules studied, with the exception of MHC class I. Each column represents the mean ± SEM of, at least, 4 experiments. *p<0.05; **p<0.01, relatively to the control.

[0096] Figure 9A e Figure 9B shows that Giardia EVs strongly increase T cells proliferation (a) with a Thl profile. DCs were stimulated for 24 h with the indicated stimuli and then co-cultured with autologous T cells in a 1:10 ratio. The proliferation of T cells was determined after 5 days of co-culture by analyzing the percentage of the total CD4+ (b) and CD8+ T (c) cells presenting a decrease in CFSE fluorescence. The polarization of T cells towards Thl (CD4+Tbet+) (d), Th2 (CD4+GATA3+) (e) and "natural" Treg (CD4+CD25+FoxP3+) (f) induced by DCs was evaluated by flow cytometry. The percentageof T cells activation was assessed by the expression of the cell marker CD25 (g). The results are expressed as the percentage of cells within T lymphocytes (h-i). Polyinosinic:polycytidylic acid (Poly l:C) and Resiquimod (R848) were used as positive controls. Each column represents the mean ± SEM of, at least, three independent experiments.

[0097] Figure 10 shows the effect of Giardia EVs on matured DCs cytokine production. Giardia EVs significantly increased the secretion of IL-1 (A), IL-10 (B) and IFN-y (C) and cytokines by 0.56 and 0.68- fold in comparison to the control, respectively. Meanwhile, the level of IL-12 (p70) (D) was significantly down-regulated (1.6-fold decrease in the protein level). The ELISA results further confirmed no significant alterations in the secretion of IL-4 (E) after the treatment with the EVs. Overall, the Giardia EVs modulate the matured DCs immune response.

[0098] Figure 11 shows that Giardia EVs induce antigen-specific neutralizing antibodies IgG against trophozoites and EVs proteins in immunized mice, along the vaccination schedule. At day 14, with only the vaccine prime administration, no mice developed detectable antibodies against G. lamblia. After the first vaccine boost, at day 28, all mice immunized with the positive control (trophozoites lysates) had detectable IgG against the parasite. However, only one mouse immunized with EVs was able to generate detectable specific IgG. At day 42, all mice from both groups had considerable IgG titers against G. lamblia, with slightly higher values for the group immunized with the trophozoites' lysates. This difference has to be carefully considered since we were analyzing the specificity of the antibodies using the trophozoites' lysates as capture antigens, which could be to a certain extent beneficial for the LYS immunized mice. Specific serum IgG levels of mice immunized on days 0, 14 and 28 with 30 pg / dose of Giardia EVs and 60 pg / dose of G. lamblia trophozoites lysate (LYS). Blood was collected on day 14, 28 and 42 and antibody levels were determined by ELISA either using trophozoites lysates as capture antigens. The end-point titer in the results represents the antilog of the last log 2 dilution for which the OD were at least two-fold higher than the value of the naive sample equally diluted. Numbers above bars represent the number of mice on which antibody levels were detected; Data (mean — SEM) represents groups of 3 mice each.

[0099] Figure 12 shows that Giardia EVs induce antigen-specific neutralizing antibodies IgG isotypes IgGl, lgG2a against trophozoites and EVs proteins in immunized mice. In Figure 12A, IgGs detected were specific against proteins present in the EVs, while in Figure 12B, IgGs detected were specific against proteins present in G. lamblia trophozoites. In both scenarios, all mice immunized with EVs and LYS presented specific IgG titers. In concordance with the results from Figure 11, mice immunized with trophozoites lysates (LYS) presented slightly higher IgG titers, even when specificity was assessed with EVs as capture antigens. This indicate that the immunogenic proteins are indeed conserved in excreted EVs. The results on IgG subclasses showed other differences between using LYS or EVs to capturespecific immunoglobulins. When reactivity was assessed with EVs (A), not only the IgG titers were higher, but the IgG subclasses showed a mixed Thl / Th2 response. In fact, lgG2a titers were detected in 2 out of 3 mice immunized with EVs, while all mice immunized with LYS (B) presented this Thl type antibody. Nonetheless, results showed that IgG 1 was the predominant antibody produced both by mice immunized with EVs and LYS. Specific serum IgG, IgG 1 and lgG2a levels of mice immunized on days 0, 14 and 28 with 30 pg / dose of Giardia EVs and 60 pg / dose of G. lamblia trophozoites lysate (LYS). Blood was collected on day 42 and antibody levels were determined by ELISA either using EVs (A) or trophozoites lysates (B) as capture antigens. The end-point titer in the results represents the antilog of the last log 2 dilution for which the OD were at least two-fold higher than the value of the naive sample equally diluted. Numbers above bars represent the number of mice on which antibody levels were detected; Data (mean — SEM) represents groups of 3 mice each.

[0100] Figure 13 shows the comparison of the capture antigen specificity when analyzing the immune response of SC immunized mice. All IgGs titers achieved when specificity was assessed with EVs were higher than the ones assessed with LYS Since the concentration of the capture antigen was the same (10 pg / well) one can hypothesize that immunogenic proteins are concentrated in EVs, increasing the reactivity with IgGs present in the mice serum. Serum anti-Giardia lambia trophozoites IgG and Serum anti-Giardia lambia EVs IgG was quantified on the blood of mice immunized with 30 pg / dose of EVs (purple) and 60 pg / dose of G. lamblia trophozoites lysate (LYS, green) at day 0, 14 and 28. Blood was collected on day 42 and antibody levels were determined by ELISA, either using the lysate as capture antigen or the EVs as capture antigen (10 pg / well). The end-point titer in the results represents the antilog of the last log 2 dilution for which the OD were at least two-fold higher than the value of the naive sample equally diluted. Each group comprised 3 animal; horizontal dash represents the group mean.

[0101] Figure 14 shows the characterization of antigenic proteins found in Giardia EVs and in trophozoites' lysates (LYS) by SDS-PAGE (A) and Western blot (B). For trophozoites total protein content (LYS) it is possible to observe innumerous proteins that run through the gel at the most diverse molecular weights (MW) (A). EXO and MVs on their turn, did not show such amount of diverse proteins. However, when the proteins reactivity was assessed against serum for mice immunized with EVs, two main bands, around 47 kDa and 22 kDa, were accentuated on MVs and EXO, while a third band was visible in LYS (~ 70 kDa) (B). Detection of proteins in MVs, EXO and LYS (10, 30 and 20 pg / well, respectively) using a 10% SDS-PAGE, stained with Coomassie Brilliant blue G-250 (A). Molecular weight (MW) of proteins was compared with a pre-stained protein marker. Western blot identification of antigenic proteins reactive to the serum of mice previously immunized with EVs. Separated proteins through SDS-PAGE were transferred to nitrocellulose membranes, blocked with milk (5%) and incubatedwith serum from immunized mice at a dilution of 1:50. Anti-Giardia lamblia EVs IgG bound to specific proteins was further detected by HRP-IgG and ECL substract (B).

[0102] Figure 15 shows the capture of encapsulated Giardia EVs by macrophage cells.

[0103] Table 1 shows the identification of antigenic proteins in Giardia EVs. The proteomic characterization was done by mass spectrometry in EVs proteins fractions that bounds to specific anti- EVs antibodies present in serum of immunized mice (22 KDa and 50 KDa). We were able to identify 14 proteins in exosomes; 7 with 47-50 KDa and 7 with 22-30 KDa. We highlight the presence of Elongation factor 1-alpha, Alpha-7.3 giardin, tubulin and Variant Surface Proteins (VSP), known as antigenic proteins in Giardia infections.Table 1. Identification of antigenic proteins in Giardia EVs

[0104] The results herein achieved showed that Giardia immunogenic proteins are conserved in excreted EVs and showed a great capacity of Giardia EVs to elicit a specific immune response and allow the production of vaccines against G. lamblia infection. Indeed, Giardia EVs (exosomes and microvesicles) show pro-inflammatory activity in macrophages and DCs, and mice achieved interesting specific antibody titers and a mixed Thl / Th2 immune response without the necessity of any additional adjuvant. Moreover, a formulation for oral administration was developed with success, specifically the encapsulation of Giardia EVs into glucan particles (GPs), further envisaging the oral administration of the vaccine.

[0105] The Giardia EVs are a cell-free product that is cheap and affordable to produce. Even more, the burden of EVs encompasses a "natural" antigenic mixture of proven immunogenic proteins, with no need of recombinant proteins production.

[0106] Besides this application, Giardia EVs could also be incorporated in any other vaccines formulations already commercialized on the market, allowing the claim of adjuvant once EVS inducedimmunostimulatory effects that can help the initiation, amplification and guidance of an appropriate adaptive immune response to a certain antigen.

[0107] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0108] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above described embodiments are combinable.

[0109] Where ranges are provided, the range limits are included. Furthermore, it should be understood that unless otherwise indicated or otherwise evident from the context and / or understanding of a technical expert, the values which are expressed as ranges may assume any specific value within the ranges indicated in different achievements of the invention, at one tenth of the lower limit of the interval, unless the context clearly indicates the contrary. It should also be understood that, unless otherwise indicated or otherwise evident from the context and / or understanding of a technical expert, values expressed as range may assume any sub-range within the given range, where the limits of the sub-range are expressed with the same degree of precision as the tenth of the unit of the lower limit of the range.

[0110] The following dependent claims further set out particular embodiments of the disclosure.References• Anderson KA et al.. Impact of Giardia vaccination on asymptomatic Giardia infections in dogs at a research facility. Can Vet J. 2004. 45(11): 924-30.• Cabrera-Licona A, Solano-Gonzalez E, Fonseca-Linan R, Bazan-Tejeda ML, Raul A-G, Bermudez-Cruz RM, et al. Expression and secretion of the Giardia duodenalis variant surface protein 9B10A by transfected trophozoites causes damage to epithelial cell monolayers mediated by protease activity. Exp Parasitol. 2017;179:49-64.• Dorrington MG, Fraser IDC. NF-kappaB signaling in macrophages: dynamics, crosstalk, and signal integration. Front Immunol. 2019; 10:705. https: / / doi.org / 10.3389 / fimmu.2019.00705 PMID: 31024544• Evans-Osses I, Mojoli A, Monguio-Tortajada M, Marcilla A, Aran V, Amorim M, et al. Microvesicles released from Giardia intestinalis disturb host-pathogen response in vitro. Eur J Cell Biol. 2017.• Feng XM et al. Vaccination with Bivalent DNA Vaccine of al-Giardin and CWP2 Delivered by Attenuated Salmonella typhimurium Reduces Trophozoites and Cysts in the Feces of Mice Infected with Giardia lamblia. PLoS One. 2016; ll(6):e0157872.• Gavinho B, Rossi IV, Evans-Osses I, Lange S, Ramirez ML Peptidylarginine Deiminase inhibition abolishes the production of large extracellular vesicles from Giardia intestinalis, affecting host-pathogen interactions by hindering adhesion to host cells. https: / / doiorg / 101101 / 586438. 2020.• Huda Nurul & Nurunnabi Md. Potential Application of Exosomes in Vaccine Development and Delivery. Pharm Res. 2022 13: 1-37. doi: 10.1007 / sll095-021-03143-4.• Jimenez JC et al. Antibody and cytokine responses to Giardia excretory / secretory proteins in Giardia intestinalis-infected BALB / c mice. Parasitol Res. 2014 Jul; 113 (7) :2709-18.• Khosravi M et al. Isolation and Functions of Extracellular Vesicles Derived from Parasites: The Promise of a New Era in Immunotherapy, Vaccination, and Diagnosis. Int J Nanomedicine. 2020; 15:2957-2969. doi: 10.2147 / IJN.S250993.• Lee HY et al. Giardia lamblia binding immunoglobulin protein triggers maturation of dendritic cells via activation of TLR4-MyD88-p38 and ERK1 / 2 MAPKs. Parasite Immunol. 2014 Dec; 36(12):627-46.• Lopez-Romero G et al.. Characterization of BIP protein of G. lamblia as a potential immunogen in a mouse infection model. Immunobiology. 2017 Aug; 222(8-9):884-891• Martin-J et al. Exosomes from Plasmodium yoelii-infected reticulocytes protect mice from lethal infections. PLoS One 2011, 6, e26588.• Mauricio, T.; Aveiro, S.; Guedes, S.; Lopes, D.; Melo, T.; Neves, B.M.; Domingues, R.; Domingues, P. 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Claims

• Wu 1 et al. Extracellular Vesicle-Mediated Communication Within Host-Parasite Interactions. Front Immunol 2019, 9, 3066.• Zhao P et al. Extracellular vesicles secreted by Giardia duodenalis regulate host cell innate immunity via TLR2 and NLRP3 inflammasome signaling pathways. PLoS Negl Trap Dis 2021, 15(4): e0009304. https: / / doi.org / 10.1371 / journal. pntd.0009304• Zhao, et al..Giardia duodenalis extracellular vesicles regulate the proinfammatory immune response in mouse macrophages in vitro via the MAPK, AKT and NF-KB pathways. Parasites Vectors 2021. 14:358 https: / / doi.org / 10.1186 / sl3071-021-04865-5.C L A I M S Extracellular vesicles of Giardia spp for use in the prevention or treatment of an infection, in particular an infection with a parasite, bacterium, or virus; more in particular an infection with Giardia spp. The extracellular vesicles of Giardia spp for use according to the previous claim wherein the extracellular vesicles are of Giardia lamblia. The extracellular vesicles of Giardia spp for use according to any of the previous claims wherein the extracellular vesicles of Giardia spp comprises exosomes and microvesicles. The extracellular vesicles of Giardia spp for use according to any of the previous claims comprising a particle size ranging from 1-1000 nm; preferably 1-700 nm; more preferably ranging from 80-230 nm. The extracellular vesicles of Giardia spp for use according to any of the previous claims for use as a vaccine adjuvant. A capsule with a shell and a core, preferably for use in medicine or veterinary, wherein the core comprises extracellular vesicles of Giardia spp according to any of the previous claims. The capsule according to the previous claim wherein the shell comprises a polysaccharide; wherein the polysaccharide is selected from a list consisting of: glucan, chitosan, chitin, laminarin, xylan, mannan, chrysolaminarin, arabinoxylan, fucoidan and galactomannan, amylopectin, amylose, or mixtures thereof. The capsule according to any of the previous claims 6-7 wherein the capsule is glucan. The capsule according to any of the previous claims 6-8 wherein the amount of extracellular vesicles of Giardia spp in the core ranges from 10-100 pg / mL;preferably 10-60 pg / mL; more preferably preferably 30- 60 pg / mL; even more preferably 60 pg / mL. The capsule according to any of the previous claims 6-9 wherein the size of the capsule ranges from 0.5 - 20 pm.The capsule according to any of the previous claims 6-10 for use as an adjuvant for improving the immune response of other vaccines to treat infection diseases. A composition comprising a therapeutically effective amount of the extracellular vesicles of Giardia spp or of the capsule according to any of the previous claims and a pharmaceutical acceptable carrier, for use in medicine or veterinary. A vaccine comprising a therapeutically effective amount of the extracellular vesicles of Giardia spp or of the capsule according to any of the previous claims and a pharmaceutical acceptable carrier. The composition / vaccine according to any of the previous claims 12-13 wherein the amount of extracellular vesicles of Giardia spp ranges from 30- 60 pg. The composition / vaccine for use according to any of the previous claims 12-14 for use in the prevention of infections from Giardia spp. The composition / vaccine according to any of the previous claims 12-15 for use in the prevention or treatment of a disease that response positively to the increase of innate cells and / or T cells. The composition / vaccine for use according to any of the previous claims 12-16 for use as an immunogenic agent. The composition / vaccine for use according to any of the previous claims 12-17 wherein the composition / vaccine is an oral, an intravascular, an intravenous or an intranasal composition / vaccine; preferably an oral composition / vaccine. A kit for use in medicine comprising the extracellular vesicles of Giardia spp according to any of the previous claims 1-5, the capsule according to any of the previous claims 6-11 and / or the composition / vaccine according to any of the previous claims 12-18. Method for producing a capsule comprising extracellular vesicles of Giardia spp as described in any of the previous claims 6-11 comprising the following steps: diluting Giardia spp extracellular vesicles with aqueous solvent; more preferably water or saline buffer; preferably PBS; mixing the obtained diluted Giardia spp extracellular vesicles with a polysaccharide; preferably wherein the polysaccharide is glucan;incubating the previous mixture, in a period ranging from 1-3 h and in a temperature ranging from 2-6 °C; more preferably incubating for 2 h at 4 °C; freeze-drying the incubated mixture; wherein the temperature of freeze-drying ranging from - 20 °C to +20 °C in a period ranging from 4-14 h; preferably for 12 h at an automatic program that gradually increases the temperature 80 °C; mixing said freeze-dried mixture with a first solution of Torula yeast RNA; preferably said Torula yeast RNA is solubilized in 50 mM Tris-HCI, pH 8, 2 mM EDTA, 0.15 M NaCI; incubating the previous mixture, preferably in a period ranging from 5 min - 2 h and a temperature ranging from 20 - 80 °C; more preferably incubating during 30 min at 50°C; adding a second solution of Torula yeast RNA to the mixture obtained in the previous step; incubating the previous mixture, preferably in a period ranging from 5 min - 2 h and in a temperature ranging from 20 °C to 80 °C; more preferably incubating during 1 h at 50°C; centrifugating the previous mixture; preferably 3 times during 10 min at 2000 x g; collecting the precipitated residue to obtain capsules comprising extracellular vesicles of Giardia spp.

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