WO2018087644A2 - Vésicules extracellulaires géantes immunogènes (veg) de protozoaires parasites et procédés pour les induire et les purifier - Google Patents

Vésicules extracellulaires géantes immunogènes (veg) de protozoaires parasites et procédés pour les induire et les purifier Download PDF

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WO2018087644A2
WO2018087644A2 PCT/IB2017/056917 IB2017056917W WO2018087644A2 WO 2018087644 A2 WO2018087644 A2 WO 2018087644A2 IB 2017056917 W IB2017056917 W IB 2017056917W WO 2018087644 A2 WO2018087644 A2 WO 2018087644A2
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trophozoites
vesicles
ehvegs
erythrocytes
extracellular vesicle
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WO2018087644A3 (fr
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Francisco SIERRA LÓPEZ
Luis Antonio CARREÑO SÁNCHEZ
José Luis ROSALES ENCINA
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Centro De Investigación Y De Estudios Avanzados Del Instituto Politécnico Nacional
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/10—Protozoa; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis

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  • the present invention pertains to the field of medical parasitology and immunology, and vaccines design, particularly to methods for inducing and isolating giant protozoa vesicles with immunogenic and immunoprotective properties, more particularly from Entamoeba histolytica, Giardia lamblia and Trypanosoma cruzi. Specifically, providing methods for inducing such vesicles, referred to herein as EhVEGs and describing their potential medical properties and uses.
  • Amoebiasis is a human-only gastrointestinal infection caused by Entamoeba histolytica, a parasite characterized by two stages in its life cycle; the cysts that are infectious and the trophozoite [1 ,2].
  • the trophozoite may reside in the human intestine, which occasionally results in invasive amoebiasis, characterized by its phagocytic capacity (red blood cells (RBC) and bacteria), degradation of components of the host's extracellular matrix, and destruction of human cells [3 4], which may greatly degrade the intestinal mucosa, penetrate the tissue and spread through the bloodstream to migrate to and form abscesses in other organs such as the liver, lungs or brain [5].
  • the main clinical manifestations of invasive amoebiasis are colitis and amebic hepatic abscess (AHA) [6].
  • trophozoites accomplish cell lysis in contact-dependent form in AHA, but trophozoites have rarely been detected in direct contact with hepatocytes and are therefore attributed to extensive damage and destruction of tissue distant to the localization of trophozoites and to the release of proteases and toxins by cells of the immune system, mainly by neutrophils lysed by contact with trophozoites [8-1 1 ]. It has recently been reported that Kupffer cells and monocytes also contribute substantially to hepatic destruction [10].
  • E. histolytica trophozoites such as that carried out on human erythrocytes
  • the phagocytic qualities of E. histolytica trophozoites is one of the most recognizable behaviors of this parasite, which has been used as a diagnostic indicator of invasive infection, and has been proposed as an indicator of qualitative pathogenicity and virulence indicator [1 1 -13].
  • numerous proteins, glycoproteins, enzymes, and molecules have been studied that are involved since the contact with the target cell up to the course of the phagocytosis process [1 1 ,14].
  • the trophozoites of E. histolytica are capable of binding extracellular matrix components (ME) [3,15].
  • the interaction of trophozoites with fibronectin (FN) promotes adhesion, as they are capable of recognizing it via proteins such as integrin ⁇ 1 that is located on the surface of the parasite cells, known as the FN receptor (BI EhFNR) [16,17].
  • FN fibronectin
  • BI EhFNR FN receptor
  • FN induces in the trophozoite of E. histolytica the reorganization of the cytoskeleton, resulting in the formation of actin adhesion plaques and multiple focal contacts, leading later to the degradation of FN in situ [18].
  • FN has been used to stimulate E.
  • histolytica trophozoites generally in amounts ranging from 100 ⁇ g to 200 ⁇ g in glass coverslips of 20x20 mm (or smaller), and has also been used in high concentrations in plates with smaller area wells [17-24]; however, and prior to the present invention, FN had never been used with the purposed developed and described herein.
  • EhADH1 12 the protein complex formed by EhADH1 12 with the cysteine protease EhCP112 (EhCPADH complex), some tyrosine phosphatases proteins, and the secretion of tiny electron-dense granules with collagenase activity, proteases, lytic enzymes, etc.
  • the trophozoites of E. histolytica contain large numbers of vacuoles and vesicles, where some of them can secrete their contents; however, prior to the present invention, there were no reports which would demonstrate and/or prove that E. histolytica secretes extracellular vesicles into the medium.
  • Extracellular vesicles have a diameter range that generally comprises a diameter of 30-1000 nm [29] and are currently classified according to their cellular origin and/or their biological function, or based on their biogenesis, commonly classifying them in exosomes (30-120 nm diameter), micro vesicles (50-1000nm), and apoptotic bodies (500-5000 nm) [30,31 ].
  • Apoptotic bodies are observed in programmed cell death called apoptosis; they contain full organelles and large nuclear fragments with a large number of histones, which end up causing cell fragmentation so that their function and origin are very different from other classifications of VEs [31 ]. It has recently been reported that E.
  • histolytica can undergo apoptosis, which is characterized by DNA fragmentation and subsequent nucleus fragmentation where inducers are necessarily required for this type of programmed cell death, since naturally the trophozoites in a complete TYI-S-33 culture do not suffer apoptosis [32,33].
  • VEs from parasites that have been studied contain components of the parasite's body (proteins, lipids, nucleic acids, cytoplasm, cytoskeleton), what is related to these being immunogenic with a probable immune modulating function, since it has been shown that some VEs come to modulate-manipulate the environment in which they are, for the purpose of expressing phenotypes that benefit the parasite, as observed with T. vaginalis and malaria [35,38-40].
  • proteins that have been found in the VEs, they can be mentioned, for example, myosin, ⁇ -actin, cofilin, and glyceraldehyde 3- phosphate dehydrogenase (GADPH), being the presence of lipids usually associated with lipid rafts such as glycerophospholipids, sphingolipids, cholesterol and ceramide another of their characteristics [41 ,42].
  • myosin ⁇ -actin
  • cofilin cofilin
  • GADPH glyceraldehyde 3- phosphate dehydrogenase
  • VEs have been used to induce in vivo protection against the infectious agent producing them
  • OMV outer membrane vesicles
  • Vaccines with OMV are the most studied today. These OMVs have been prepared using extraction methods with detergents, which increases the release of vesicles in bacteria for their purification, decreasing-eliminating membrane molecules such as lipopolysaccharide (LPS), which causes problems with the good condition of the vesicles, since LPS is required in many bacteria to maintain vesicular structure, but is very toxic in some cases; however, in adequate amounts LPS serves as adjuvant for the vaccine [49,50]. To date, all vaccines with OMV have shown their effectiveness against the epidemic strains from which they were generated [51 ].
  • LPS lipopolysaccharide
  • DOC Sodium deoxycholate
  • SDS sodium dodecyl sulfate
  • Triton X-100 Triton X-144 are equally applied [49,52].
  • DOC is typically used in extraction buffers at 0.1-2% ([53-55], while the concentration of SDS to extract OMV usually ranges between 2-15% [56].
  • Triton X-100 is used at a concentration of 1 % (it may be accompanied by 1 mM phenethyl alcohol), while Triton X-1 14 has been used at concentrations of around 2% and to higher concentrations in the treatment of samples containing OMV [52].
  • Metronidazole is currently the drug of choice for treating amoebiasis at high doses depending on the severity of the infection [60,61 ]. However, there is a concern about increased resistance to this drug, as has been detected for some years [60,62]. MTZ is administered at a dose of 750 mg three times per day (TVD) for 5-10 days, but still, a luminal action amoebicide is necessary to eradicate amoebiasis, so that paromomycin (30 mg/kg/day/10 days) and diloxanide (500 mg/TVD/10 days) can also be administered [63].
  • Another drug that has been added for treating the disease is nitazoxanide; however, this and other drugs such as MTZ are secreted in breast milk besides generating undesirable side effects [63,64].
  • the doses of the drugs currently used against amoebiasis are very high since they are usually administered at an advance stage of the disease, which is one of the reasons for designing a vaccine against the infection is still desirable to prevent the damage caused by amoebiasis and the use of drugs at high doses when the infection is already present.
  • VEs and OMV have shown to provide effective protection against pathogenic bacteria as these vesicles contain various proteins, which together deliver a better protection than a single one of the particular proteins.
  • LMW-PTP low molecular weight tyrosine phosphatase
  • LMW-PTPs can be found in Archae, bacteria and eukaryotes [67-71] and are present in practically all parasitic protozoa (except apicomplexa) [72].
  • E. histolytica possesses in its genome 2 genes for Eh LMW-PTPs, which have a single amino acid of difference in position 85 of the 157 that composes them. So, the crystallographic structure and catalytic activity of the recombination protein of the GenBank sequence XP_656359 (Ehl_MW-PTP1 ) have been reported so far.
  • EhLMW-PTPs The expression of EhLMW-PTPs is reported at very low levels, hardly detectable in trophozoite extracts by immunoprecipitation from several million trophozoites followed by immunoblot [69], so the natural biological functions of EhLMW-PTPs and their location are unknown.
  • the present invention relates to a method for inducing in parasitic protozoa; e.g., E. histolytica, the efficient production of VEs, which in addition, are of very large dimensions, and until before the present invention had not been reported that they could be produced by E. histolytica or the size in which they also produced in the present invention for Giardia lamblia and Trypanosoma cruzi, so for the purposes of this invention, we have called them giant extracellular vesicles or VEGs, and particularly for the case of E. histolytica as giant extracellular vesicles of E. histolytica or EhVEGs.
  • E. histolytica the efficient production of VEs, which in addition, are of very large dimensions, and until before the present invention had not been reported that they could be produced by E. histolytica or the size in which they also produced in the present invention for Giardia lamblia and Trypanosoma cruzi, so for the purposes of this invention, we have called them
  • the EhVEGs that we induce and describe in the present invention have as characteristic, a high proteolytic activity, a high content of EhLMW-PTPS checked by immunodetection, and a high content of the EhCPADH complex, the BI EhFNR receptor being detectable to a lesser extent.
  • these EhVEGs can carry their content remotely from the trophozoite, so we have found that, in vitro, said EhVEGs can lyse erythrocytes after a few hours in the absence of trophozoites.
  • EhVEGs that we obtained and analyzed according to the results reported in the present invention have a diameter of 1000-4000 nm (1 -4 ⁇ ), although we also found EhVEGs with a diameter of 500-1000 nm (0.5-1 ⁇ ) and occasionally greater than 4000 nm (>4 ⁇ ).
  • mass spectrometry we identified some proteins from selected segments of SDS- PAGE gels with extracts of EhVEGs, where we mainly found proteins such as the heavy chain of myosin, Gal/GalNac lectin, clathrin, actin, peripheral membrane protein, GADPH, EhSec31 , among others.
  • VEGs of dimensions up to 1 ⁇ in diameter, which they were immunogenic when recognized by sera from human patients who had suffered the diseases caused by these parasites.
  • VEGs s obtained in the present invention we were able to obtain antigens useful for the effective in vivo treatment of the disease caused by the parasite that produced them; e.g., amoebiasis caused by E. histolytica, including its stage as AHA, through effective vaccine compositions containing such VEGs.
  • FIG. 1 Shows the comparison of the DNA sequences and amino acids of Ehl_MW-PTP1 (XM_651267, XP_656359), and Ehl_MW-PTP2 (XM_648265, XP_653357) proteins.
  • the change of a single nucleotide at position 254 is highlighted, which generates a restriction site for BsrD1 enzyme in Ehl_MW-PTP1 and only a change in amino acid no. 85, V to A.
  • the boxes indicating the amino acid sequences CLGNICRS and DPYY show the sites implicated in the catalysis of LMW-PTPs.
  • FIG. 1 Shows (a) the purification of His-Ehl_MW-PTP1 (lane 1) and His-Ehl_MW-PTP2 (lane 2) recombinant proteins; and (b) it is observed that sera from mice immunized with said recombinant proteins recognize both proteins (2 and 3 respectively).
  • Anti-His-EhLMW-PTPs serum was used to study the sub cellular distribution of EhLMW-PTPs in trophozoites and to label EhVEGs (containing a large amount of EhLMW-PTPs).
  • FIG. 3 Shows the secretion of EhVEGs induced with FN and the interaction of EhVEGs with human erythrocytes. Numerous EhVEGs (A) already secreted (black arrows) can be observed, whereas the red arrows follow the secretion of 4 EhVEGs.
  • the erythrocytes (A, f) were added to the trophozoites with EhVEGs, and the image was taken 4 minutes later (2 hours with 9 min of culture on FN), observing EhVEGs interacting with erythrocytes and in erythrophagocytosis; (B) secreted EhVEGs that were purified were placed in culture and human erythrocytes were added later, observing in red arrows the follow up of the collision of EhVEGs with human erythrocytes.
  • Figure 4 Shows the erythrocyte aggregation and lysis in mixed cultures of EhVEGs with human erythrocytes (a, b, c) and control cultures (d, e, f).
  • the culture is observed at time 0 (a, d), at 12 h at 4°C (b, e), and at 12 h at 4°C, and subsequently at 5 h at 28°C (c, f), where total lysis of erythrocytes is observed (c).
  • Erythrocytes in PBS or in TYI-S-33 medium were incubated as a control under the same conditions, but erythrocyte lysis was not observed (d, e, f).
  • Figure 5 Shows the production, secretion, and purification of EhVEGs vesicles of the invention.
  • Trophozoites were incubated on coverslips coated with fibronectin (FN). At different times, trophozoites (a-d) and purified EhVEGs (e) were fixed (using ZnS0 4 as described in Example 4) and were incubated with mouse anti-His-EhLMW-PTPs serum and subsequently with goat anti-mouse
  • IgG antibody coupled to FITC green color. F-actin was marked in red with phalloidin-rhodamine.
  • FIG. 6 Shows the production of EhVEGs of the invention associated with plasmatic membrane.
  • EhVEGs stained with anti-His-EhLMW-PTPs were counted on trophozoites adhered to the FN film at the indicated times (results from 8 independent experiments). Secreted EhVEGs were not included.
  • FIG. 7 Shows the EhVEGs of the invention secreted to the TYI-S-33 culture medium. Trophozoites were cultured in TYI-S33 medium and at 120-130 min of FN stimulation, EhVEGs of 1- 4 ⁇ were counted and clearly identified by light microscopy (8 reproductions). Only the EhVEGs secreted into the culture medium were included, which were counted from videos and photographs, and they were located within an area with 20-70 adhered trophozoites.
  • FIG. 8 Shows the production of EhVEGs of the invention by interaction with FN with erythrocyte extract and by erythrophagocytosis.
  • Trophozoites were cultured for 2h on coverslips coated with (a) sonicated blood cells and (b) FN.
  • FIG. 9 Shows the densitometric analysis of the labeling distribution of EhLMW-PTPS.
  • the densitometric analysis of the selected sections in red color of the respective images (d, e, f) is observed.
  • the labeling of EhLMW-PTPs is far more intense within the EhVEGs of the invention than in other region of the trophozoites. The details of the analysis are described in Example 13.
  • FIG. 10 Shows the detection of EhLMW-PTPs in unstimulated and stimulated trophozoites to produce the EhVEGs of the invention with FN during 120 min.
  • Total extracts of unstimulated (-) and stimulated (+) trophozoites were used in immunoblotting assays with anti-His-EhLMW-PTPs serum.
  • Monoclonal anti ⁇ -actin (C4) (Santa Cruz) was used as loading control.
  • FIG. 11 Shows the proteins associated with the EhVEGs of the invention. Is observed the 12% SDS-PAGE of purified EhVEGs from plasmatic membrane (2), secreted to the complete TYI-S-33 medium (3), and secreted to TYI-S-33 medium with 0.5% bovine serum (4), total extract of unstimulated trophozoites (1) and molecular weight markers (PM). Approximately 1 .2x10 5 EhVEGs and 6x10 4 trophozoites were analyzed. The gels were stained with Coomasie blue.
  • FIG. 12 Shows a Western blot of the EhVEGs secreted by trophozoites from E. histolytica.
  • Proteins obtained from the TYI-S-33 culture medium of the FN-induced trophozoites according to the adjusted method for an area of 78 cm 2 (+) were analyzed by immunoblotting with anti-B1 EhFNR, anti-EhCPADH, and anti-His-EhLMW-PTPs serum.
  • TYI-S-33 culture medium of non-induced trophozoites (-) was analyzed as a control. Proteins contained in 500 ⁇ of culture medium were precipitated with 2 mM ZnS0 4 and recovered by centrifugation.
  • the arrows indicate the recognition of EhLMW-PTPs, EhCPADH and BI EhFNR proteins, respectively.
  • FIG. 13 Shows the distribution of EhCPADH in trophozoites with EhVEGs.
  • A confocal microscopy
  • B densitometric analysis of a trophozoite with an EhVEGs associated with the plasmatic membrane.
  • FN-induced trophozoites (detailed in Examples 1 and 2, and in proportions adjusted to 4 cm 2 glass coverslips) were labeled with rabbit anti-EhCPADH antibody (Aa) showing the clear camp of the representative trophozoite (Ab);
  • the densitometries (Bc-Bd) corresponds to the gray and red underlined regions of the upper images, respectively.
  • Non-permeabilized samples Details of the processing of the samples and images are described in Example 13.
  • FIG. 15 Shows EhVEGs and human erythrocytes.
  • EhVEGs purified with ZnS0 4 (a) and without ZnS0 4 according to the FN induction variant method I (see details in Examples 1 and 2) were incubated with human erythrocytes for 5 min (b) and 30 min (c) in TYI-S-33 medium at 37°C. The mixture was fixed, and the presence of EhCPADH was determined with anti-EhCPADH (green) and EhLMW-PTPs with anti-His-EhLMW-PTPs (red). On the left is the clear field and on the right the merge. The co-localization EhCPADH-EhLMW-PTPs was observed in yellow.
  • FIG. 16 Shows that EhLMW-PTPs in total extracts of EhVEGs are susceptible to being degraded.
  • the degradation (A) of EhLMW-PTPs in EhVEGs is observed, where EhVEGs, after being purified, were packed with ZnS0 4 , placed in 1x loading buffer with 2x completeTM inhibitor (EDTA-Free, Roche) to boil for 8 min immediately (1 ), subsequently for 10 min (2), and later on for 15 min (3); they were afterwards cooled on ice to equilibrate the pH with NaOH and immediately loaded onto an SDS-PAGE gel to continue with Western blot;
  • (B) irregular recognition of EhLMW-PTPs in total extracts of EhVEGs extracts were made using ZnS0 4 , 1x loading buffer and 2x completeTM inhibitor were boiled immediately, equilibrated with NaOH and analyzed immediately by SDS-PAGE followed by Western blot) in which a high amount of complexes remain in the samples were
  • S/ without anti-His-EhLMW-PTPs serum.
  • 'Pre', 'S/ ', and lane 3 it used the same EhVEGs extract.
  • lanes 1 -3 we used an anti-His-EhLMW-PTPs serum revealed with goat anti-mouse IgG coupled to alkaline phosphatase.
  • FIG. 17 Shows the EhVEGs associated with the plasmatic membrane of trophozoites, before and after their extraction.
  • Trophozoites grown on FN for 120 min were treated with SDS-Triton X-100 (Method described in Examples 1 , 2, 4, induction with FN) to extract the EhVEGs associated with their plasmatic membrane.
  • Parallel cultures did not undergo treatment.
  • the presence of EhVEGs in the plasmatic membrane was reviewed by epifluorescence for counting in 80-120 trophozoites. Representative graph of 8 assays.
  • FIG. 18 Shows that the EhVEGs of the invention are immunogenic.
  • Total extracts of trophozoites (A) and extracts of EhVEGs (B) were analyzed by immunoblotting.
  • the sera used were mouse [o Pre (preimmune serum), a-ET (anti-trophozoite total extract serum), a-EhVEGs ACF (anti-EhVEGs serum with Freund's complete adjuvant), a-EhVEGs S/A (anti-EhVEGs serum without adjuvant)] and human serum [a-amib Mex (patient serum with amoebiasis in Mexico), a-amib Col (patient serum with amoebiasis in Colombia), a-amib Human pool (mixture of a-amib Mex and a-amib Col)].
  • FIG. 19 Shows the reduction of amebic hepatic abscess (AHA) in hamsters immunized with EhVEGs.
  • a group (13) of male Syrian hamsters (Mesocricetus auratus) were immunized with extracts of heat-denatured EhVEGs in PBS (50 ⁇ g of protein/Hamster) intraperitoneal ⁇ , and another group (10) was only administered with PBS by the same via. After 48 days, both groups were inoculated with 1x10 6 trophozoites in the right lobe of the liver, and after 7 days the formation of the amebic hepatic abscess (AHA) was evaluated.
  • H AHA PBS weight of liver from which the AHA was dissected in hamsters vaccinated with PBS
  • AHA PBS the weight of the AHA dissected from hamsters vaccinated with PBS
  • H AHA EhVEGs weight of liver with AHA in hamsters immunized with EhVEGs
  • H EXT AHA EhVEGs weight of liver from which the AHA was dissected from hamsters immunized with EhVEGs
  • AHA EhVEGs weight of liver from which the AHA was dissected from hamsters immunized with EhVEGs
  • AHA EhVEGs The significant difference ( * ) was determined using oneway ANOVA analysis with the Newman-Keuls multiple comparison test. The p ⁇ 0.05 was considered significant.
  • FIG. 20 Shows that by the method of the invention, FN induces the formation of VEGs in Giardia lamblia. It observe (A) trophozoites of G. lamblia that were cultured in the absence or the presence of a FN film for 120 min. VEGs (arrows) are observed in the culture medium or associated with the parasite; (B) trophozoites of G.
  • EhLMW-PTP1 (XP_656359) and EhLMW-PTP2 (XP_653357).
  • the sites implicated in the catalysis of LMW-PTPs are highlighted with a green and a blue box.
  • the residues that are 100% identical are shown with asterisks, while with two points (:) the conserved substitutions, and with a point (.) the least conserved substitutions.
  • Example 1 As a result of the induction method of the invention with FN in doses according to the calculations showed in Example 1 (2.5-5.2 ⁇ g/cm 2 and 0.5-10 ⁇ g/cm 2 ) and performed with LIT medium, a high amount of vesicles was obtained with about a 0.5-1 ⁇ diameter.
  • a vesicle of approximately 1 ⁇ in diameter has approximately 4 times more volume than a vesicle of 0.5 ⁇ .
  • Obtaining frequently secreted vesicles of higher volume using the method of the invention suggests that more of the proteins of interest secreted in them can be obtained, as was observed when the cross-reaction marking was increased with the assayed antibody.
  • FIG. 23 Shows the alignment of E. histolytica Ehl_MW-PTP1 with T. cruzi proteins.
  • We searched for T. cruzi proteins with peptide sequences in common with the sequence of Ehl_MW-PTP1 (XP_656359) using the server https://blast.ncbi. nlm.nih.gov/Blast.cgi?PAGE Proteins. Fragments of the proteins were aligned according to the peptides in common using the server http://www.ebi.ac.uk/Tools/msa/clustalo/.
  • GI71403398 hyperthetical protein of 605 aa
  • GI557861841 RAB GDP alpha dissociation inhibitor protein of 445 aa
  • GI557858597 outputative mucin associated surface protein of 145 aa
  • GI407852706 hyperthetical protein TCSYLVIO-002856 of 348 aa.
  • the residues that are 100% identical are shown with asterisks, while with two points (:) the conserved substitutions, and with a point (.) the least conserved substitutions.
  • FIG. 24 Shows that sera from patients with Chagas disease or Giardiasis recognize components of the respective VEGs obtained by the method of the invention.
  • Total extracts of G. lamblia trophozoites, T. cruzi epimastigotes, and purified VEGs from FN-stimulated cultures of G. lamblia (A and B) and T. cruzi (C and D) were analyzed by SDS-PAGE at 12% stained with Coomassie blue (A and C) and immunoblot (B and D) with sera from patients with giardiasis (B) and sera from patients with Chagas disease (D).
  • GI-FN lamblia trophozoites stimulated with FN
  • GI-VEs-FN total extract of VEs secreted by G. lamblia trophozoites stimulated with FN
  • Tc-N/E total extract of T. cruzi basal secreted VEs
  • Tc-VEs total extract of epimastigotes of T. cruzi stimulated with FN
  • Tc-FN total extract of T. cruzi VEs produced by stimulation with FN
  • Tc- VEs-FN total extract of T. cruzi VEs produced by stimulation with FN
  • the amount of unstimulated and stimulated parasites is equivalent, and the number of VEGs corresponds to that produced by the parasites of the respective lane.
  • GI-VEs-FN was extracted from the medium in the presence of bovine serum.
  • the present invention provides a method for inducing and isolating giant extracellular vesicles, also known as VEGs, from parasitic protozoa.
  • giant extracellular vesicles also known as VEGs
  • EhVEGs For giant extracellular vesicles from E. histolytica; for example, we use the acronym "EhVEGs".
  • EhVEGs the same is applicable to other parasitic protozoa of clinical interest, such as Giardia lamblia and Trypanosoma cruzi.
  • Giardia lamblia and Trypanosoma cruzi There is no precedent of these giant vesicles in the state of the art, much less how to induce and/or produce them.
  • E. histolytica has been studied for several decades, but it has thus far been demonstrated through the method described herein that trophozoites are capable of secreting large extracellular vesicles EhVEGs. Accordingly, among the first aspects of the present invention are: the evidence of the induction and/or production of the formation of VEGs; for example, EhVEGs, in trophozoites under specific culture conditions, which are described herein as embodiments of the present invention; to provide a method and some of its variants for producing them, and comprise the stimulation of trophozoites with fibronectin (FN), the culture of trophozoites in a film or coating prepared with fragments of blood cells, and the culture of trophozoites in which erytrophagocytosis is promoted.
  • FN fibronectin
  • the following steps are carried out to obtain trophozoites from virulent strains; e.g. for the preparation of EhVEGs from E. histolytica:
  • AHA amebic hepatic abscess
  • EhVEGs which have been secreted from highly virulent variants of the parasite; however, conventional methods of culturing corresponding to the protozoan parasite of interest to obtain the trophozoites may alternatively be used.
  • VEGs of the invention are cultivated according to one of the following embodiments:
  • fibronectin preferably in the presence of fibronectin films that are placed on suitable surfaces that allow the adhesion of the film and the subsequent interaction of the trophozoites of interest with said FN film in the presence of suitable culture media for the cultivation and/or growth of said trophozoites; in this case, fibronectin of any origin purified from fresh blood can be used however, it is preferred to use natural host fibronectin to infect the parasite; e.g., human fibronectin for parasitic protozoa of clinical interest that infects humans; or b) In the presence of lysed erythrocytes, preferably in the presence of films made with lysed erythrocytes, which are placed on suitable surfaces that allow the adhesion of the film and subsequent interaction of the trophozoites of interest with the specified erythrocyte film, in the presence of the suitable culture medium for cultivation and/or growth of said trophozoites.
  • FN fibronectin
  • erythrocytes of any origin obtained from fresh blood may be used.
  • it is preferred to used erythrocytes from the natural host infected by the parasite e.g., human erythrocytes for parasitic protozoa of clinical interest that infect humans.
  • the trophozoites are allowed to be cultured for determined times and temperatures to induce the production of VEGs; e.g.; for a time of 30 to 120 min, preferably during the time needed for the adequate production of VEGs, and at a temperature of 37°C, either in the presence or not of bovine serum.
  • VEGs are obtained from:
  • VEGs are obtained, they are purified through one of the following embodiments:
  • the resulting solution where VEGs are found ZnS0 4 may optionally be added, bringing said compound to a final concentration of 2-5 mM for further centrifugation at 1 ,500 to 2,000 x g for 10 min (in a tilt rotor at 500-800 x g for 5-10 min), recovering in this way purified VEGs.
  • This procedure can substitute for or complement the process of obtaining VEGs by centrifugation, which is described at the end of each purification process.
  • EhVEGs immunogenic E. histolytica VEGs
  • Variant method I Induction with FN.
  • the trophozoites of interest are cultured in TYI-S-33 medium with or without 5-10% bovine serum at 37°C [75,76] on a "film” or fibronectin coating (purified human FN of fresh blood as indicated in the literature) [75,77] made on a plastic or glass surface (e.g., cell culture boxes, Petri dishes, coverslips, slides, or other surface that serve the same purpose) with 2.5-5.2 ⁇ g of FN per cm 2 of surface; e.g., for a surface of about 78 cm 2 , it is necessary to pre incubate about 15 ml of TYI- S-33 medium for about 15-20 min at 37°C to subsequently seed the trophozoites dispersing in the pre-incubated medium.
  • a plastic or glass surface e.g., cell culture boxes, Petri dishes, coverslips, slides, or other surface that serve the same purpose
  • 2.5-5.2 ⁇ g of FN per cm 2 of surface e.g., for a surface
  • 2x10 6 -3.5x10 6 viable trophozoites suspended in TYI-S-33 medium should be seeded and spread over an area of about 78 cm 2 of the culture vessel (e.g., in a 10 cm diameter plastic Petri dish), so that they spread homogeneously throughout all the area to remain in 2x10 4 -4.5x10 4 trophozoites per cm 2 and continue the culture at 37°C; technical aspects of the culture are detailed in Examples 1 and 2.
  • the EhVEGs of the invention are produced, some of which are secreted into the culture medium, and others remain associated with the outer membrane of trophozoites, which are purified according to the procedures detailed in Examples 3 and 4.
  • the culture supernatant is collected (in case it needed to remove contaminating trophozoites, the supernatant is centrifuged at 120 x g for 5 min to discard the pellet and then filter in 5 ⁇ pore filters), and a solution of 25 mM ZnS0 4 in PBS is added to have a final concentration of 2-5 mM of ZnS0 4 ; the mixture is homogenized by inversion and then centrifuged at 500-800 x g in a tilt rotor for 10 min or more (2000 x g in a fixed-angle rotor).
  • the EhVEGs of different sizes will be obtained in the pellet with some cellular debris.
  • the culture supernatant obtained (in case it needed to remove contaminating trophozoites, the supernatant is centrifuged at 120 x g for 5 min to discard the pellet and then filter in 5 ⁇ pore filters) should be centrifuged at more than 10,000 x g in 1.5 ml conical tube centrifuges for about 15-20 min to precipitate the larger EhVEGs.
  • stronger centrifugal forces will be used to form the EhVEGs pellets; e.g., 15,000 x g for 20 min for filled tubes in a JA-20 rotor (Beckman Coulter, Inc.)
  • the methodology for the purification of EhVEGs comprises the following: A. To obtain EhVEGs in density gradient centrifugation with sucrose in PBS.
  • EhVEGs At the interfaces between 70-60% and 60-50%, it is possible that few trophozoites contaminants are found with EhVEGs being secreted, whereas at the interface 40-30% they will be observed mainly the EhVEGs, which are collected, and PBS is added to dilute the sucrose to a concentration of about 10%; then ZnS0 4 in PBS is added to have a final concentration of 2-5 mM ZnS0 4 , and the EhVEGs are collected at 1500-2000 x g for 10 minutes (at lower density interfaces, at the interface 40-30% EhVEGs are also observed, and they can be collected using the same procedure with ZnS0 4 ). A great quantity of EhVEGs will be obtained in the pellet, and the contaminating trophozoites are removed through centrifugation with the gradients. B. To obtain EhVEGs by stages in sucrose gradients.
  • a button will correspond mainly to trophozoites that did not stably adhere to the culture boxes, which will carry a certain amount of EhVEGs (possibly associated and disassociated from the trophozoites during the process); this and the 50% sucrose phase should be discarded if they are not to be analyzed. Homogenize the collected by gently inverting the tube about 10 times (it will contain EhVEGs and some contaminants such as small trophozoites of lower density).
  • EhVEGs To the homogenized with EhVEGs, PBS is added in a 1 :1 ratio to dilute the sucrose; (if it needed to obtain EhVEGs without ZnS0 4 , centrifuge at 15,000 x g for 15 min) then add ZnS0 4 in PBS to have a final concentration of 2-5 mM of ZnS0 4 (subject to cold incubation for 15 min), and collect the EhVEGs at 1500-2000 x g for 10 min. A great quantity of EhVEGs will be obtained in the pellet. By centrifugation with the gradient, trophozoites contaminants are eliminated. C. For purification of EhVEGs associated with outer membrane of trophozoites of E. histolytica with gradient solutions of SDS-Triton in PBS.
  • the trophozoites who were removed from the supernatant that has the EhVEGs secreted are placed in petri dishes or coverslips, then add 10 ml of 4-2% of p-formaldehyde in PBS at 37°C for Petri dishes with an area of 78 cm2 or 2 ml for coverslips in a 3.5 cm diameter Petri dish; after incubating the dishes for 40-60 min at room temperature, place them on an orbital shaker with temperature control at 50 rpm 26-28°C for 20 sec (or, make carefully 20 orbital movements manually, placing the boxes on a flat surface).
  • Solution 1 (SDS 0.0015% + Triton X100 0.0006%, in PBS) at 26-28°C for 10 min; place on the orbital shaker at 50 rpm, 26-28°C for 20 sec. Recover the supernatant from Solution 1 SDS-Triton X100 in a 15 ml conical Falcon tube and place on ice.
  • EhVEGs aggregates are formed that are visible if they are in high numbers (millions), which is observed in white color.
  • Centrifuge at 500-800 x g in tilt rotor because of the formation of aggregates of EhVEGs, it is possible to form the button that is little compacted) for 5-10 min in Falcon conical tubes of 15 ml.
  • the samples are processed as soon as they are obtained; and for the protein analysis, the samples should be processed (by adding protease inhibitors) immediately to analyze the proteins in acrylamide gels, in denaturalizing conditions (SDS-PAGE) with Coomassie stain and Western blot.
  • Variant method II Induction with blood cells.
  • sonicated human blood cells take about 40 ⁇ of blood from a finger or venous puncture, wash 2 times with PBS by centrifuging at 180 x g, place about 2.5x10 6 erythrocytes in 25 ml of PBS to sonicate in an amplitude of 90 for 10 sec) on a culture surface, preferably of 1x10 3 to 5x10 3 sonicated erythrocytes /cm 2 .
  • the trophozoites should preferably be seeded by spreading 2 ml with 1x10 6 to 1 .7x10 6 trophozoites/ml suspended in TYI-S-33 medium for an area of about 78 cm 2 (e.g., in a 10 cm diameter plastic Petri dish), so that they spread homogeneously throughout the area to result in 2x10 4 -4.5x10 4 trophozoites per cm 2 .
  • the technical aspects of the culture are detailed in Examples 1 and 3.
  • the purification of EhVEGs is carried out by: 1) centrifugation by density gradients with sucrose in PBS; 2) by stages in sucrose gradients; and 3) the EhVEGs associated with the outer membrane are purified with SDS-Triton gradient solutions in PBS, following exactly the same steps as described for the variant method I.
  • the technical aspects of the purification are detailed in Example 4. It is important to consider that it is possible that the EhVEGs obtained have contamination by remains of blood cells, therefore, therefore in case the analysis detects high amount of contamination, the EhVEGs should be purified in times greater than 120 minutes; the blood type should also be considered if necessary. The filtration of supernatants with EhVEGs in filters with 5 ⁇ pores should be considered to obtain them from the filtrate.
  • Variant method III Induction of EhVEGs by phagocytosis of human erythrocytes in suspension.
  • trophozoites of interest Cultivate the trophozoites of interest in TYI-S-33 medium with or without 5-10% bovine serum at 37°C; add the suspension of human erythrocytes washed and resuspended in PBS at a ratio of 1 :20 to 1 :50 (trophozoite:erythrocyte) to the culture of trophozoites. Incubate at 37°C for 30-60 min, and then remove the remaining non-phagocyted erythrocytes in the medium after two washes with TYI- S-33 S/SB (without bovine serum) at 37°C.
  • the EhVEGs produced by the trophozoites following induction with human erythrocytes can be purified from the supernatant or from those associated with outer membrane.
  • the essential steps include the following:
  • the culture boxes are shaken at 50 rpm at 37°C for 20 sec; the supernatant medium (it contains EhVEGs with some contaminants such as trophozoites that were not stably adhered) into a 50 ml Falcon conical tube on ice and proceed with the separation methods of EhVEGs in density gradient centrifugation with sucrose in PBS, which are described in steps A or B of variant method I. It is recommended to carry out the purification of EhVEGs from the supernatant by prior filtering with a 5 ⁇ pore filter.
  • a 25 mM solution of ZnS0 4 in PBS will be added to the collected sample to provide a concentration of 2-5 mM of ZnS0 4 incubated on ice for 10-15 minute and centrifuged at 2000 x g in a fixed-angle rotor (also 500-800 x g in a tilt rotor) for 5- 10 min. Wash the EhVEGs to reduce the concentration of the detergents used.
  • Example 4 Technical details of the methods for obtaining EhVEGs from the TYI-S-33 medium supernatant are described in Example 4 from step 8.1 , where contamination by erythrocyte fragments that remained after erythrophagocytosis events in the supernatant should be considered.
  • the production and purification of the EhVEGs of the present invention has proved to be more controllable with the induction with FN and sonicated erythrocyte films, as a way to illustrate the present invention, we show the characterization of the EhVEGs produced using said films.
  • the present invention provides the first method of purification of EhVEGs, with or without the use of detergents, and their use as immunogens for the development of vaccines as well as laying the foundations for further studies on EhVEGs as carriers of important proteins in the pathogenesis and virulence of the parasite of interest.
  • the components of EhVEGs have shown to be antigenic for both animal models and humans who have suffered from amoebiasis.
  • EhLMW- PTPs low molecular weight protein tyrosine phosphatase of E. histolytica
  • Figure 1 shows the comparison of the amino acid and DNA sequences of the proteins Ehl_MW-PTP1 and Ehl_MW-PTP2, where it is seen that only one amino acid is different (position 85) between them, implying that both proteins are recognizable by the same anti-His-EhLMW-PTP serum in immunoblot assays with the recombinant proteins describes in Example 8.
  • Figure 3 shows representative images of different moments of secretion of EhVEGs (induced by FN) (A) and the co-culture of EhVEGs purified with erythrocytes (B). It also shows induced trophozoites (Af) whereto human erythrocytes were added, observing erythrophagocytosis (performed in a culture with TYI-S-33 medium at 37°C where once a high amount of secreted EhVEGs was observed, human erythrocytes in PBS were added prepared as described in Examples 12 and 15), and the interaction of EhVEGs with erythrocytes, whereas in (B) shows the secreted purified EhVEGs and placed in cultivation with human erythrocytes in which the EhVEGs collision with erythrocytes is promoted (the EhVEGs obtained from supernatant without ZnS0 4 were pilled and placed in TYI-S- 33 medium to add then human erythrocytes and
  • FIG. 4 shows the effect of purified EhVEGs on erythrocytes, which showed agglutinating (b) and lytic (c) capacity.
  • Figure 5 shows representative confocal microscopy images of the production, secretion, and purification of EhVEGs.
  • EhVEGs In the present invention, at 2 hours of induction with FN or erythrocyte lysate, most trophozoites produce an EhVEG, which is associated with the outer membrane ( Figure 6) or is released into the culture medium ( Figure 7).
  • Figure 8 shows images displaying the production of the EhVEGs of the invention with the three methods of induction and/or production described as embodiments of the invention, and the phenomenon of erytrophagocytosis of induced trophozoites in which the labeling with the Anti-His- EhLMW-PTPs antibodies surround (coat) the erythrocytes.
  • EhLMW-PTPs This labeling of EhLMW-PTPs is more intense in EhVEGs associated with the outer membrane of induced trophozoites than in any other subcellular region as well as in the purified EhVEGs, as shown in Figure 9.
  • the protein pattern of the EhVEGs observed by SDS-PAGE depends from the origin of them, either associated with the trophozoite outer membrane or purified from the culture medium with high or low concentrations of bovine serum as shown in Figure 1 1.
  • the EhLMW-PTPs are enriched in the induced and purified EhVEGs according to the present invention ( Figure 12), as well as the adhesin EhCPADH and the BEhFNR receptor for fibronectin.
  • FIG 16 show that in EhVEG extracts, EhLMW-PTPs are susceptible to strong degradation (A) being proportional as time passes even in the presence of cOmpletTM 2x protease inhibitor cocktail (EDTA-Free, Roche).
  • EhLMW-PTPs are associated with a large amount of proteins so it is necessary to properly denature and boil the extracts for 8 min to avoid irregular electrophoretic shift as observed in the immunoblot in lanes 1-3 (B).
  • the protective effect of the immune response induced by them against the development of amebic hepatic abscess (AHA) in the hamster model was analyzed.
  • hamsters were immunized 3 times with 50 ⁇ g doses of denatured EhVEGs (heat inactivated) and were then inoculated 1x10 6 trophozoites intrahepatically as described in Example 1 1 (no adjuvant was used).
  • Immunized hamsters (13) showed a 70% reduction in AHA development when compared to animals that were not immunized ( Figure 19).
  • Clathrin heavy chain partial 1 1 .95 BAE94781 ; Gl: 103484580 193.03
  • Peripheral membrane protein 4.5 AAA291 10.1 ; Gl :158967 31 .17
  • EhVEGs induce a protective immune response with the development of antibodies when they are inoculated in laboratory animals (vaccine) without an adjuvant.
  • vaccine laboratory animals
  • the EhVEGs are an ideal candidate for the development of a vaccine against amoebiasis (intestinal and hepatic).
  • the present invention provides a method for obtaining and isolating EhVEGs quantifying them and determining their size (1-4 ⁇ ). We verified that these are vesicles that can be induced and obtained by the methods described herein (to a lesser extent EhVEGs of 0.5-1 ⁇ in diameter are also observed by confocal microscopy).
  • EhVEGs can be obtained by extraction with detergents (EhVEGs associated with trophozoites) and without detergents (EhVEGs, which have been secreted).
  • EhVEGs or other similar vesicles Prior to the present invention, EhVEGs or other similar vesicles have not been reported to be secreted by trophozoites from E. histolytica or from other protozoa such as G. lamblia or T. cruzi (although for T. cruzi, the diameter of VEs is commonly less than 0.5 ⁇ ) are known, and their existence was unknown until prior to the present invention (under non-apoptotic conditions).
  • VEGs as vaccines against infectious diseases caused by the pathogen agent from which they are derived or obtained (protozoan of clinical importance).
  • inactivated EhVEGs may be potential candidates for vaccines due to the immune response that they induced in animal models, for example, as shown in the results we obtained in the amebic hepatic abscess (AHA) model in hamsters immunized with them, where their use caused the significant decrease of the AHA.
  • AHA amebic hepatic abscess
  • Vaccine pharmaceutical compositions comprising the giant extracellular vesicles (VEGs) of the invention or extracts derived from them, from pathogenic protozoa including, for example, Entamoeba histolytica, Giardia lamblia, and Trypanosoma cruzi are also embodiments of the invention; the pharmaceutically acceptable excipients and vehicles are also considered within such embodiments.
  • VEGs giant extracellular vesicles
  • EhVEGs and VEGs are not the product of an apoptosis event.
  • the large size of the EhVEGs and VEGs of the invention facilitates the obtaining of a good amount of their proteins, vesicular components, and their observation by light microscopy to identify their presence during the process.
  • E. histolytica trophozoites during the induction and secretion of EhVEGs was evaluated, resulting in more than 90% during the 20-120 min of incubation with FN, blood cell film, and in erytrophagocytosis events. The viability was also evaluated 8h after the induction trials started, and it was also more than 90%.
  • the integrity of the nuclei was observed with DAPI by confocal microscopy of 20 to 180 min to broadly confirm their good condition. The results of the nuclei observation by confocal microscopy are shown in Figures 5 and 8. Based on what was found in E.
  • the present invention was tested in other protozoan parasites to determine if the induction and secretion process of VEGs is also present.
  • extracellular vesicles were obtained with the parasites Giardia lamblia and Trypanosoma cruzi. These species were selected with the intention of covering a wide spectrum of species where secretion of giant VEs could be conserved, and especially the clinical interest species as pathogenic protozoa.
  • Giardia Some species of Giardia can produce secretory vesicles during processes involved with the encystment stage, showing dimensions smaller than 1000 nm [82-84]. Additionally, in the Giardia culture supernatant, proteins have been found that suggest the presence of VEs similar to exosomes [39,85], of which no images have yet been found in the literature because the massive induction of secretion and purification of these has not yet been resolved.
  • T. cruzi parasite epimastigotes which were cultured for 30-120 min in LIT (Liver Infusion Triptose) medium on glass coverslips, coated with 2.5-5.2 ⁇ g doses of FN per cm 2 of surface, large VEs were obtained, which could be observed by confocal microscopy using anti-His-EhLMW-PTP polyclonal antibodies ( Figure 22). Trypanosoma cruzi does not theoretically have a protein that complies with the enzymatic function of LMW-PTP, and we did not find a protein with the homologous enzymatic function when searching in "Blast" software, taking as references the sequences EhLMW-PTPs.
  • LIT Liver Infusion Triptose
  • the embodiment of the method of the present invention for inducing VEs with the FN film is usable for the production of large vesicles of other protozoan parasites, where it is further required to adjust the culture medium of the parasite to be studied.
  • the culture medium was TYI-S-33 supplemented with bile
  • E. histolytica the medium used was TYI-S-33
  • T. cruzi the medium was LIT.
  • Table 3 shows the diameters or evidences reported in the literature up to the present invention of E. histolytica, T. cruzi, and Giardia lamblia VEs.
  • VES volume and mass obtained from the VES
  • TYI-S-33 easily filtered with 220 nm pore filters (0.22 micron) detected by
  • VEs of different dimensions have been observed by diverse techniques with commonly observed dimensions of between 20 nm to approximately 500 nm; however, VEs were found within the classification of
  • Example 1 Induction and/or production of immunogenic giant extracellular vesicles of Entamoeba histolytica.
  • trophozoites of the strain E. histolytica HM1 :IMSS considered as international reference, with which the AHA can be reproduced in an in vivo model in hamsters.
  • the trophozoites were cultivated under axenic conditions in TYI-S-33 medium [76,89,90] and were harvested during the log phase of growth by centrifugation at 200 x g.
  • the cell pack was washed several times with PBS pH 7.4 or with TYI-S- 33 medium.
  • the culture surface is coated with a FN "film" with con 0.5-10 ⁇ g de FN per cm 2 where the trophozoites will be cultivated, preferably with 2.5-5.2 ⁇ g of FN per cm 2 , either the culture in plastic or glass containers, such as plastic or glass Petri dishes of 10 cm in diameters and sterile coverslips of 20x20 mm, preferably drying the FN during the coating of the surface under a continuous flow of air and then placing them under UV light during 10 min for sterilization.
  • the vesicles can be observed without difficulty from around 60 min from starting the induction, with a good yield to perform their extraction in about 2 hours after induction and in continuous hours.
  • Human fibronectin used to carry out the present invention can be purified by the modified gelatin- Sepharose column affinity chromatography method [75,77] and quantified using a molar extinction coefficient of 1 .28 at 280 nm for 1 mg/ml.
  • a coating or "film” For inducing the formation and secretion of EhVEGs with the use of the preparation of fragments of human blood cells, it is necessary to prepare a coating or "film". It is important to maintain the proportion of erythrocytes as they will be sonicated and will be placed in relation to each cm 2 ; a simple way of preparing the surface coating comprises obtaining blood via the bloodstream and immediately placing it in sterile PBS 1x (e.g., 40 ⁇ of blood in 30-50 ml of 1x PBS), homogenize, place on ice, quantify the erythrocytes, sonicate such erythrocytes in PBS for about 10 sec (in an "ultrasonic processor” equipment is recommended at amplitude of 90), spread on a plastic or glass surface 1x10 3 -5x10 3 of the sonicated erythrocytes per cm 2 , preferably dry under a continuous flow of air, and place for 10 min under UV light.
  • PBS 1x e
  • EhVEGs For inducing the formation, secretion, and purification of EhVEGs with the use of complete human erythrocytes, 5-100 erythrocytes per trophozoite will be required to be first cultured in TYI-S-33 medium, removing the erythrocytes that did not interact with trophozoites at 30-60 min after starting the culture (those that were not phagocytosed and are free in the medium.
  • Example 2 Variant method I. Induction of EhVEGs with FN. For obtaining the EhVEGs, the following steps were performed:
  • the culture will be maintained at 37°C. Time will start counting immediately after the seeding of trophozoites is finished;
  • Example 7 Preferably at 120 min after starting the induction of cultures at 37°C, place the culture boxes on a temperature-controlled orbital shaker such as MaxQTM 4000 (Thermo ScientificTM MaxQTM) at 50 rpm, 37°C for 20 sec. Collect the supernatant medium already containing EhVEGs with some contaminants such as trophozoites that were not stably adhered, and place it in a 50 ml conical tube on ice.
  • Example 3 Variant method II. Induction of EhVEGs on coated surfaces (film) of fragments of blood cells. For obtaining the EhVEGs, the following steps were performed:
  • Example 4 Purification of the EhVEGs produced after induction either with FN (variant method I) or by culturing on coated surfaces (film) of blood cells (variant method II). Obtaining the EhVEGs from the supernatant medium and those associated with the outer membrane of trophozoites can be performed according to the following steps, which are subsequently carried out to obtaining the EhVEGs:
  • EhVEGs should be enriched by centrifuging at low speed, it will be added to the collected the necessary amount of a 25 mM solution of ZnS0 4 in PBS, to have a final concentration of 2-5 mM of ZnS0 4 . Homogenize by inversion about 10 times. Place the tube on ice for 10-15 min then centrifuge at 2000 x g in a fixed-angle rotor (also 500-800 x g in a tilt rotor) for approximately 10 min. A large quantity of EhVEGs of different sizes will be obtained in the pellet.
  • the supernatant with EhVEGs is filtered through 5 ⁇ pore filters before adding the ZnS0 4 . If you want to obtain a pellet of EhVEGs without using ZnS0 4 , the supernatant should be centrifuged at more than 10,000 x g for 15-20 min.
  • the ZnS0 4 favors the agglomeration of EhVEGs, which is useful in several circumstances, and to precipitate them more easily.
  • Samples stored at this stage of the procedure should rapidly resuspend for the required EhVEGs in 2-4% p-formaldehyde in PBS; incubate for 20 min in ice and then allow the desired amount to dry on the lamellae or glass coverslips to continue the immunofluorescence process.
  • a button will correspond mainly to trophozoites that did not stably adhere to the culture boxes, which will carry a certain amount of EhVEGs (were possibly associated with the trophozoites and disassociated themselves from them during the process); this and the phase of 50% sucrose will be discarded if it is not desired to be analyzed. Homogenize the collected by gently inverting about 10 times (it will contain EhVEGs and some contaminants such as small size trophozoites with less density to those previously discarded). If necessary, filter the collected in a 5 ⁇ pore filter.
  • EhVEGs To the collected with EhVEGs (dilute these with PBS up to ensure a sucrose percentage less than 20%) will be added ZnS0 4 in PBS to have a final concentration of 2-5 mM ZnS0 4 if it is desired to precipitate them easily. Homogenize by inversion about 10 times. Place the tube on ice for 10-15 min. Centrifuge at 2000 x g for about 10 min. A large amount of EhVEGs will be obtained in the pellet. Gradient centrifugations remove the contaminating trophozoites. 8.2. Purification of EhVEGs associated with the outer membrane of E. histolytica trophozoites with gradient solutions of SDS-Triton in PBS.
  • Solution 1 (SDS 0.0015% + Triton X100 0.0006%, in PBS) at 26-28°C for 10 min, place at the orbital shaker at 50 rpm, 26-28°C for 20 sec; recover the supernatant of Solution 1 SDS-Triton X100 in a conical tube of 15 ml and place it on ice.
  • loading buffer optionally placing 1 % formaldehyde in final concentration
  • boiling for 8 min in bain-marie cool at room temperature in water wash, place on ice, add 4-8 ⁇ of ⁇ - mercaptoethanol, 14.3 M per 100 ⁇ of sample; equilibrate with NaOH to obtain a pH of approximately 6.8, distinguishable by the blue color indicator of the loading buffer and immediately charge the SDS-PAGE gel.
  • Example 5 Variant method III. Induction and purification of EhVEGs by phagocytosis (erythrophagocytosis). According to this variant method, the following steps were performed to obtain the EhVEGs:
  • erythrocytes will be removed, performing smooth orbital movements for 10 sec to decant the supernatant; then, 2 washes are carried out with 8 ml per approximately each 78 cm 2 of area with TYI-S-33 S/SB medium at 37°C; move smoothly with orbital movements for 15 sec, decant the medium, and collect the reminder with a Pasteur pipette from the walls of the culture box trying to do it quickly. 7. Add 15-20 ml of TYI-S-33 medium per each 78 cm 2 to continue the culture, coating the surfaces well.
  • Obtaining and purifying the EhVEGs from the supernatant medium and those associated with the outer membrane of trophozoites can be performed as described in Example 4, taking care to filter with 5 ⁇ pore filters after being obtained from the supernatant, and to do the washing of EhVEGs to decrease the concentration of the detergents used.
  • Example 6 Isolation of AHA trophozoites.
  • AHA amebic hepatic abscess
  • 1 *10 6 trophozoites were injected into a volume of 100 ⁇ in the right hepatic lobe of male hamsters (Mesocricetus auratus) as has been reported [75,91 ,92].
  • the animals were sacrificed 7 days after inoculation, and the livers were completely removed under sterile conditions. Subsequently, the areas covering the lesion were cut into slices, transferred to TYI-S-33 medium, and the recovered trophozoites were incubated in culture tubes under anaerobic conditions for 14 days. Log-phase growth trophozoites were harvested for use in later assays.
  • Example 7 Cloning and expression of His-EhLMW-PTPs (His-EhLMW-PTPs, (His-EhLMW- PTP1 and His-Ehl_MW-PTP2) recombinant proteins.
  • Ehl_MW-PTP1 genes GenBank XM_651267 encoding the XP_656359 protein
  • Ehl_MW-PTP2 GenBank XM_648265 encoding the XP_653357 protein
  • BamH1 5'- CGGATCCATGAAGTTGTTGTTTGTATGTTTAGGC-3'
  • the LMWR antisense designed to contain the recognized restriction site by EcoR1 enzyme (5'- CAGGAATTCTTAATTAATAAGTTTTCCTTCTTCTAG-3').
  • the PCR product was cloned into CR®4-TOPO plasmid. Plasmids with the inserts were used to transform E. coli DH5aTM bacteria. Colonies were selected for growth in Luria Bertani medium. By restriction with the enzymes EcoR1 , BamH1 and BsRD1 , the plasmid was determined with the insert corresponding to each culture of bacteria. After identifying the plasmids with the inserts corresponding to Ehl_MW-PTP1 and Ehl_MW-PTP2, bacterial transformation assays were performed with E.
  • coli TOPI O'F the inserts were released with EcoR1 and BamH1 , and subcloned into plasmids pRSET A (InvitrogenTM), which adds an amino acid sequence containing a 6 histidine (His) tag.
  • the inserts were subjected to automated sequencing using the Taq Fluorescence-Based Dye Terminator Cycle Sequencing kit (Perkin-Elmer/Applied Biosystems 377 - DNA sequencer 18Eln), by which the sequences were checked.
  • E. coli cells strains BL21 (DE3) pLysS were transformed with the recombinant expression vectors containing the mentioned inserts, inoculated into LB medium containing 100 mg/l ampicillin (Sigma), and incubated at 37°C. Protein production was induced by addition of isopropyl ⁇ -thiogalactoside (IPTG) (1 mM) for 150 min.
  • IPTG isopropyl ⁇ -thiogalactoside
  • Example 8 Western blot.
  • the purified His-EhLMW-PTPs recombinant proteins, the purified trophozoites lysates, and the extract of purified EhVEGs were analyzed on 12-15% SDS-PAGE gels. A duplicate of the samples was visualized by Coomassie blue or silver staining. The proteins on the SDS-PAGE gels were transferred by electrophoresis onto a sheet of nitrocellulose paper as described in the literature [93].
  • WB histolytica amoebae (1 :300), EhVEGs-vaccinated mouse serum in complete Freund adjuvant (1 :300), vaccinated-EhVEGs mouse serum (1 :300), and sera from patients from Colombia and Mexico who suffered from amoebiasis (1 :300).
  • WB was developed with NBT/BCIP (tetrazolium nitro-blue and 5-bromo-4-chloro-3'-indolyphosphate, Sigma).
  • Example 9 Immunization protocol of mice for obtaining anti-His-EhLMW-PTPs serum. 8-10 week old Balb/C mice housed in controlled microenvironments were managed according to Cinvestav institutional guidelines for animal care. A pre-immune serum was obtained. Eight mice were treated with recombinant EhLMW-PTPs protein once weekly for 4 weeks (10-15 ⁇ g per dose of each mouse, intraperitoneally). The first immunization of each mouse was performed with Freund's complete adjuvant emulsion (Sigma), and the following immunizations with incomplete Freund's adjuvant (Sigma). Blood was drawn to obtain immune serum within six weeks of the first immunization, and every subsequent month.
  • the main purpose of the serum obtained with anti-His- EhLMW-PTP polyclonal antibodies in the present invention is to detect the considerable amount of EhLMW-PTPs that exist and which we found within the EhVEGs of the invention, which allows to identify them, since the labeling of EhLMW-PTPs in any other area of the size of the EhVEGs in the body of the trophozoites is much smaller.
  • Example 10 Immunization protocol of mice with EhVEGs. 8-10 week old Balb/C mice housed in controlled microenvironments were managed according to Cinvestav institutional guidelines for care. A pre-immune serum was obtained. Three mice for each group were treated; the groups were those immunized with EhVEGs (group 1), immunized with EhVEGs-Complete Freund Adjuvant (group 2), and immunized with total extract of trophozoites (group 3). Immunizations were performed on days 0, 7 and 15 with approximately 50 ⁇ g of EhVEGs extract boiled for 8 min in 100 ⁇ of PBS per dose, intraperitoneal ⁇ , while 100 ⁇ g per dose were used with the total extract of trophozoites. Blood was drawn to obtain immune serum within 40 days of the first immunization. The main purpose of these immune sera was to know the immunogenic property of the EhVEGs of the invention, seen in the banding pattern recognized in the EhVEGs extracts.
  • Example 11 Sham hamster (Mesocricetus auratus) immunization protocol with EhVEGs and AHA challenge. 4-5 week old golden Syrian hamsters (Mesocricetus auratus) (CINVESTAV, Mexico) were used. The animals were housed in controlled microenvironments and were managed according to the institutional guidelines for animal care (adhered to the Mexican norm NOM-062- ZOO-1999). A pre-immune serum was obtained. Two groups of 6 animals each were formed. The groups were those immunized with EhVEGs (group 1) and those inoculated with sterile PBS (group 2).
  • Immunizations were performed on days 0, 16 and 32 with approximately 50 ⁇ g of EhVEGs extract boiled for 8 min in 100 ⁇ of PBS per dose, injecting via intraperitoneally; for the controls was inoculated 100 ⁇ of PBS on the same days. Blood was drawn to obtain immune serum within 40 days of the first immunization.
  • the hamsters were challenged with 1x10 6 virulent trophozoites of E. histolytica, by injection into the right lobe of the liver.
  • At 7 days were sacrificed, and analyzed the percentage by weight of AHA with respect to the total weight of the liver with AHA.
  • the main purpose of the experiment was to know the protective effect of vaccinating hamsters with denatured EhVEGs.
  • Example 12 Secretion of EhVEGs in real-time, erythrocyte interaction with trophozoites- EhVEGs and isolated EhVEGs.
  • the procedure was performed in 10 cm diameter plastic Petri dishes following the same procedures of Example 2 up to step 5 and of Example 3.
  • the secretion of the EhVEGs was observed; photographs were taken and videos shot at different times. Videos were mainly shot at 1 :50:00 hours after starting the cultivation. Between 2:00:00 and 2:05:00 hours, 1-20 human erythrocytes were added for each trophozoite and video was shot to choose the moment when several erythrocytes settle and interact with the trophozoites-EhVEGs.
  • EhVEGs purified secreted EhVEGs were taken and placed in PBS or TYI-S-33, and human erythrocytes were added; the collision of the EhVEGs with human erythrocytes was followed by video.
  • EhVEGs were used and incubated in 4.5 x 1 .2 x 1.0 cm plastic cuvettes with TYI-S-33 medium (the presence of EhVEGs was tested by observing it in an inverted microscope and by analysis in confocal microscopy); subsequently, approximately 1-10 human erythrocytes were added for each EhVEG; this was incubated for 20 min at 20-26°C and then placed in a 12 h incubation at a tilt of 15°-20° into a refrigerator (REB800A Nieto, Metalfrio) at 4°C; the EhVEGs cuvettes were subsequently placed vs.
  • a refrigerator REB800A Nieto, Metalfrio
  • erythrocytes at 28°C for 5 hrs.
  • erythrocytes were incubated alone in TYI-S-33 medium or in PBS and photographs were taken at different times of the EhVEGs vs. erythrocytes experiment.
  • Example 13 Immunofluorescence. E. histolytica trophozoites adhered at a 37°C on glass coverslips were cultured at different times and in about 120 min under different conditions to induce the secretion of EhVEGs per cm 2 , as described above or only in glass. The times to be analyzed were fixed with 2-4% p-formaldehyde in PBS. The fixed parasites were not permeabilized to observe the EhVEGs on the surface of the trophozoites. To observe the recognition of proteins in the cytoplasm of the cells and VEGs in the plasmatic membrane, permeabilization at low concentrations of detergents (SDS 0.0015-0.005% + Triton X100 0.0006-0.002%, in PBS) for 3-10 min were used.
  • Blocking was performed with 5% bovine serum in PBS for 1 h. Incubation with mouse serum anti- His-EhLMW-PTPs and rabbit pAb anti-EhCPADH1 12 was for 1 h. Secondary antibodies were goat anti-mouse IgG coupled to FITC (fluorescein isothiocyanate) (ZIMED), goat anti-rabbit IgG coupled to FITC (ZIMED), and Alexa 594 coupled to goat anti-mouse IgG (Molecular Probes). Slides were washed with abundant PBS after correspondingly incubating each series of antibodies.
  • FITC fluorescein isothiocyanate
  • ZIMED goat anti-rabbit IgG coupled to FITC
  • Alexa 594 coupled to goat anti-mouse IgG
  • the films were mounted in Vectashield solution with or without DAPI [95] on coverslips for further analysis by fluorescence microscopy and analysis in Confocal ZEISS LSM 700.
  • the distribution of F-Actin during the formation of the EhVEGs was taken as the highest level of emission captured at the maximum emission obtained from the actin filaments in the trophozoites (to avoid sweeping), falloidin-rhodamine (1 :50 dilution) [96] was used incubating for 1-120 min.
  • For immunofluorescence of EhVEGs 35,000-70,000 purified vesicles were placed in coverslips and allowed to dry to subsequently follow the entire immunofluorescence process as described, being careful of the washes.
  • the eluted fractions were automatically mixed with a solution of 5 mg/ml a-cyano-4-hidroxycinnamic acid (CHCA) in 50% ACN with 0.1 % TFA as matrix and dripped ("spotted") into a 384-well MALDI stainless steel plate using MALDI Ekspot (Eksigent, Redwood City CA) at a rate of 30s per spot with a matrix flow of 1 .6 L/min.
  • the generated spots were analyzed on a MALDI-TOF/TOF 4800 plus mass spectrometer (AB Sciex, Framingham MA). Each MS spectrum was acquired by accumulating 1000 shots in a mass range of 850-4000 Da with a laser intensity of 3100.
  • MS/MS spectra of the selected precursors were fragmented by activating the Collision Induced Dissociation (CID) cell, and acquired by a cumulative of 3000 shots with a laser intensity of 3800.
  • CID Collision Induced Dissociation
  • the generated MS/MS spectra were compared through Protein Pilot v. 2.0.1 software (AB Sciex, Framingham MA) against Entamoeba histolytica protein data base (downloaded from NCBI, 48060 protein sequences) using the Paragon algorithm.
  • the search parameters were modification in the cysteines by iodoacetamide (carbamidomethylcysteine), trypsin cutting enzyme and all the biological modifications set by the algorithm.
  • the Unused Score was set at 1.3 to acquire 95% confidence.
  • the identified proteins were grouped by ProGroup algorithm in the software to minimize redundancies. Protein identification was performed in the LaNSE-CINVESTAV Genomic, Proteomic and Metabolic Unit.
  • Example 15 Preparation of erythrocytes for challenge with trophozoites and EhVEGs.
  • Human erythrocytes were obtained from venipuncture or finger puncture using sterile syringe and around 5- 10 ⁇ of blood was immediately placed for every 1 ml of PBS. The erythrocytes were then washed three times with PBS by centrifugation at 180-200 x g and resuspended in TYI-S-33 medium without serum or in PBS as needed. The concentration was counted. In all experiments, fresh erythrocytes were used for their interaction with trophozoites.
  • Example 16 Induction and purification of VEs-VEGs of Giardia lamblia.
  • the induction method of VEs-VEGs for Giardia lamblia was designed taking reference and adapting the method designed for the induction and secretion of EhVEGs.
  • the culture medium was TYI-S-33 with bile and with or without bovine serum. The procedure was followed similarly to Example 2. In 3.5 cm diameter Petri dishes, a film of 2.5-5.2 ⁇ g FN per cm 2 was placed, then 2 ml of TYI-S-33 medium with bile was placed to pre-incubate for 15-20 min.
  • Giardia lamblia cultures were placed on ice for about 10 min; the supernatant was centrifuged at 180-200 x g for 5 min; the supernatant was discarded, and TYI- S-33 medium was added to the pellet with the trophozoites, to seed them subsequently in culture boxes pre-incubated with the FN film; this was subsequently incubated at different times, including a stimulation at 120 min to detect the supernatants; the trophozoites were fixed with p-formaldehyde for fluorescence analysis and/or protein extraction.
  • the supernatant of the stimulated parasites was centrifuged for 5 min at 180 x g to precipitate non- adhered parasites and discard them; if only need to enrich the VEs-VEGs centrifuging at low speed, this will be added to the collection from a 25 mM solution ZnS0 4 in PBS, as needed to reach a final concentration of 2-5 mM of ZnS0 4 ; to homogenize, place the tube on ice for 10-15 min and then centrifuge at 2000-4000 x g for approximately 10 min. A large amount of VEs-VEGs of different sizes will be obtained from the pellet.
  • the supernatant should be centrifuged at more than 15,000 x g for 15-20 min. Samples were analyzed by SDS-PAGE and Western blot.
  • the trophozoites fixed in the FN-induced culture surfaces can be treated with the method of Example 4 (step 8.2) using SDS-Triton solutions in PBS, to obtain samples and analyze them by microscopy, fluorescence, SDS-PAGE and Western blot.
  • Example 17 Induction and purification of VEs-VEGs of Trypanosoma cruzi.
  • the induction and production method of Trypanosoma cruzi of VEs-VEGs was designed by taking reference and adapting the one designed for the induction and secretion of EhVEGs with FN.
  • the culture medium used was LIT with or without bovine serum, following the procedure similarly to Example 2.
  • 2.5-5.2 ⁇ g of FN per cm 2 and then 2 ml LIT medium (to coat the surfaces) were placed in 24-well plastic cell culture plates, 3.5 diameter Petri dishes or glass surfaces to pre-incubate for 15-20 min.
  • the epimastigote cultures of Trypanosoma cruzi were resuspended and then centrifuged for 200-1000 x g for 5-10 min; the supernatant was discarded, and the pellet with the parasites was added LIT medium to resuspend them and subsequently seed them into the culture sites pre-incubated with FN film for subsequent incubation at different times, including 20-120 min stimulation to collect the supernatants, and the parasites were fixed with 2% p-formaldehyde for fluorescence analysis.
  • the supernatant of the stimulated parasites was centrifuged for 5-10 min at 500-1000 x g to precipitate not adhered parasites and discard them to enrich the VEs-VEG-s by centrifugation at low speed; a 25 mM solution of ZnS0 4 in PBS was added to the collected to reach a final concentration of 2-5 mM of ZnS0 4 to homogenize, placing it on ice for 10-15 min and then centrifuged at 4,000- 10,000 x g for about 10 min. A large quantity of VEs-VEGs of different sizes were obtained in the pellet. Samples were analyzed by SDS-PAGE, Western blot and confocal microscopy.
  • Parasites recovered in FN-stimulated supernatants can be treated with SDS-Triton solutions to promote the dissociation of VEGs-VEs in the membrane.
  • Epimastigotes induced with FN and centrifuged at 1000 x g, gradually placing them in SDS-Triton solutions in PBS the pellet was placed in Solution 1 (SDS 0.0015% + Triton X100 0.0006%, in PBS) for 10 min and then centrifuged at 1000 x g, later the supernatant was stored and the pellet was then placed in Solution 2 (SDS 0.01 % + Triton X100 0.004%, in PBS), and subsequently in Solution 3 (SDS 0.25% + Triton X100 0.01 %, in PBS) performing the same procedure as in Solution 1.
  • Example 18 Immunofluorescence of Giardia lamblia and Trypanosoma cruzi. Cultures of Giardia lamblia trophozoites (TYI-S-33 medium with bile) and cultures of trophozoites and epimastigotes of Trypanosoma cruzi (LIT medium), were cultured at 37°C in glass coverslips at different times (between 30, 60 and 120 min) with adequate doses of the FN film to induce the secretion of VEs and VEGs.
  • the times to be analyzed were fixed with 2-4% p-formaldehyde in PBS.
  • the Giardia lamblia trophozoites adhere well to glass coverslips (and to plastic surfaces); however, the Trypanosoma cruzi epimastigotes do not adhere strongly so the medium was carefully removed from the sides after incubating the fixative and then let the samples dry for 5 min.
  • the fixed parasites were not permeabilized to observe the VEs-VEGs on the surface.

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Abstract

La présente invention concerne des vésicules (extracellulaires) sécrétées géantes (appelées VEG) qui sont associées à des espèces de protozoaires parasites, qui ont des dimensions de 500 à 400 nm de diamètre, et des procédés pour permettre leur induction, leur sécrétion et leur purification. Ces vésicules possèdent une activité protéolytique et sont composées d'une grande quantité de protéines; en particulier, elles ont été obtenues à partir de trophozoïtes d'Entamoeba histolytica (appelés EhVEG) et d'autres protozoaires parasites, tels que Giardia lamblia et Trypanosoma cruzi. L'invention concerne également l'utilisation de telles vésicules, par exemple des EhVEG, pour immuniser des mammifères susceptibles d'être infectés par Entamoeba histolytica. La présente invention concerne en outre des compositions pharmaceutiques immunogènes vaccinales efficaces comprenant les VEG induites et isolées, par exemple des EhVEG, qui sont utiles pour le traitement et la prévention de l'abcès hépatique amibienne. La présente invention représente le premier rapport qui démontre et/ou vérifie que E histolytica sécrète des vésicules extracellulaires de grandes dimensions dans le milieu de culture.
PCT/IB2017/056917 2016-11-11 2017-11-06 Vésicules extracellulaires géantes immunogènes (veg) de protozoaires parasites et procédés pour les induire et les purifier WO2018087644A2 (fr)

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