BRPI1005909A2 - attenuated transgenic strain of trypanosoma cruzi as a vaccine vector - Google Patents

Abstract

attenuated transgenic strain of trypanosoma cruzi as a vaccine vector. The present invention describes an attenuated transgenic trypanosoma cruzi strain and its use as a vaccine vector. In addition, a method of transforming attenuated trypanosoma cruzi strain with exogenous genes to obtain this vaccine vector is described. This vaccination strategy can be used to treat diseases that require induction of a cell-mediated immune response, such as cancer and diseases caused by intracellular microorganisms such as toxoplasmosis, malaria, tuberculosis, among others.

Description

Attenuated TRYPANOSOMA CRUZI TRANSGENIC CEPA AS A VACCINAL VECTOR The present invention describes an attenuated transgenic strain of Trypanosoma cruzi and its use as a vaccine vector. In addition, a method of transforming attenuated Trypanosoma cruzi strain with exogenous genes to obtain this vaccine vector is described. This vaccination strategy can be used to treat diseases that require induction of a cell-mediated immune response such as cancer and diseases caused by intracellular microorganisms such as toxoplasmosis, malaria, tuberculosis and others.

In the prior art, some patent documents demonstrate the use of attenuated Trypanosoma cruzi strains as a vaccine: US 2005244437 - Live antenuated parasite vaccine; WO 8303199 - Method for obtaining reduced infectivity variants from protozoan parasites, thus obtained variants and utilization thereof; BE 892553 - Protozoal strains of reduced infectivity prepared by treating virulent strain with mutagen, useful as vaccine preparation. However, these strains are not transgenic, ie, do not have exogenous DNA, nor are they used for prophylaxis or therapy of diseases other than that caused by the wild parasite itself. Moreover, the methodology they use for strain attenuation is not used in the present invention.

Given the increasing incidence of cancer worldwide and the few preventive and even therapeutic medical interventions available, it is necessary to develop alternative therapies capable of preventing and curing cancer.

In the late 1940s, studies using live Trypanosoma cruzi or its lysed extract demonstrated that this protozoan has a tumoricidal effect (Roskin Gl. 1946 Toxin therapy of experimental cancer. Cancer Res. 6: 363-5; Hauschka TS, Goodwin MB. 1948 Trypanosoma cruzi endotoxin (KR) in the treatment of malignant mouse tumors (Science, 107: 600-2.). Recently, Batmonkh and colleagues (Batmonkh Z, Kallinikova VD, Pakhorukova LV, Kravtsov EG, Karpenko LP, Dalin MV. In vivo anticancer activity of trypanosoma cruzi lysates of different genetic groups. Bull Exp Biol Med. 2006 Oct; 142 (4) : 470-3. English, Russian) have shown that Epimastigote lysate from different T. cruzi genetic groups (P209-1, Gambal, Sp104-1, MASu, Y7 / 1, MN12, CL-Brener, 86/2036, Y7 / 2-1) inhibits Erlich adenocarcinoma growth in rats. While the basis of this antitumor activity is still unknown, our group suggests that the initial activation of innate immune system cells by parasite components may contribute to the development of protective immunity against the parasite and other host aggressions. The possibility of using the T. cruzi parasite in cancer treatment opens a new perspective in the development of vaccine vectors. Combining the technology of transformation of Trypanosomatids with exogenous antigens (DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, Macedo AM, Vasquez MP, Levin MJ, Teixeira SMR 2004 Expression of exogenous genes in Trypanosoma cruzi. Parasitol Res., 92: 113-20) and the use of attenuated strains such as CL-14 (Lima MT, Jansen AM, Rondinelii E, Gattass CR. 1991 Trypanosoma cruzi: properties of a clone isolated from the Parasitol Res, 77: 77-81; Lima MT, Lenzi HL, Gattass CR 1995 Negative tissue parasitism in mice injected with a non-infective clone of Trypanosoma cruzi Parasitol Res, 81: 6-12) , the development of viable transgenic parasites becomes possible for use in vaccine strategies. The attenuation and less infectious phenotype of the T. cruzi CL-14 strain is probably due to the lower expression of glycoprotein 82 (gp82), which is responsible for the parasite interaction with host cell surface molecules and subsequent invasion. to host cell (Ruiz RC, Favoretto SJr, Dorta ML, Oshiro MEM, Ferreira AT, Manque PM, Yoshida N. 1998 Infectivity of Trypanosoma cruzi isolates is associated with differential expression of surface glycoproteins with differential Ca2 + signalling activity. Biochem. J. , 330: 505-11), making the cited strain four times less infectious than its parental CL (Atayde VD, Neira I, Cortez M, Ferreira D, Freymuller E, Yoshida N. 2004 Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14. Inter J. J. Parasitol., 34: 851-60.). Previous studies show that inoculation of CL-14 trypomastigote mice, rather than inducing disease, is able to induce protection against a subsequent challenge with the virulent CL strain, preventing parasitemia, mortality, and the development of Chagas disease-related symptoms. (Lima MT, Jansen AM, Rondinelli E, Gattass CR. 1991 Trypanosoma cruzi: properties of a clone isolated from the CL strain. Parasitol Res., 77: 77-81; Lima MT, Lenzi HL, Gattass CR. 1995 Negative tissue parasitism in mice injected with a non-infective clone of Trypanosoma cruzi. Parasitol. Res. 81: 6-12.). In addition, it was observed that mice immunized with CL-14 and later challenged with CL had IFN-γ, lgG1, lgG2a and lgG2b production (Pyhrro AS, Moraes JLC, LMT, Gattass CR, Trypanosoma cruzi 1998 Trypanosoma cruzi: lgG1 and lgG2 are the main immunoglobulins produced by vaccinated mice Parasitol Res., 84: 333-7; Soares MBP, Gonçalves R, Pyhrro AS, Costa DA, Paiva CN, Gattass CR 2003 Balanced cytokine-producing pattern in mice immunized with an avirulent Trypanosoma cruzi, Annals of the Brazilian Academy of Sciences, 75 (2): 167-72). Therefore, due to its low virulence and high immunogenicity, this strain has great potential to be used as a vaccine vector.

By inserting exogenous genes into the T. cruzi CL-14 genome, this vaccine vector will carry the antigen to the host cell, favoring MHC class I antigen presentation and CD8 + T lymphocyte activation, which are the cells. effector against intracellular pathogens and tumors.

Vaccine formulations currently being tested against tumors and other diseases have been made by combining protein antigens with TLR (Toll-like receptors) agonists as immunological adjuvants, which increases the ability to activate DCs (dendritic cells) and thus favor , the development of strong T cell and Th1 lymphocyte-mediated immunity (Wille-Reece U, Flynn BJ, Lore K, Koup RA, Kedl RM, Mattapallil JJ, Weiss WR, Roederer M, Seder RA. HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CP8 T cell responses in nonhuman primates (Proc. Natl. Acad. Sci. USA. 2005, 102: 15190-4).

The most promising protein antigens to induce tumor cell-specific immune response are cancer / testis antigens, a family of antigens that are not expressed in normal cells, with the exception of testis germ cells, which are not recognized by the immune system ( Kirkin, AF, Dzhandzhugazyan, K.N., and Zeuthen, J. (2002) Cancer / testis antigens: structural and immunobiological properties Cancer Invest 20, 222-236; Zendman, AJ, Ruiter, DJ, and van Muijen, GNP (2003) Cancer / testis-associated genes: Identification, expression profile, and putative function J. Cell Physiol 194, 272-288 The therapeutic potential of this group of tumor antigens has been demonstrated by several studies (van Pel, A Van der Bruggen, P., Coulie, PG, Brichard, VG, Lethé, B., Van den Eynde, BJ, Uyttenhove, C., Renauld, JC, and Boon, T. (1995). antigens recognized by cytolytic T. lymphocytes Immunol Rev. 14 5, 229-250 .; Thurner, B., Haendle, lt. Röder, C., Dieckmann, D., Keikavoussi, P., Jonuleit, H., Bender, A., Maczek, C., Schreiner, D., von den Driesch, P., Brocker, EB, Steinman, RM, Enk, A., Kampgen, E., and Schuler, G. (1999). Vaccination with Mage-3A1 peptide-pulsed mature, monocyte-derived dendritic cells expands specific cytotoxic T cells and induces regression of some metastases in advanced stage 1V melanoma. J. Exp. Med. 190, 1669-1678; Reynolds, S.R., Zeleniuch-Jacquotte, A., Shapiro, R.L., Roses, D.F., Harris, Μ. N., Johnston, D., and Bystryn, J. C. (2003). Vaccine-induced CD8 + T-cell responses to MAGE-3 correlate with clinical outeome in patients with melanoma. Clin. Cancer Res. 9, 657-662). Due to the high heterogeneity in the expression of the various members of this family, this group of antigens is still little used as an immunological target in relation to differentiation antigens or overexpressed antigens.

An anti-tumor vaccine formulation is disclosed in the present invention. capable of inducing anti-NY-ESO-1 immune response and host resistance against tumors expressing such antigen. This formulation involves the use of an attenuated Trypanosoma cruzi recombinant strain capable of expressing the tumor protein NY-ESO-1, a cancer-testis antigen that exhibits high spontaneous humoral and cellular immunogenicity. The NY-ESO-1 protein belongs to the cancer / testis family of tumor antigens and is known as one of the most immunogenic tumor antigens (Scalan MJ, et al, 2002 Identification of cancer / testis genes by database mining and mRNA expression analysis. Int Cancer 98: 485-92). NY-ESO-1 is found in most tumor types, but its expression varies from individual to individual. The frequency of expression in some tumor types, such as melanoma, lung, esophagus and synovial sarcomas, can be as high as 80% of patients (Gnjatic S, Nishikawa H, Jungbluth AA, Güre AO, Ritter G, Jiger E, Knuth A, Chen YT, Old L. NY-ESO-1: Review of an immunogenic tumor antigen (Adv Cancer Res. 2006; 95: 1-30). Its function in germ cells and tumor development remains unknown, as is that of most cancer / testis antigens. However, due to its high immunogenicity and distribution in various types of tumor tissue, NY-ESO-1 constitutes a strong antigen candidate to be applied to cancer immunotherapy.

In addition to the NY-ESO-1 antigen, the present invention also demonstrates the elaboration and characterization of transgenic parasites expressing the following proteins or protein combinations: MAGE-A3, CTSP-1, NY-ESO-1 + Thymidine kinase (TK), CTSP-1 + MAGE-A3 + NY-ESO-1 + TK. Like NY-ESO-1, MAGE-A3 and CTSP-1 proteins belong to the cancer / testis family. The idea of using different antigens on the same parasite is that this increases the chance of vaccination success, since each "cancer / testis" has different expression from individual to individual. Herpes simplex-derived thymidine kinase can be used in this system in combination with cancer / testis proteins or other protein antigens to function as a biosecurity gene. After tumor elimination, the patient administers a nucleoside analogous substance, such as guanosine found in the drug Acyclovir (Elion GB, Furman PA, Fyfe JA, Miranda P, Beauchamp L, Schaeffer HJ. Selectivity of action of an antiherpetic agent, 9- (2-hydroxyethoxymethyl) guanine Proc Natl Acad Sci USA 1977 Dec; 74 (12): 5716-20), Ganciclovir or other drugs with similar mechanisms of action which will eliminate all parasites. This drug can also be used to circumvent any overreaction of the immune system. US 2010092498 - "Anti-tumor vaccine derived from normal chemically modified cells" describes the use of a composition capable of inducing immune response in mammals comprising lymphoid cells in which the expression of cancer / testis tumor antigens is induced. The cancer / testis antigens used were MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, GAGE-3-7, NY-ESO-1 and BORIS. This document demonstrates a process for preparing normal cells capable of presenting antigens without the use of any external agent. However, using the patient's own cells makes treatment very expensive. The present invention describes the use of a recombinant strain of Trypanosoma cruzi capable of expressing cancer / testis proteins as a vaccine vector. An advantage of using T. cruzi as a vaccine vector when compared to the use of other cells, such as the one cited in the previous document, is that T. cruzi is produced in vitro on a large scale and can be inoculated in any patient. . In addition, recombinant strains are more stable and can be transported at room temperature. Finally, it is noteworthy that the administration of the vaccine of the present invention is done in few doses, which is less inconvenient for the patient.

In addition to cancer, the present invention demonstrates that the attenuated transgenic T. cruzi strain can also be used in the therapy or prophylaxis of any disease that requires induction of a Th1-type immune response, such as diseases caused by intracellular microorganisms (toxoplasmosis, malaria, tuberculosis, among others).

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - Monitoring of parasitemia and mortality of animals infected with CL-14 and CL Brener strains. (A) C57BL / 6 mice infected with metacyclic trypomastigote forms of the CL-14 and CL Brener strains were monitored for 30 days for parasitemia and mortality. (B) C57BL / 6 mice vaccinated with the CL-14 strain were challenged with the blood trypoosmigote form of the CL Brener strain. These were followed for 30 days for parasitemia and mortality.

Figure 2 - Western blot to detect expression of NY-ESO-1 protein by transgenic parasites.

Anti-NY-ESO-1 and anti-Histag Western blot. 105 parasites were used for each construct or control (0) E. coli expressing NY-ESO-1; (1) CL-14 WT; (2) CL-14 NY-ESO-1 His (+), (3) CL-14 NY-ESO-1 His (-); (4) CL-14 NY-ESO-1 gp63 concentrated 10X culture supernatant; (5) CL-14 NY-ESO-1 gp63.

Figure 3 - Expression of NY-ESO-1 protein in the three transgenic strains.

Confocal microscopy of CL-14 NY-ESO-1 His (-) transgenic parasites CL-14 NY-ESO-1 His (+), CL-14 NY-ESO-1 gp63, compared with CL-14 WT strain in infective forms metacyclic trypomastigote. Preparations using anti-NY-ESO-1 and anti-Histag antibodies were performed to distinguish constructs with or without histidine tail. In green the protein NY-ESO-1 is observed, in blue the nucleus stained with Dapi, in gray the transmitted image (Dic) and, finally, the overlap of the three images (Merge).

Figure 4 - Expression of NY-ESO-1 protein in the four phases of the parasite cell cycle.

Confocal microscopy of the CL-14 NY-ESO-1 His (+) transgenic parasite in epimastigote, metacyclic trypomastigote, extracellular amastigote, and culture trypomastigote forms. In green, NY-ESO-1 protein, in blue, Dapi-stained nucleus in gray, the transmitted image (Dic) and, finally, the overlap of the three images (Merge).

Figure 5 - Anti-NY-ESO-1 humoral response induced by vaccination with transgenic parasites In order to evaluate the production of anti-NY-ESO-1 antibodies induced by vaccination protocols with transgenic parasites or wild control, animal serum. Vaccines were subjected to total anti-IgG, IgG1 and IgG2c ELISA against recombinant NY-ESO-1 protein (A) and Western blot (B).

Figure 6 - Anti-NY-ESO-1 cell response induced by vaccination with transgenic parasites In order to evaluate IFN-γ production by splenocytes from animals vaccinated with transgenic parasites, an anti-INF-y ELISA was performed after in vitro stimulation with specific CD4 + and CD8 + T-NY-ESO-1 T peptides and TSKB-18 as a control of the anti-CL-14 response.

Figure 7 - Tumor growth follow-up after vaccination with transgenic parasites Animals vaccinated with two doses of 107 metacyclic trypomastigote parasites were challenged with 5 x 104 B16 melanoma cells expressing or not NY-ESO-1.

Figure 8 - Anti-NY-ESO-1 and anti-TSKB20 cell response in deficient animals Splenocytes from iNos (nitric oxide synthase) deficient animals, IL-12 (interleukin-12) or Myd88 (myeloid factor 88) and wild control C57BL / 6 who received immunization protocols on days 0 and 30 were compared for IFN-γ production by anti-INF-γ ELISA after restimulation with CD4 + and CD8 + NY-ESO-1- T peptides TSKB-18 as control of the anti-CL-14 response.

Figure 9 - Evaluation of the percentage of CD8 + and tetramer + cells for NY-ESO-1 and TSKB20 in wild and deficient animals.

Splenocytes from animals deficient for iNos (nitric oxide synthetase), IL-12 (interleukin-12) or Myd88 (myeloid factor 88) and vaccinated C57BL / 6 wild-type control were subjected to flow cytometry to assess the percentage of CD8 positive cells. as well as positive for CD8 + cell-specific NY-ESO-1 peptide. To this end, they were labeled with anti-CD3 PerCP, anti-CD8 FITC and NY-ESO-1 PE tetramer antibodies. The readings were taken in FACScalibur and analyzed in the FlowJo program.

Figure 10 - Western blot for analysis of NY-ESO-1 and HSV-1 TK expression by the transformed parasite. Cell lysate from the transformed parasites, or from wild type CL-14, was Western blotted for detection of HSK-1 and NY-ESO-1 TK. For this, NY-ESO-1 or TK HSV-1 specific monoclonal antibodies were used. For construct B2, which secretes the NY-ESO-1 antigen, 10X concentrated culture supernatant was used instead of cell lysate.

Figure 11 - Parasite death curve induced by Aciclovir administration. L6 cells were plated in 24-well plates and incubated at 37 ° C, 5% CO2 for one day. The next day, the cells were infected with 10 parasites / cell, these being in the metacyclic trypomastigote form of each construct or wild-type CL-14. Different concentrations of the drug Aciclovir were added to the culture medium. On days 5, 7 and 9 after infection, the culture supernatants were collected for parasite counting. Chart values represent the sum of the three counts.

DETAILED DESCRIPTION OF THE TECHNOLOGY EXAMPLE 1 - Method for Preparation of Attenuated Trypanosoma cruzi Transgenic Strain Aiming to use the CL-14 strain as a vaccine vector, initially C57BL / 6 mice and CD8_ / 'mice were infected intraperitoneally with 107 CL parasites. -14 or 5 x 105 CL Brener strain parasites in the metacyclic trypomastigote form for evaluation of parasitemia and mortality compared to the virulent CL Brener strain (Figure 1 A). The CL-14 strain is not observed to cause parasitemia and mortality in C57BL / 6 or CD8'7 'animals.

C57BL / 6 animals previously infected with 107 CL-14 strains were subjected 30 days after this vaccination to a challenge of 5 x 103 CL Brener parasites in the blood trypomastigote form to monitor parasitemia and mortality (Figure 1 B). Only vaccinated animals were able to control the parasitemia and prevent its mortality.

Once the low virulence of the CL-14 strain was established, transgenic strain preparation was then initiated as follows.

Parasitemia and mortality Animals infected with 7. cruzi strain CL-14 and strain CL Brener were monitored for the presence of blood trypomastigote parasites for 30 days. To this end, 5 pl of blood were taken from the tail of the animals and 50 fields were analyzed on a 20x20 microscopy slide with 40 X magnification. The value of the parasitemia was given by the Brener method, which multiplies the number of parasites by the factor. of microscope correction (Figure 1).

Cloning of protein antigen In order to generate Trypanosoma cruzi strain CL-14 capable of expressing the protein antigen of interest (NY-ESO-1, for example), the gene coding for this antigen is cloned into plasmid pRockneo ( DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, Macedo AM, VazquezMP, Levin MJ, Teixeira SM Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols Parasitol Res. 2004 Jan; 92 (2) : 113-20.).

In this example with NY-ESO-1 protein, three constructs were developed: (a) pRockNY-ESO-1 without histidine syrup for expression in the parasite cytoplasm (b) pRockNY-ESO-1 with histidine syrup for expression in the parasite cytoplasm (c) pRockNY-ESO-1 with glycoprotein 63 signal peptide (gp63) to direct secretion of the transgenic protein.

Trypanosoma cruzi culture The 7. cruzi CL-14 strain is maintained in culture in LIT (Liver Infusion Tryptose) supplemented with 10% SFB, 100 U / ml penicillin and 100 pg / ml streptomycin in BOD a 28 ° C. Transgenic parasites are maintained as well as the wild strain, but in the presence of 250 pg / ml geneticin.

To obtain extracellular trypomastigote or amastigote forms, LLCMK2 or L6 cells were maintained in RPMI supplement with 5% SFB and penicillin / streptomycin are infected with metacyclic trimostigotes in the proportion of 5 parasites / cell. They are kept in an oven at 34 ° C and 5% CO2. The following day, the cultures are treated for 24 hours with horse serum to eliminate possible epimatigote forms of the culture. Subsequently, SFB culture is reestablished until extracellular amastigote forms appear around 7 to 8 days after infection. T. cruzi transfection Parasites of the CL-14 strain are transfected by electroporation, in which 108 parasites in the log phase are washed with 1X PBS, centrifuged and resuspended in electroporation buffer. To the 0.2 cm electroporation cuvette is added 50 pg of the plasmid previously digested with Not \ or water as mock and then the parasites are added. The parasites are electroporated at 0.3 KV and 500pF with two pulses. at 10 second intervals using a Bio-Rad gene pulser (Bio-Rad) The electroporation product is transferred to 10% SFB supplemented LIT culture medium 24 hours after electroporation, 250pg / ml of geneticin for selection of transgenic parasites The selection period lasts from 3 to 6 weeks Transgenics were evaluated for the integration of expression cassettes in the parasite genome by Southern blot assay (data not shown).

Western Blot For Western blotting, 107 parasites in epimastigote form are washed in 1X PBS and resuspended in 1X sample buffer and resolved on 15% SDS PAGE gel electrophoresis. After the run, the samples were transferred to nitrocellulose membrane, blocked and then incubated with primary antibodies (anti-NY-ESO-1, anti-His tag or immunized animal serum) for 2 hours at room temperature with shaking. They are then washed and incubated for 1 hour with secondary anti-mouse total IgG antibody - HRP. After incubation, the membranes are washed and developed after ECL plus is applied to the membrane according to the manufacturer's instructions and then scanned in Scaner Storm (GE).

Figure 2 shows the expression of NY-ESO-1 protein by transgenic parasites by Western blot of the parasite lysate for the histidine and non-tailed constructs and the supernatant analysis of the gp63 signal peptide-containing construct.

Confocal Microscopy Transgenic parasites, as well as the wild form, are subjected to confocal microscopy to detect protein antigen (NY-ESO-1) expression by the parasite. For this purpose, epimastigote forms, metacyclic trypomastigotes, extracellular amastigotes and cell culture trypomastigotes are fixed with 2% paraformaldehyde, washed with 1X PBS and applied to poly-L-lysine slides for cell adhesion. The following day the samples are rehydrated, permeabilized with 0.1% Triton X-100 and blocked for 1 hour with 1X PBS with 5% SFB. They are then incubated with primary antibodies (anti-NY-ESO-1 or anti-His tag) for 1 hour. Before and after incubation with the Alexa 488 secondary IgG anti-mouse secondary antibody, slides are washed 5 times with blocking buffer. As a last step, Vectashieid (Vector Laboratories) is added prior to placement of the coverslip.

Slides were analyzed on a LS88 Zeiss confocal laser microscope with 488 qm laser for NY-ESO-1 protein detection, 358 qm for DAPI stained nucleus and DiC image visualization. Images were acquired and formatted by software Zeiss LSM Image Browser.

Figure 3 shows protein expression by confocal microscopy technique.

In addition to identifying protein expression in the metacyclic trypomastigote infective form, confocal microscopy was also performed at the four stages of parasite differentiation, in which it was possible to prove persistent protein expression during the parasite's cell cycle (Figure 4). EXAMPLE 2 - Evaluation of the ability of transgenic parasites to induce immune response Infection in vivo with T. cruzi C57BL / 6 mice and deficient mice that received inoculum of 107 parasites per animal in the intraperitoneal metacyclic trypomastigote were infected with CL-14 or your transgenics. At the time of infections for immunization purposes, two equivalent infections were performed, the second 30 days after the first. Serum from immunized animals was used to check for the production of anti-NY-ESO-1 specific antibodies (Figure 5). The cellular response induced by the immunization protocol was measured 21 days after the second immunization (Figure 6). These data show that transgenic parasites were able to induce NY-ESO-1 antigen specific immune response.

B16 WT and B16 melanoma tumor line NY-ESO-1 Cultivation B16 melanoma cell line cells were cultured in RPMI supplemented with 10% SFB and penicillin / streptomycin in an oven at 37 ° C, 5% CO2. To the NY-ESO-1-expressing transgenic strain, 300 pg / ml of geneticin was added to maintain the expression plasmid.

B16 Melanoma Challenge Immunized mice were challenged with B16 melanoma whether or not expressing the NY-ESO-1 tumor antigen. Each animal received 5 x 104 cells subcutaneously in the posterior dorsal region. Tumor growth was followed twice a week for a period of 40 days. Tumor measurement was given as the area in mm2 (Figure 7). This data shows that transgenic parasites were able to induce tumor inhibition specific to NY-ESO-1 antigen.

Flow cytometry for CD8 + / tetramer + lymphocyte evaluation Once it is established that the living transgenic parasite is effective both in inducing the specific immune response and in inhibiting tumor growth, the question arises as to the mechanism involved in inducing this response. In order to answer this question, immunizations were performed in IL-12, Myd88, CD8 and iNos deficient animals, all in parallel with the wild control. Spleen-derived cells from animals immunized with T. cruzi were subjected to flow cytometry to assess the CD8 + population responsive to tetramers containing TSKB18 or NY-ESO-1 CD8 peptides. To this end, the cells were labeled with the tetramers, anti-CD3 and anti-CD8. The readings were taken in FACScalibur and analyzed using FlowJo software.

Cell response data (Figure 8) and cytometry (Figure 9) show that IL-12 and Myd88 deficient animals have a decrease in IFN-γ production when compared to wild animal C57BL / 6 and iNos deficient animal . Although Myd88 and IL-12 deficient mice do not have a lower percentage of CD8 + / NY-ESO-1 tetramer + cells, it is suggested that these cells are not capable of producing IFN-γ, suggesting the crucial role of the Myd88 adapter molecule in induction of specific immune response following vaccination protocols with transgenic parasites, as expected for the previously characterized IL-12 - / - animals for low IFN-γ production. EXAMPLE 3 - Transfection and characterization of T. cruzi CL-14 strain with NY-ESO-1 and Thymidine Kinase. The Herpes simplex type - 1 Thymidine Kinase (HSV-1 TK) gene is widely used as a negative selective marker in mammalian cells (Moolten, FL 1986.) Chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy Cancer Research 46, 5276 5281.). This is an example of a suicide gene which, when expressed, causes the transfected cell to commit metabolic suicide in the presence of an appropriate drug (Ganciclovir or Aciclovir). HSV-1 TK, in contrast to mammalian thymidine kinase, is capable of phosphorylating specific nucleoside analogs such as Ganciclovir and Aciclovir to their monophosphate form. This monophosphate is then converted by cellular kinases to a very toxic nucleoside triphosphate, which is incorporated into DNA leading to inhibition of DNA synthesis and cell death (Elion, GB 1983. The biochemistry and mechanism of action of acyclovir. Journal of Antimicrobial Chemotherapy 12, 9-17.). The high specificity of HSV-1 TK for these nucleoside analogs makes it possible to selectively kill HSV-1 TK-expressing cells, not reaching other dividing cells. The use of HSV-1 TK in this system, in combination with the cancer / testis proteins, allows the patient to eliminate the acyclovir drug after elimination of the tumor and to eliminate all parasites. This drug can also be used to circumvent any overreaction of the immune system. Thymidine Kinase Cloning Two constructs were selected for insertion of the HSV-1 TK gene: pROCKNYESO-1 His + and pROCKNYESO-1GP63. Initially, the HSV-1 TK gene was PCR amplified using specific primers containing the XbaI and XhoI restriction sites at the ends. After amplification, the PCR product was digested with Xbal and Xhol and the digestion product was inserted into plasmid pROCKHygro (DaRocha WD, Silva RA, Bartholomeu DC, Pires SF, Freitas JM, Macedo AM, VazquezMP, Levin MJ, Teixeira SM. Expression of exogenous genes in Trypanosoma cruzi: improving vectors and electroporation protocols Parasitol Res. 2004 Jan; 92 (2): 113-20. The presence of the insert was confirmed by digestion with the same enzymes and 1% agarose gel analysis. The pROCKHygroTK construct was then digested with BamH1 and Spel for HSV-1 TK insert removal and cloning into selected plasmids. The presence of the insert was confirmed by Xbal and Xhol digestion and 1% agarose gel analysis. The constructs were digested with the restriction enzyme Notl and the product was electroporated into T. cruzi CL-14.

Western Blot For analysis of NY-ESO-1 and HSV-1 TK protein expression by the transformed parasites, total lysates of these and the culture supernatant of the construct were subjected to Western Blot as described above, but using HSV-specific monoclonal antibody. 1 TK, in addition to anti-NY-ESO-1 (Figure 10).

Drug susceptibility analysis Aciclovir L6 cells supplemented with RPMI with 5% SFB and penicillin / streptomycin are infected with metacyclic trypomastigotes at a rate of 5 parasites / cell. The cells are kept in an oven at 34 ° C and 5% CO2. The following day, the cultures are treated for 24 hours with horse serum to eliminate possible epimastigote forms of the culture. Subsequently, the culture with SFB is reestablished with the addition of the drug Aciclovir in different concentrations. On days 7, 8 and 9 after infection, the parasites present in the culture supernatant were collected and counted. The sum of the three days for each drug concentration was considered. Figure 11 shows the ability of parasites to proliferate following administration of Aciclovir. This result demonstrates that by combining HSV-1 TK and the protein antigen of interest, the parasite can be specifically eliminated at the end of treatment.

Claims (10)

Method for the preparation of attenuated transgenic Trypanosoma cruzi strain, comprising the following steps: (a) gene cloning for protein antigen in plasmid pRockneo, pRockHygro or the like; b) Electroporation transfection of the attenuated T. cruzi CL-14 strain with the plasmid containing one or more protein antigen genes. 2. Attenuated Trypanosoma cruzi strain, characterized in that it is transgenic, containing at least one exogenous gene that codes for a polypeptide antigen. Attenuated Trypanosoma cruzi strain according to claim 2, characterized in that the polypeptide antigen is selected from a group comprising preferably NY-ESO-1, MAGE-A3, CTSP-1 antigens or part thereof, alone or in combination. . Attenuated Trypanosoma cruzi strain according to claims 2 and 3, characterized in that it optionally contains a gene encoding HSV-1 thymidine kinase. Attenuated strain of Trypanosoma cruzi according to claims 1 to 4, characterized in that it is used as a vaccine vector. Attenuated Trypanosoma cruzi strain according to Claims 1 to 4, characterized in that it is used in the preparation of a live vaccine for the treatment or prophylaxis of diseases which require induction of a lymphocyte-mediated immune response selected from the group comprising: tumor diseases and diseases caused by intracellular microorganisms. Live vaccine, comprising an attenuated transgenic Trypanosoma cruzi strain as defined in claims 2 to 5. Live vaccine according to claim 7, characterized in that it is therapeutic and / or prophylactic. Live vaccine according to claim 7, characterized in that it is administered in a reduced number of subsequent doses. Live vaccine according to claim 7, characterized in that it can be used in combination with heterologous vaccination protocols.