BRPI0801906A2 - genetically modified trypanosoma cruzi ts antigen sequence, recombinant ts protein and genetically modified viruses expressing recombinant ts antigen - Google Patents

Abstract

GENETICALLY MODIFIED SEQUENCE OF TRYPANOSOMA CRUZI ANTIGEN TS, PROTEIN RECOMBINANT TS AND GENETICALLY MODIFIED VIRUSES EXPRESSING RECOMBINANT ANTIGEN TS. The present invention relates to the construction of a genetically modified Trypanosoma cruzi TS antigen sequence, a recombinant protein encoded by said sequence and genetically modified viruses expressing the recombinant TS antigen. The invention is useful for mammalian immunization against Chagas disease.

Description

"GENETICALLY MODIFIED SEQUENCE OF ANTIGEN TS DETRYPANOSOMA CRUZI, RECOMBINANT MODIFIED PROTEIN AND VIRUSGENETICALLY MODIFIED EXPRESSING TSRECOMBINANT ANTIGEN"

The present invention relates to the construction of a genetically modified Trypanosoma cruzi TS antigen sequence, a recombinant protein encoded by said sequence and genetically modified viruses expressing the recombinant TS antigen. The invention is useful for the immunization of Chagas disease.

Infection with the parasitic protozoan Trypanosoma cruzi causes Chagas disease, a neglected disease that affects more than 16 million people in Latin America. More than 300,000 new patients become infected with T. cruzian every year, although drug use has significantly decreased the rates of mortality and morbidity associated with this disease. Drugs used to treat Chagas disease in chronically infected individuals are not effective and naturally multiresistant chemotherapy parasites have been described in several regions of Latin America (URBINA. JA Specific treatment of Chagasdisease: Current status and new developments. Curr. Opin. Infect. Dis 14, 733-741 (2001).

Immunological protection against this parasite appears to be highly dependent on the induction of CD8 + and interferon gamma (IFN-gamma) T-cell responses, similar to that found for pre-erythrocytic stages for Plasmodium species or for infections caused by Toxoplasma gondii. CD4 + T cells with cytokine secretion profile of T helper type 1 (Th1) lymphocytes and antibodies have been described as mediators of protection against all the aforementioned diseases. Therefore, the development of a T. cruzi vaccine would help in inducing broad immunity against parasite antigens (Arch. Allergy Immunol. 1997. 114,103-110) (BRENER Z. andGAZZINELLI RT Immunological control of Trypanosoma cruzi infection and pathogenesis of Chagas disease. .

Several studies have focused on the development of a vaccine using attenuated T. cruzi separasites (MENEZES H. Active immunization of mice with the strain Y strain of Trypanosoma cruzi against experimental infection of mice. Rev. Inst.Med. Trop. Sao Paulo. 1968. 10, 1-4) or killed (BASOMBRIO MA Trypanosomacruzi: Partial prevention of the natural infection of guinea pigs with a killed parasitevaccine. Exp. Parasitol. 1990. 71,1-8). Unfortunately, most immunizations can only delay disease manifestations or, in the best of circumstances, decrease the associated pathology. Effective protection against T. cruzi could be achieved when adjuvants directing a Th1 response were used. (HOFT DF & EICKHOFF CS Type 1 immunity provides both optimal mucosal and systemic protection against mucosally invasive, intracellular pathogen. Infect. Immun.2005. 73, 4934 -4940) and / or DNA subunit vaccines that encode certain parasite antigens (SEPULVEDA P., HONTEBEYRIE M., LIEGEARD P., MASCILLIA., NORRIS KA DNA-based immunization with Trypanosoma cruzi complementregulatory protein elicits complement Iytic antibodies and confers protection against Trypanosoma cruzi infection, Immune Infect 2000. 68, 4986-4991. SCHNAPP AR, EICKHOFF CS, SCHARFSTEIN J., HOFT DF Induction of B and T-cell responses tocruzipain in the murine model of Trypanosoma cruzi infection Microbes Infect. 2002. 4,805-813 FRALISH BH & TARLETON RL Genetic immunization with LYT1 or a poolof trans-sialidase gene protects mice from Iethal Trypanosoma cruzi infection Vaccine. 2003. 21, 3070-3080).

When plasmid DNA vaccines were used, two antigens were successfully tested: trans-sialidase (TS) and amastigote surface protein-2 (ASP-2) (COSTA F., FRANCHIN G., PEREIRA-CHIOCCOLA VL, RIBEIRAOM. M. , SCHENKMAN S., RODRIGUES, MM Immunization with a plasmid DNA containingthe gene of transsialidase reduces Trypanosoma cruzi infection in mice Vaccine.1998 16, 768-774 FUJIMURA AE, KINOSHITA SS, PEREIRA-CHIOCCOLA VL, RODRIGUES MM DNA sequencing encoding CD4 + and CD8 + T-cell epitopes are important for efficient protective immunity induced by DNA vaccination with a Trypanosoma cruzi gene Infect Immun 2001, 69, 5477-5486 GARG N. & TARLETON RL Genetic immunization antigen-specific protective elicits immunoresponses and decreases disease severity in Trypanosoma cruzi infection Infect.Immune 2002. 70, 5547-5555 BOSCARDIN SB, KINOSHITA SS, FUJIMURA AE, RODRIGUES MM Immunization with cDNA expressed by amastigotes of Trypanosoma cruzi elicits protecti ve immune response against experimental infection.Infect. Immun. 2003. 71, 2744-2757. FRALISH B.H. & TARLETON R.L. Geneticimmunization with LT1 or a pool of trans-sialidase genes protects mice from IethalTrypanosoma cruzi infection. Vaccine. 2003. 21, 3070-3080. VASCONCELOS J.R., HIYANE M.I., MARINE C.R., CLASER C., AX. A.M., GAZZINELLI R.T., BRUNA-ROMERO O., ALVAREZ J.M., BOSCARDIN S.B., RODRIGUES M.M.Protective immunity against Trypanosoma cruzi infection in a highly susceptible mousestrain after vaccination with genes encoding the amastigote surface protein-2 andtrans-sialidase. Gene Ther. 2004. 15, 878-886)

TS is a trypomastigote expressed antigen that catalyzes the transfer of glycoconjugate sialic acids from the host to the acceptor of parasite plasma molecules (SCHENKMAN S., EICHINGER D., PEREIRA ME, NUSSEN-ZWEIG V. Structural and functional properties of Trypanosoma trans-sialidase, Annu, Rev. Microbiol, 1994. 48, 499-523).

Recombinant viruses have been shown to be efficient for devacin development. Clinical trials with recombinant virus-based vaccine candidates for malaria, tuberculosis, and AIDS demonstrate unprecedented levels of cellular immune response against recombinant virus-expressed products, when compared to vaccine formulations based on purified recombinant proteins or plasmid DNA vaccines (ROCHA C., CAETANO B., AX A., BRUNA-ROMERO O. Recombinant viruses as tools to induce protective cellularimmunity against infectious diseases (Int. Microbiol. 2004, 7, 89-94).

An advantage in the use of recombinant viruses is that the viral particle itself has an adjunct effect by itself, which makes it possible to avoid the use of other adjuvant compounds or cytokines. Therefore, recombinant adenoviruses, influenza viruses, vaccinia viruses (MVA) stand out for their immunomodulatory properties (GUILLOT L., LE GOFFIC R., BLOCK S., ESCRIOUN., AKIRA S., SIGNHAR M. Involvement of toll-like receptor 3 in the immune response to Iung epithelial cells to double-stranded RNA and influenza A virus (J. Biol. Chem. 2003. 280, 5571-5580).

The mechanism of action of recombinant adenovirus-based vaccines is infection of host cells, resulting in the expression of heterologous antigens at the cytoplasm level of infected cells. The antigen thus expressed may still be secreted. Recombinant adenoviruses are highly immunogenic due to their ability to infect immune cells specialized in antigen presentation, such as dendritic cells. In addition, the immunostimulatory effect exerted by some proteins of the viral capsid, which results in the enhancement of the immune response, as well as a better targeting of the cytokine profile produced by effector cells to a typeTh1, which is important in protecting against agents. parasitic species such as T. cruzi.

The vast majority of recombinant poxvirus construction techniques, including the MVA virus (Modified Vaccinia Ankara Virus), involve homologous DNA recombination in infected cells. Generally, recombinant virus production employs transfer plasmid vectors containing an expression cassette where the exogenous gene is controlled by a strong, usually early and late, promoter of the poxviruses. This cassette is flanked by DNA sequences homologous to sequences present in the virus genome, and will serve to direct recombination to the desired locus.

In our constructs, transfer plasmids direct insertion of exogenous genes into sites called the Deletion Region II and / or Deletion Region III of the MVA virus genome. These are genomic regions whose present genes were altered, removed or mutated during the virus generation process about 40 years ago. Thus, these naturally-modified (or mutated) regions are suitable for the insertion of exogenous degenerate expression cassettes, since the insertion of these elements does not alter any vital MVA virus function. The recombination rate is relatively high, around 0.1%, and recombinant viruses can be selected, plaque purified and amplified.

The safety of the MVA-based vaccine vector has been experimentally demonstrated when artificially immunosuppressed monkeys were inoculated with high doses of the virus (109 PFUs - a 100-fold amount greater than that normally used for poxvirus immunization). Throughout the follow-up period the animals showed no hematological or pathological abnormalities. These non-replicative features of MVA make the virus workable under Tier 1 Biosafety conditions, and also prevent the virus from spreading to unvaccinated individuals or the environment. Despite extensive replicative limitation in certain cells, such as mammalian cells, MVA has retained all the peculiar features that make Poxviruses attractive eukaryotic expression vectors, including their enormous ability to accommodate exogenous DNA in their genome (up to 25 Kb); precise viral control of the expression of proteins encoded by viral or recombinant genes, absolute absence of viral persistence or integration into the host genome; and ease for the production of recombinant vectors and vaccines.

In the present invention, transfer plasmids basically consist of cDNAs encoding the proteins of vaccine interest cloned into plasmid LW44 (Figure 1) or the like. Next to it, and in opposite orientation, is cloned the marker gene encoding the green fluorescent protein (GFP). Co-expression of this protein is used to facilitate selection of recombinant viruses. These two genes are finally flanked by DNA sequences from the MVA virus homologous to the inclusion in the viral vector genome, called Deletion Region III.

In Figure 2, in the 5 '- 3' direction, the insertion cassette consists of: Nucleotide sequence homologous to the 5'-portion of the Deletion Region II and / or Illusion Region of the MVA genome (1); Early / late modified promoter of Vaccinia virus (2); marker protein genecoder - Fluorescent Green Protein (GFP) or Fluorescent Red Protein (RFP) (3); cDNA / Gene cloning site of interest (4), Modified early / late Vaccinia virus promoter (5); nucleotide homologue to the 3 'portion of the Deletion Region II and / or Deletion Region III of the MVA genome (6). Although both the MVA virus II and III deletion regions can also be used for insertion of exogenous gene expression cassettes, the constructs described in this patent were created from the insertion of different expression cassettes only in the Deletion III region, leaving the insertion in the Ilcomo region as a feasible methodological alternative to be used in due course.

In the following, the present invention will be described in detail by way of examples. It should be noted that the invention is not limited to these examples, but also includes variations and modifications within the limits in which it operates.

EXAMPLE 01: Construction of Transfer Plasmids

The construction of the transfer plasmids containing the cDNA encoding the wild-type WSN virus neuraminidase (NA) segment contains the negatively orientated NAclonate segment in the transfer plasmid pPR7 between the truncated human polymerase I promoter (pol I) sequence and the sequence. hepatitis virus daribozyme δ. This type of construct allows the synthesis of a synthetic vRNA molecule in transfected cells. Plasmid pPRNA38 was constructed from plasmid pPRNA35. It encodes a recombinant NA segment, in which the NA ORF is followed by duplication of the 3 'promoter region, a Xho \ / Nhe \ cloning site, a duplicate of the last 42 nucleotides of the NA ORF, and the 5'original promoter region (Figure 03).

The transfer plasmid pPRNA38-dTS was constructed for expression of a 660 nucleotide fragment of the TS gene, which contains epitopes recognized by CD4 + and CD8 + T lymphocytes. Briefly, for the pPRNA38-TS plasmid construct, the TS fragment was obtained by PCR using the GGG CTC GAG CAT GAG CAT GAA AAC AGT CGT TGG antisense oligonucleotides and antisense GGG GCT AGC TAA ATG GAT ACT TGC CCA ACG TTA TAA ATA CCG CCAAGA AAT TGA TTT GT. The latter contains the sequence coding for TS epitopes recognized by CD8 + T lymphocytes. The amplification product obtained was purified and subsequently cloned into plasmid pPRNA38 between the Xho \ eNhe I sites.

EXAMPLE 02: Production of Recombinant Influenza Viruses by Reverse Genetics

The expression strategy used by the researchers of the present invention was based on the studies demonstrated that the influenza virus polymerase complex is capable of recognizing the 3 'and 5' promoters even when they are located within a pseudo-viral segment. Thus, the researchers of the present invention exploited these observations to construct recombinant influenza viruses, stably carrying a bicistronic segment of NA1 in which a heterologous sequence is under the control of a doubled 3 promoter region. Such bicistronic segments can be transcribed and replicated in two distinct ways following interaction of the 5 'promoter region with each of the two 3' promoter regions. In this type of construct, the NA coding region is located at the proximal 3 'position and the heterologous sequence is located near the 5' end. Thus, since NA protein expression is essential for viral viability, we ensured that a complete bicistronic segment was maintained throughout the spread of recombinant viruses. Influenza viruses were obtained by reverse genetics according to the technique previously described by Fodore collaborators (Figure 04).

Briefly, sub-confluent monolayers of MDCK and HEK 293T cell coculture cells grown in Dulbecco's modified minimal medium (DMEM) supplemented with 10% fetal bovine serum (SFB) were transfected by plasmids encoding the wild-type or recombinant NA segment, as well as the plasmids encoding the remaining 7 segments of the influenza virus, along with the plasmids encoding the four influenza virus replication complex proteins (pcDNA-PB1, pcDNA-PB2, pcDNA-PA and pcDNAA-NP) using the Fugene transfection reagent. 6 (Roche) (Figure 05).

Thus, eight ribonucleoprotein complexes were reconstituted in vivo allowing transcription and replication of all viral segments and synthesis of new virions. After 24 hours incubation at 35 ° C, transfected cells were incubated for a further two days in 2% SFB supplemented DMEM. The recombinant viruses thus obtained were amplified once in MDCK cells and twice purified by MDCK1 lysis plates before undergoing final amplification in MDCK cells. Viral stocks will have infectious titers determined on MDCK cells by the lysis plate method under an agarose layer in DMEM medium with 2% SFB. The presence of the heterologous sequence in the viral genome was analyzed by PCR and sequencing techniques.

EXAMPLE 03: Obtaining and characterizing recombinant influenza viruses carrying a heterologous sequence.

Viruses carrying a fragment of Trypanosomacruzi TS protein (vNA38-TS) were obtained by the technique of obtaining recombinant viruses entirely from plasmids (as described in Figure 01). The genomes of the above mentioned bistristronic virus carriers were analyzed by RT-PCR using oligonucleotides that allow the insertion region of the heterologous sequence to be analyzed. Expected size amplification fragments were obtained (data not shown), indicating that they had conserved the heterologous sequence in their genomes.

EXAMPLE 04: Production of a heterologous TS protein fragment in MDCK cells infected by recombinant influenza viruses.

Confluent monolayers were infected with 2 m.o.i of wild-type vNA virus or two recombinant vNA38-TS virus clones. 24 hours after infections, cell extracts were prepared and the TS protein was analyzed by western blot. As shown in Figure 05, significant levels of TS proteins could be detected in MDCK cells, demonstrating the ability of influenzarecombinant viruses to express these heterologous sequences.

EXAMPLE 05: Production of Parental and Recombinant Viruses in Cell Cultivation.

Multiplication of parenteral (MVA) or recombinant viral vectors should be done in chicken embryo fibroblast (CEF) cells produced by primary culture of embryos of about 10 days of laying. Briefly, embryos are dissected under sterile conditions for removal of internal organs. The cells are then dispersed mechanically by syringing, and also chemically, by trypsin incubation. The cells are washed to remove red blood cells and other contaminants, and then suspended and sedimented in plastic vials containing E-MEM or DMEM medium supplemented with 10% fetal bovine serum. The construction of poxviral vectors is based on homologous combining of viruses in infected cells in the presence of transfer plasmids containing the target antigen-encoding DNAS.

The recombinants are then generated by transfecting the transfer plasmids into chicken embryo (CEF) cells previously infected with the MVA virus. Briefly, CEFs are infected with MOI of 0.1 to 1 of the parental MVA viruses. After one hour of adsorption, a solution containing 2 pg of the transfer plasmid and 5μΙ of the transfectant Lipofectamine (Invitrogen, USA) is added to the infected cells. The cells are incubated for 5 hours when the transfection medium is removed and replaced with cultures supplemented with 2.5% fetal bovine serum. The cells are then incubated for an additional 48 hours and collected at the end of the period. Recombinant viruses are selected and purified in successive rounds of plaque purification by microscopic visualization of the presence of the GFP marker protein (green fluorescence). It is noteworthy that, through the way the transfer vector was designed, only recombinant viruses containing the gene expression cassette should also express the GFP protein. Following purification cycles of recombinant clones, these titers are amplified in successive cycles of increasing cell number infection. When titers of the order of 109 PFUs / ml are achieved, the viral suspensions are purified on sucrose mattress and homogenized in 10mM Tris-HCI, aliquoted and stored at -70 ° C.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: A) Plasmid pLW44, transfer vector for construction of recombinant MVA viruses. The cloning site of the protein coding gene of interest is cloned between restriction sites for the enzymes Smal and Xbal. The GFP protein genecoder is represented. Promoters are not represented. B) Example of ready-to-use plasmid, where the A2 protein genecoder, for example, from Leishmania, has been inserted. Figure 02 - (A) Transfer plasmid for construction of recombinant MVA viruses. B) Detail of the insertion and expression cassette containing the following gene elements: Nucleotide sequence homologous to the 5 'portion of the Deletion Region II and / or Deletion Region III of the MVA genome (1); Early / late modified promoter of Vaccinia virus (2); gene encoding protein-Fluorescent Green Protein (GFP) or Fluorescent red protein (RFP) (3); cDNA / Gene cloning site of interest (4), Early / late modified Vaccinia virus promoter (5); nucleotide homologous to the 3 'portion of the Deletion Region II and / or Deletion Region III of the MVA genome (6).

Figure 03: Schematic representation of parasynthetic transfer plasmids of NA bistristronic segments derived from influenza A / WSN / 33. Plasmid pPR-NA35 is derived from plasmid pPR-NA which contains the full-length NA negative-segment cDNA under the control of the human polymerase I promoter truncate. It contains the cDNA of the negatively orientated recombinant segment of NA, in which the NA ORF is followed by the 3 'dopromotor duplication, an XhoI / Nhel cloning site and the original 5'promoter. PlasmidopPR-NA38 is derived from plasmid pPR-NA35. It was built to conserve 5 'end integrity throughout its last 70 nucleotides. This was achieved by inserting a duplicate of the last 42 NA ORF nucleotides (white square) which were inserted between the 5 'promoter and the Xhol / Nhel.pb linker: base pairs, SMC: multiple cloning site.

Figure 04: Reverse genetics of the A / WSN / 33 neuraminidase coding segment without helper virus (Plasmid only). Alternatively, recombinant viruses may be obtained by reverse genetics. Thus, HEK 293T cells (7x10 5 cells in 35 mm2 plates, respectively) will be co-transfected with oplasmid encoding the wild-type or recombinant NA segment (500 ng), since one of the plasmids encoding the other influenza virus segments (500ng of each plasmid); and by the plasmids that allow the expression of the polymerase complex proteins (PA, PB1 and PB2: 500 ng each) and the nucleoprotein NP (500 ng) in 10 μΙ of Fugene 6 (Roche), allowing the reconstitution of 8 functional ribonucleoprotein complexes in vivo. and thus transcription and replication of the viral segments. Supernatants will be collected 72 hours after transfection. After an amplification step in MDCK1 cells the transfecting viruses will be cloned and then amplified in MDCK cells. * 4 expression plasmids for proteins PA1 PB1 and PB2 and nucleoproteinNP. ** 8 transfer plasmids from orecombinant wild influenza virus segments. *** recombinant viruses.

Figure 05: Production of TS proteins in recombinant influenza virus infected MDCK cells. Confluent monolayers of MDCK cells were infected with an m.o.i of greater than 1 of the vNA38-TS or recombinant influenza viruses by wild-type vNA virus. 48 hours after infection, MDCK cell extracts were prepared in SDS protein gel sample buffer. After electrophoresis, the samples were transferred to nitrocellulose membranes and incubated with a pool of mouse sera immunized with recombinant TS proteins. Membrane-bound antibodies were detected by incubation of immunoblot membranes with peroxidase mouse anti-IgG antibodies. The chemiluminescence reaction was visualized with a chemiluminescence amplifier, followed by exposure on film.

Figure 06: Reverse genetics for obtaining an influenza virus encoding a T. cruzi trans-sialidase (TS) fragment. Recombinant viruses were obtained by reverse genetics without helper virus. Briefly, HEK 293T cells (7x10 5 cells in 35 mm 2 plates, respectively) were co-transfected with pPRNA38-TS oplasmids (500 ng) by each of the plasmids encoding the other influenza virus segments (500 ng of each plasmid); and by plasmids that allow the expression of the proteins of the polymerase complex (PA, PB1 and PB2: 500 ng each) and of the nucleoprotein NP (500 ng) in 10 μΙ of Fugene 6 (Roche). Cell supernatants were collected 72 hours after transfection. Viruses were subjected to an amplification step in placard lysis cloned MDCK1 cells and subjected to further amplification in MDCK cells. The results depicted here illustrate the phenotype of 2 independent viral clones for one of the recombinant viruses. SEQUENCE LISTING

SEQ ID No. 01 - TS Fragment

1 ATG GGG AAA ACA GTC GTT GGG GCC AGT AGG ATG TTC TGG CTA ATG 45

Met Gly Lys Thr Val Val Gly Ala Ser Arg Met Phe Trp Leu Met4 6 TTT TTC GTG CCG CTT CTT CTT CTG CTC TGC CCC AGC GAG CCC GCG 90

Phe Phe Val Pro Leu Leu Leu Leu Leu Cys Pro Be Glu Pro Ala91 CAT GCC CTG GCA CCC GGA TCG AGC CGA GTT GAG CTG TTT AAG CGG 135

His Wing Read Wing Pro Gly Be Ser Arg Val Glu Read Phe Lys Arg

136 CAA AGC TCG AAG GTG CCA TTT GAA AAG GGC GGC AAA GTC ACC GAG 180 Gln Being Ser Lys Val Pro Phe Glu Lys Gly Lys Val Thr Glu 181 CGG GTT GTC CAC TCG TTC CGC CTC CTC GTT AAT GTG GAC 225 Arg Val Val His Ser Phe Arg Leu Pro Ala Leu Val Asn Val Asp 226 GGG GTG ATG GTT GCC ATC GCG GAC GCT TAC GAA ACA TCC AAT 270 Gly Val Met Val Ala Ile Wing Asp Gly Arg Tyr Glu Thr Be Asn 271 GAC AAC TCC CTC ATT GAT ACG GTG GCG AAG TAC AGC GTG GAC GAT 315 Asp Asn Ser Leu Ile Asp Thr Val Wing Lys Tyr Ser Val Asp Asp 316 GGG GAG ACG TGG GAG ACA ATT GCC ATC AAG AGC CGT GCA 360 Gly Glu Thr Trp Glu Thr Gln Ile Ala Ile Lys Asn Ser Arg Ala 361 TCG TCT GTT TCT CGT GTG GAT CCC ACA GTG ATT GTG AAG GGC 405 Ser Ser Val Ser Arg Val Val Asp Pro GGA AGC TAC AAC AGT TCG AGG 450 Asn Lys Leu Tyr Val Leu Val Gly Ser Tyr Asn Ser Cys Arg 451 TAC TGG ACG TCG CAT GGT GAT GCG AGA GAC TGG GAT ATT CTG CTT 495 Tyr Trp Thr Be His Gly Asp Ala ArgAsp Trp Asp Ile Leu Leu 496 GCC GTT GGT GAG GTC ACG AAG TCC ACT GCG GGC AAG ATA ACT 540 Ala Val Gly Glu Asp Thr Lys Ser Thr Ally Gly Lys Ile Thr 541 GCG AGT ATC AAA TGG GGG CTC CCC GTG TCA AAG GAA TTT TTC 585 Wing Ser Ile Lys Trp Gly Ser Pro Val Ser Leu Lys Glu Phe Phe 586 CCG GCG GAA ATG GAA GGA ATG CAC ACA AAT CAA TTT CTG GGC GGT 630 Pro Ala Glu Met Glu Met His Thr Asn Gln Phe Leu Gly Gly 631 ATT TAT AAC GTT GGG CAA GTA TCC ATT TAG 660

Claims (5)

1. "GENETICALLY MODIFIED SEQUENCE OF ANTIGENS TS DETRYPANOSOMA CRUZI characterized by the nucleotide sequence comprises Seq. ID No (1). 2. RECOMBINANT TS PROTEIN characterized by the amino acid sequence comprising Seq. ID at (1). 3. GENETICALLY MODIFIED VIRUS THAT EXPRESSES RECOMBINANT ANTIGENOTS characterized by comprising Seq. ID at (1). GENETICALLY MODIFIED VIRUS THAT EXPRESSES RECOMBINANT ANTIGENOTS according to claim 03, characterized by the serum adenovirus virus. GENETICALLY MODIFIED VIRUS EXPRESSING THE RECOMBINANT ANTIGENOTS according to claim 03, characterized by the modified Ankara serum virus (MVA).