BRPI0806285A2 - genetically modified sequence of trypanosoma cruzi asp-2 antigen, recombinant asp-2 protein, and genetically modified viruses expressing recombinant asp-2 antigen - Google Patents

Abstract

GENETICALLY MODIFIED SEQUENCE OF THE TRYPANOSOMA CRUZI ANTIGEN ASP-2, ASP-2 RECOMBINANT PROTEIN AND GENETICALLY MODIFIED VIRUSES EXPRESSING THE RECOMBINANT ANTIGEN. The present invention relates to the construction of a genetically modified sequence of the Tiypanosoma cruzi ASP-2 antigen, a recombinant protein encoded by said sequence and genetically modified viruses that express the recombinant ASP-2 antigen. The invention is useful for immunizing mammals against Chagas disease.

Description

"GENETICALLY MODIFIED Sequence Of ASP-2 ANTIGEN DETRYPANOSOMA CRUZI1 RECOMBINATING PROTEIN ASP-2 AND VIRUSGENETICALLY MODIFIED EXPRESSING ASPECT-2 RECOMBINATING"

The present invention relates to the construction of a genetically modified sequence of Trypanosoma cruzi ASP-2 antigen, a recombinant protein encoded by said sequence and genetically modified viruses expressing recombinant ASP-2 antigen. The invention is useful for mammalian immunization against 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 various 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 T lymphocyte cytokine secretion profile type 1 (Th1) 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 that direct a Th1 response are 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 encoding 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 genes protects mice from Iethal Trypanosoma cruzi infection.Va ccine. 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 elicits antigen-specific protective immuneresponses 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 protectiv and immune response against experimental infection.Infect. Immun. 2003. 71, 2744-2757. FRALISH B.H. & TARLETON R.L. Geneticimmunization with LT1 or a pool of trisisialidase 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)

ASP-2 is a surface amastigote antigen with a known function (LOW H.P., SANTOS M.A., WIZEL B. & TARLETON R.L. Amastigotesurface proteins of Trypanosoma cruzi are targets for CD8 + CTL. J. Immunol. 1998.160, 1817-1823).

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 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 Thh 1 type, which is important in protecting against parasitic agents 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 mammalian cells, MVA has retained all the peculiar characteristics 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 the 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 Flick and Hobom's studies, which demonstrated that influenza virus complexopolymerase is capable of recognizing 3 'and 5' promoters even when they are located within a viral pseudo-segment. . Thus, the researchers of the present invention exploited these observations to construct recombinant influenza viruses, stably carrying a NA-segmenticistronic, in which a heterologous sequence is under the control of a duplicate 3d promoter region. Such bistristronic segments can be replicated transcripts in two different ways after interaction of the 5 'promoter region commenced with one of the two 3' promoter regions. In this type of construction, the NA coding region is in the proximal 3 'position and the heterologous sequence is located near the 5' end. Thus, since expression of NA protein is essential for viral viability, we ensured that a completeistronicistronic segment was maintained throughout the spread of recombinant viruses. Influenza viruses were obtained by reverse genetics according to the technique previously described by Fodor. and 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. Recombinant viruses thus obtained were amplified once in MDCK cells and twice purified by MDCK cell lysis plates before being subjected to 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 TS protein fragment (vNA38-TS), the n-terminal (VNA38-ASP2-PTN1) or media (vNA38-ASP2-PTN2) portion of the Trypanosoma cruzi ASP-2 protein were obtained by the virus-obtaining technique. recombinantly from plasmids (as described in Figure 01). The results illustrated in Figure 06 represent vNA38-ASP2-PTN1 and vNA38-ASP2-PTN2 viruses after cloning and complementary amplification in MDCK cells. The genomes of the viruses carrying the above-mentioned bicistronic segments were analyzed by RT-PCR using oligonucleotides to analyze the insertion region of the heterologous sequence. Expected size amplification fragments were obtained (data not shown), indicating that they had retained 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 infection, 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 parental (MVA) or recombinant viral vectors should be done in chicken embryo fibroblast (CEF) 1 cells produced by primary embryo culture 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 µg of transfer plasmid and 5 µl of lipofectamine transfectant (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 pl_W44, 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, was 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 MDCK cells, the transfecting viruses will be cloned and then amplified in MDCK cells. * 4 expression plasmids for proteins PA, PB1 and PB2 and nucleoproteinNP. ** 8 transfer plasmids from orecombinant wild influenza virus segments. *** recombinant viruses.

Figure 05: Recombinant viruses were obtained by reverse genetics helper virus. Briefly, HEK 293T cells (7x10 5 cells in 35mm 2 plates, respectively) were co-transfected with pPRNA38-TS plasmids (500ng) by each of the plasmids encoding the other influenza virus segments (500 ng of each plasmid); and by plasmids allowing expression of the polymerase complex proteins (PA, PB1 and PB2: 500 ng each) and dan nucleoprotein NP (500 ng) in 10 μΙ Fugene 6 (Roche). Cell supernatants were collected 72 hours after transfection. Viruses were subjected to an amplification step in MDCK1 cells cloned by lysis plate and subjected to complementary amplification in MDCK cells. The results shown here illustrate the phenotypes of 2 independent viral clones of each recombinant virus. SEQUENCE LIST. ID N. 01 - ASP Fragment

ASP2-PTN1 fragment

1 ATG GGA ATG CAG GTG CAG ATC CAG AGC CTG TTT CTG CTC CTC CTG TGG CCG GGG TCC. 60

Met Gly Met Gln Val Gln Ile Gln Ser Leu Phe Leu Leu Leu Leu Leu Trp Val Pro Gly Ser61 AGA GGA GGT ACC GCC GCT AGG AAG TCC GGG GAT GCG CCG CTG CCA CAA TGG GTC 120

Arg Gly Gly Thr Wing Wing Val Glu Be Lys Be Gly Asp Wing Pro Leu Pro Gln Trp Val

121 GAT ATT TTG GTG CCG GAA AAG ACG CAG GTG CTG CCG AAA GAG GCT TCT AAA AGT GGA GTG 180 Asp Ile Phe Val Gly Lys Thr Gln Val Leu Pro Lys Glu Gly Ser Lys Ser Gly Val 181 AAG AAA GCA TTT GCT GCG CCT TCT CTC GTC AGT GCT GGT GGA GTA ATG GTT GCC TTT GCT 240 Lys Ala Phe Ala Pro Wing Ala Will Be Ala Gly Gly Val Met Val Ala Phe Ala 241 GAA AGC TTG TTC ATT GTC CAT GAA CAT TTG TTT GGG ATT AAG CCT TAC GAA 300 Glu Ser Leu Phe Val Tyr Ile Go HiS Gly His Asn Leu Phe Gly Ile Lys Pro Tyr Glu 301 ATT GTG GCC GGG TAC ATC GCT GCG TGA CCG TCC ATT GTC GCT GTC GAT AAT 360 Ile Val Gly Wing Tyr Ile Lys Wing GlU Wing Tr Tr Pro Pro Ile Val Glu Wing Val Asn 461 GCT ACT ACA TGG AGA ACC CAC ACT GTT ATT GGC AGA AAT GGC AAT GAT CGT TTG TGT 420 Wing Thr Thr Trp Arg Thr HiS Thr Val Ile Gly Ser Arg Asn Gly Asn Asp Arg Leu Cys 421 TAT TTG CAA CGG CCA ACA GCA GTT GCA AGG GAC AGT AAG GTG TTT CTA CTT GTG GGA AGC 480 Tyr Leu Gln Arg Pro Wing Ala Arg Asp Ser Lys Val Phe Leu Leu ValGly Ser 481 GAT ACT ACA AGA TAT GAC AGC GAT GAT AAT ATG TGG GTG AAA GAT GGC TGG GAT ATT CAA 540 Asp Thr Thr Arg Tyr Asp Asp Asn Met Trp Val Lys Asp Gly Trp Asp Ile Gln 541 CTG GTT GAG GGT GTG GCC ACG CAG TCC AAG GAC GGC GTG CAG AGT AAA CTG GTC AGC TGG 600 Leu Val Glu Gly Val Ala Thr Gln Ser Lys Asp Gly Val Gln Ser Lys Leu Val Ser Trp 601 GGT GAA CCC AAG TCG TTA AAA CAG ATA CTC AAT CAC ACT CAA GAT CAG CTG AGG TAG 660 Gly Glu Pro Lys Ser Leu Lys Gln Ile Leu Asn His Thr Gln Asp Gln Leu Arg STOP

ASP2-PTN2 fragment

1 ATG GGA ATG CAG GTG CAG ATC CAG AGC CTG TTT CTG CTC CTC CTG TGG GTG CCC GGG TCC 60

Met Gly Met Gln Val Gln Ile Gln Ser Leu Phe Leu Leu Leu Leu Leu Trp Val Pro Gly Ser61 AGA GGA GGT ACC GAT GTT ACT GCT GGT TCA GCA ATT GTG ATG CAG AAT GAC ACG 120

Arg Gly Gly Thr Asp Val Val Thr Wing Gly Gly Ser Gly Ile Val Met Gln Asn Asp Thr

121 CTT GTG TTT CCT CTG ATG GTG AAT GGG CAA AAT TAC CCT TTT TCC TCG ATC ACT TAC TCG 180 Read Val Phe Pro Read Met Val Asp Gly Gln Asn Tyr Pro Phe Ser Ser Ile Thr Tyr Ser 181 ACG GAC AAA GGC AAT ACC TGG GTG TTC CCA GAG GGC ATT TCT CCT GTA GGA TGC CTT GAT 240 Thr Asp Lys Gly Asn Thr Trp Val Phe Pro Glu Gly Ile Ser Pro Val Gly Cys Leu Asp 241 CCC CGC ATC ACA GAA TGG GAG CAA ATT CTC ATG GTT CAG TGT AAA GAT GAC 300 Pro Arg Ile Thr Glu Trp Glu Thr Gly Gln Ile Leu Met Ile Val Gln Cys Lys Asp Asp 301 CAG AGT GTG TTC GAG TCG GAC ATG GGG AAA ACG TGG ACC GGG ACC CTC 360 Gln Ser Val Phe Glu Ser Arg Asp Met Gly Lys Thr Trp Thr Glu Ala Ile Gly Thr Leu 361 TCA GGC GTG TGG ATG TCA CAA CCA GGA GTT CTA TAC AAA ATT TTT CGT GTG GGA 420 Ser Gly Val Trp Val Met Ser Gln Pro Gly Val Arg Leu Tyr Lys Ile Phe Arg Val Gly 421 GCC CTC ATC ACC GCC ACC ATT GAG GGA AAG GTC ATG CTT TAC ACT CAG AGA GGG TAC 480 Wing Leu Ile Thr Wing All Thr ThrGly Tyr 481 ACC TCG GGG GAG AAA GAG GCC ACT GCA CTC TAC CTT TGG GTC ACG GAC AAC AAC CGC ACG 540 Thr Be Gly GlU Ala Thr Ala Leu Tyr Leu Trp Go Thr Asp Asn Arg Thr 541 TTT CAT GTT GGA CCG CTT TTT TTG GAG GAT AAT GTG AAT GAG ACG CTT GCC AAC GCC CTA 600 Phe His Val Gly Pro Leu Phe Leu Glu Asp Asn Val Asn Glu Thr Leu Asn Ala Leu 601 CTG TAC TCG GAT GGT GCG TTG CAC CTT TTA AAG GAG AGG GCC AAT GAA AAA GAC GAG TAG 660 Leu Tyr Ser Asp Gly Gly Leu His Leu Read Lys Glu Arg Wing Asn Glu Lys Asp Asp STOP

Claims (5)

1. "GENETICALLY MODIFIED SEQUENCE OF ANTIGEN ASP-2 DETRYPANOSOMA CRUZI characterized by the nucleotide sequence comprised of Seq. ID no (1). 2. ASP-2 RECOMBINANT PROTEIN characterized by the amino acid sequence comprising Seq. ID at (1). 3. GENETICALLY MODIFIED VIRUS THAT EXPRESSES RECOMBINANT ANTIGENOAS characterized in that it comprises Seq. ID at (1). GENETICALLY MODIFIED VIRUS EXPRESSING THE RECOMBINANT ANTIGENOASP-2 according to claim 03, characterized in that the virus is an influenza virus. GENETICALLY MODIFIED VIRUS THAT EXPRESSES RECOMBINANT ANTIGENOASP-2 according to claim 03, characterized in that the virus is a modified Ankara virus (MVA).